Heparosan-producing bacterium and heparosan manufacturing method
09975928 ยท 2018-05-22
Assignee
Inventors
- Shunsuke Yamazaki (Kanagawa, JP)
- Tomoko Shimizu (Kanagawa, JP)
- Kenichi Mori (Kanagawa, JP)
- Naoto Tonouchi (Kanagawa, JP)
Cpc classification
C12P19/04
CHEMISTRY; METALLURGY
C08B37/0075
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
C12P19/26
CHEMISTRY; METALLURGY
International classification
C12N15/00
CHEMISTRY; METALLURGY
C12P19/34
CHEMISTRY; METALLURGY
C12P19/26
CHEMISTRY; METALLURGY
C12P19/04
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
C07H1/00
CHEMISTRY; METALLURGY
C07K1/00
CHEMISTRY; METALLURGY
Abstract
A method for producing heparosan is provided. Heparosan is produced by culturing an Escherichia bacterium having a heparosan-producing ability and modified so that expression of one or more genes, such as rbsR, rbsK, rbsB, hsrA, glgB, glgX, micF, rcsD, rcsB, ybiX, ybiI, ybiJ, ybiC, ybiB, rfaH, nusG, pcoR, pcoS, pcoE, yhcN, yhcO, aaeB, aaeA, aaeX, g1455, alpA, g1453, yrbA, mlaB, mlaC, mlaD, mlaE, mlaF, yrbG, norW, ybjI, ybjJ, ybjK, rybB, yjjY, yjtD, thrL, thrA, thrB, fruA, psuK, ytfT, yjfF, fbp, yagU, paoA, paoB, gsiC, gsiD, yliE, irp2, irp1, bhsA, ycfS, lepB, rnc, era, dapA, gcvR, bcp, hyfA, rpoE, nadB, yfiC, srmB, g1414, g1413, nuoE, nuoF, nuoG, glmZ, hemY, hemX, hemD, rlmL, artQ, artM, artJ, rlmC, ybjO, yejO, yejM, yejL, rpoS, ygbN, ygbM, ygbL, g3798, g3797, g3796, g3795, g3794, g3793, g3792, ryjA, soxR, soxS, yjcC, yjcB, efeU, and efeO, is/are increased in a medium, and collecting heparosan from the medium.
Claims
1. A method for producing heparin, the method comprising: A) culturing an Escherichia coli bacterium having a heparosan-producing ability in a medium to produce and accumulate heparosan in the medium; B) treating the heparosan using chemical methods, enzymatic methods, or both chemical and enzymatic methods to produce heparin; and C) collecting the heparin; wherein the bacterium has been modified so that expression is increased of an Escherichia coli rpoE gene; wherein the bacterium has been further modified so that expression of an Escherchia coli heparosan biosynthesis enzyme gene is increased.
2. The method according to claim 1, wherein the bacterium has been further modified so that expression of an Escherichia coli rfaH gene is increased.
3. The method according to claim 2, wherein the bacterium has been further modified so that expression is increased of an Escherichia coli nusG gene.
4. The method according to claim 1, wherein said expression is increased by increasing the copy number of the gene(s), modifying a gene expression control sequence of the gene(s), or both.
5. The method according to claim 3, wherein: the rfaH gene is a DNA comprising the nucleotide sequence of SEQ ID NO: 46, or a DNA comprising a nucleotide sequence having an identity of 90% or higher to the nucleotide sequence of SEQ ID NO: 46 and is able to increase heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount thereof is increased in the bacterium; the nusG gene is a DNA comprising the nucleotide sequence of SEQ ID NO: 48, or a DNA comprising a nucleotide sequence having an identity of 90% or higher to the nucleotide sequence of SEQ ID NO: 48 and is able to increase heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount thereof is increased in the bacterium; the rpoE gene is a DNA comprising the complementary sequence of the nucleotide sequence of positions 355 to 930 of SEQ ID NO: 116, or a DNA comprising a nucleotide sequence having an identity of 90% or higher to the complementary sequence of the nucleotide sequence of positions 355 to 930 of SEQ ID NO: 116 and is able to increase heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount thereof is increased in the bacterium.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
DETAILED DESCRIPTION OF THE INVENTION
(3) Hereafter, the present invention will be explained in detail.
(4) <1> Bacterium of the Present Invention
(5) The bacterium of the present invention is an Escherichia bacterium having a heparosan-producing ability that has been modified so that expression of one or more of the genes depicted in Tables 1 to 3 is increased.
(6) <1-1> Bacterium Having Heparosan-Producing Ability
(7) the phrase bacterium having a heparosan-producing ability can refer to a bacterium having an ability to produce and accumulate heparosan in a medium in such a degree that heparosan can be collected, when the bacterium is cultured in the medium. The bacterium having a heparosan-producing ability may be a bacterium that is able to accumulate heparosan in a medium in an amount larger than that obtainable with a non-modified strain. Examples of the non-modified strain include wild-type strains and parent strains of the bacterium. The bacterium having a heparosan-producing ability may be a bacterium that is able to accumulate heparosan in a medium in an amount of, for example, 50 mg/L or more, 100 mg/L or more, 200 mg/L or more, or 300 mg/L or more.
(8) The Escherichia bacterium is not particularly limited, and examples thereof include those classified into the genus Escherichia according to the taxonomy known to those skilled in the field of microbiology. Examples of the Escherichia bacterium include, for example, those described in the work of Neidhardt et al. (Backmann B. J., 1996, Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, pp. 2460-2488, Table 1, In F. D. Neidhardt (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology, Second Edition, American Society for Microbiology Press, Washington, D.C.). Examples of Escherichia bacterium include, for example, Escherichia coli. Specific examples of Escherichia coli include, for example, Escherichia coli K-12 strains such as Escherichia coli W3110 strain (ATCC 27325) and MG1655 strain (ATCC 47076); Escherichia coli K5 strain (ATCC 23506); Escherichia coli B strains such as BL21(DE3) strain; and their derivative strains.
(9) These strains are available from, for example, the American Type Culture Collection (Address: 12301 Parklawn Drive, Rockville, Md. 20852, P.O. Box 1549, Manassas, Va. 20108, United States of America). That is, registration numbers are given to the respective strains, and the strains can be ordered by using these registration numbers (refer to atcc.org/). The registration numbers of the strains are listed in the catalogue of the American Type Culture Collection. The BL21(DE3) strain is also available from, for example, Life Technologies (product number C6000-03).
(10) The bacterium of the present invention may be a bacterium inherently having a heparosan-producing ability, or may be a bacterium modified so that it has a heparosan-producing ability. The bacterium having a heparosan-producing ability can be obtained by, for example, imparting a heparosan-producing ability to such a bacterium as mentioned above.
(11) A heparosan-producing ability can be imparted by introducing a gene encoding a protein that participates in heparosan production. Examples of such a protein that participates in heparosan production include glycosyltransferase and heparosan efflux carrier protein. In the present invention, one gene or two or more different genes may be introduced. A gene may be introduced in the same manner as that of the method of increasing copy number of gene described later.
(12) The term glycosyltransferase referred to herein means a protein having an activity for catalyzing a reaction of adding N-acetyl-D-glucosamine (GlcNAc) and/or glucuronic acid (GlcUA) to a non-reducing end of a sugar chain to thereby extend a heparosan chain. This activity is also referred to as glycosyltransferase activity. Examples of the gene encoding glycosyltransferase include the kfiA gene, kfiC gene, and pmHS1 gene.
(13) Examples of the kfiA gene and kfiC gene include the kfiA gene and kfiC gene of the Escherichia coli K5 strain. The KfiA protein encoded by the kfiA gene of the Escherichia coli K5 strain adds GlcNAc to a non-reducing end of a sugar chain by using UDP-GlcNAc as a substrate. The KfiC protein encoded by the kfiC gene of the Escherichia coli K5 strain adds GlcUA to a non-reducing end of a sugar chain by using UDP-GlcUA as a substrate. The kfiA and kfiC genes of the Escherichia coli K5 strain constitute the kfiABCD operon (also referred to as region 2) together with the kfiB and kfiD genes. The nucleotide sequence of a region containing the kfiABCD operon of the Escherichia coli K5 strain is shown as SEQ ID NO: 24. In the nucleotide sequence of SEQ ID NO: 24, the kfiA, kfiB, kfiC, and kfiD genes correspond to the sequence of the positions 445 to 1,164, the sequence of the positions 1,593 to 3,284, the sequence of the positions 4,576 to 6,138, and the sequence of the positions 6,180 to 7,358, respectively. The amino acid sequences of KfiA, KfiB, KfiC, and KfiD proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 25 to 28, respectively.
(14) Examples of the pmHS1 gene include the pmHS1 gene of the Pasteurella multocida type D strain. The PmHS1 protein encoded by the pmHS1 gene of the Pasteurella multocida type D strain alternately adds GlcNAc and GlcUA to a non-reducing end of a sugar chain by using both UDP-GlcNAc and UDP-GlcUA as substrates. The nucleotide sequence of the pmHS1 gene of the Pasteurella multocida type D strain and the amino acid sequence of the protein encoded by this gene can be obtained from public databases such as the NCBI database (ncbi.nlm.nih.gov/).
(15) The phrase heparosan efflux carrier protein can mean a protein having the activity of excreting a heparosan chain out of a cell through cell membranes. This activity can also referred to as heparosan efflux activity. Examples of a gene encoding the heparosan efflux carrier protein include the kpsC, kpsD, kpsE, kpsM, kpsS, and kpsT genes. Examples of the kpsC, kpsD, kpsE, kpsM, kpsS, and kpsT genes include the kpsC, kpsD, kpsE, kpsM, kpsS, and kpsT genes of the Escherichia coli K5 strain and Escherichia coli B strain. The kpsC, kpsD, kpsE, and kpsS genes of these strains constitute the kpsFEDUCS operon (also referred to as region 1) together with the kpsF and kpsU genes. The kpsM and kpsT genes of these strains constitute the kpsMT operon (also referred to as region 3). The nucleotide sequences of the kpsC, kpsD, kpsE, kpsM, kpsS, and kpsT genes of these strains, and the amino acid sequences of the proteins encoded by these genes can be obtained from public databases such as the NCBI database (ncbi.nlm.nih.gov/).
(16) The gene to be introduced can be appropriately chosen according to type of the bacterium to be used, and so forth. For example, the Escherichia coli B strain has genes encoding a heparosan efflux carrier protein, but it does not have genes encoding glycosyltransferase. Therefore, a heparosan-producing ability can be imparted to the Escherichia coli B strain by introducing gene(s) encoding glycosyltransferase. Furthermore, for example, the Escherichia coli K-12 strain does not have either genes encoding glycosyltransferase or genes encoding a heparosan efflux carrier protein. Therefore, a heparosan-producing ability can be imparted to the Escherichia coli K-12 strain by introducing both gene(s) encoding glycosyltransferase and genes encoding a heparosan efflux carrier protein.
(17) Thus, examples of the Escherichia bacterium having a heparosan-producing ability include, for example, Escherichia coli K5 strain; Escherichia coli B strain such as BL21(DE3) strain introduced with the kfiA gene and kfiC gene of the Escherichia coli K5 strain; Escherichia coli K-12 strain such as W3110 strain and MG1655 strain introduced with the kfiA gene and kfiC gene of the Escherichia coli K5 strain, as well as the kpsC, kpsD, kpsE, kpsM, kpsS, and kpsT genes of the Escherichia coli K5 strain or Escherichia coli B strain; and derivative strains thereof. Specific examples of Escherichia coli B strain introduced with the kfiA gene and kfiC gene of the Escherichia coli K5 strain include, for example, the Escherichia coli BL21(DE3)/pVK9-region2 described in Examples.
(18) The bacterium having a heparosan-producing ability may also be a bacterium that has been modified so that expression is enhanced of a gene encoding a protein involved in the heparosan production and that is inherently present in the bacterium. That is, for example, the Escherichia coli K5 strain may be modified so that expression of one or more genes encoding a protein that participates in the heparosan production is enhanced. Furthermore, for example, the Escherichia coli B strain may be modified so that expression of one or more genes encoding a heparosan efflux carrier protein is enhanced.
(19) The bacterium having a heparosan-producing ability may have been further modified in other ways so long as the heparosan-producing ability is not degraded. For example, the bacterium having a heparosan-producing ability may have been modified so that expression of one or more genes, such as kfiB, kfiD, kpsF, and kpsU, is enhanced. That is, when genes encoding glycosyltransferase are introduced, for example, region 2 may be entirely introduced, and when genes encoding glycosyltransferase and genes encoding a heparosan efflux carrier protein are introduced, regions 1 to 3 may be entirely introduced.
(20) The gene used to modify a bacterium, so that, for example, impartation of a heparosan-producing ability, is not limited to the genes exemplified above or genes having a known nucleotide sequence, but may be a variant of such genes, so long as the variant encodes a protein that maintains the original function. The expression protein maintains the original function means that, in the case of the glycosyltransferase, for example, the variant of the protein has the glycosyltransferase activity, or in the case of the heparosan efflux carrier protein, the variant of the protein has the heparosan efflux activity. For example, the gene used for modification of the bacterium such as impartation of a heparosan-producing ability may be a gene encoding a protein having a known amino acid sequence including substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions. To variants of genes or proteins, the descriptions for conservative variants of the genes depicted in Tables 1 to 3 and the proteins encoded by them can be similarly applied.
(21) <1-2> Increase in Expression of Genes Depicted in Tables 1 to 3
(22) The bacterium of the present invention has been modified so that expression of one or more genes such as those depicted in Tables 1 to 3 is increased. The bacterium of the present invention can be obtained by modifying a bacterium having a heparosan-producing ability so that expression of one or more genes such as those depicted in Tables 1 to 3 is increased. The bacterium of the present invention can also be obtained by modifying a bacterium so that expression of one or more genes such as those depicted in Tables 1 to 3 is increased, and then imparting a heparosan-producing ability to the bacterium. The bacterium of the present invention may be a bacterium that has acquired a heparosan-producing ability as a result of the modification for increasing expression of one or more genes such as those depicted in Tables 1 to 3. In the present invention, modifications for constructing the bacterium of the present invention can be performed in an arbitrary order.
(23) The genes depicted in Tables 1 to 3 are, specifically, rbsR, rbsK, rbsB, hsrA, glgB, glgX, micF, rcsD, rcsB, ybiX, ybiI, ybiJ, ybiC, ybiB, rfaH, nusG, pcoR, pcoS, pcoE, yhcN, yhcO, aaeB, aaeA, aaeX, g1455, alpA, g1453, yrbA, mlaB, mlaC, mlaD, mlaE, mlaF, yrbG, norW, ybjI, ybjJ, ybjK, rybB, yjjY, yjtD, thrL, thrA, thrB, fruA, psuK, ytfT, yjfF, fbp, yagU, paoA, paoB, gsiC, gsiD, yliE, irp2, irp1, bhsA, ycfS, lepB, rnc, era, dapA, gcvR, bcp, hyfA, rpoE, nadB, yfiC, srmB, g1414, g1413, nuoE, nuoF, nuoG, glmZ, hemY, hemX, hemD, rlmL, artQ, artM, artJ, rlmC, ybjO, yejO, yejM, yejL, rpoS, ygbN, ygbM, ygbL, g3798, g3797, g3796, g3795, g3794, g3793, g3792, ryjA, soxR, soxS, yjcC, yjcB, efeU, and efeO. The genes depicted in Tables 1 to 3 are also referred to as the genes of Tables 1 to 3.
(24) The rbsR, rbsK, and rbsB genes are genes encoding factors that participate in uptake of D-ribose. The rbsR gene encodes the repressor of the rbs operon. The rbsK gene encodes a ribokinase. The rbsB gene encodes one of the subunits of the ribose ABC transporter. The rbsR, rbsK, and rbsB genes of the Escherichia coli K-12 MG1655 strain correspond to the sequence of the positions 3,936,250 to 3,937,242, the sequence of the positions 3,935,317 to 3,936,246, and the sequence of the positions 3,934,301 to 3,935,191 of the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The RbsR, RbsK, and RbsB proteins of the MG1655 strain are registered as GenBank accession NP 418209 (version NP_418209.1 GI: 16131621), GenBank accession NP_418208 (version NP_418208.1 GI: 16131620), and GenBank accession NP_418207 (version NP_418207.1 GI: 16131619), respectively.
(25) The hsrA gene encodes an inner membrane protein presumed to be a member of the major facilitator superfamily (MFS). The hsrA gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the sequence of the positions 3,937,208 to 3,938,635 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The HsrA protein of the MG1655 strain is registered as GenBank accession NP_418210 (version NP_418210.1 GI: 16131622).
(26) The nucleotide sequence of a region containing the rbsB, rbsK, rbsR, and hsrA genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 29. In the nucleotide sequence shown as SEQ ID NO: 29, the rbsB, rbsK, and rbsR genes correspond to the sequence of the positions 800 to 1,690, the sequence of the positions 1,816 to 2,745, and the sequence of the positions 2,749 to 3,741, respectively. In the nucleotide sequence shown as SEQ ID NO: 29, the hsrA gene corresponds to the complementary sequence of the sequence of the positions 3,707 to 5,134. The amino acid sequences of RbsR, RbsK, RbsB, and HsrA proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 30 to 33, respectively.
(27) The glgB gene encodes a glycogen branching enzyme (1,4--glucan branching enzyme). The glgX gene encodes a glycogen debranching enzyme. The glgB and glgX genes of the Escherichia coli K-12 MG1655 strain correspond to the complementary sequence of the sequence of the positions 3,569,339 to 3,571,525, and the complementary sequence of the sequence of the positions 3,567,369 to 3,569,342 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The GlgB and GlgX proteins of the MG1655 strain are registered as GenBank accession NP_417890 (version NP_417890.1 GI:16131306) and GenBank accession NP_417889 (version NP_417889.1 GI: 16131305), respectively.
(28) The nucleotide sequence of a region containing the glgB and glgX genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 34. In the nucleotide sequence shown as SEQ ID NO: 34, the glgB and glgX genes correspond to the sequence of the positions 989 to 3,175, and the sequence of the positions 3,172 to 5,145, respectively. The amino acid sequences of the GlgB and GlgX proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 35 and 36, respectively.
(29) The micF gene encodes an antisense RNA that participates in the expression inhibition of OmpF. The micF gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 2,311,106 to 2,311,198 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990).
(30) The rcsD and rcsB genes encode a transcription factor. The rcsD and rcsB genes of the Escherichia coli K-12 MG1655 strain correspond to the sequences of the positions 2,311,510 to 2,314,182, and the positions 2,314,199 to 2,314,849 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The RcsD and RcsB proteins of the MG1655 strain are registered as GenBank accession NP_416720 (version NP_416720.1 GI:16130153) and GenBank accession NP_416721 (version NP_416721.1 GI: 16130154), respectively.
(31) The nucleotide sequence of a region containing the rcsB, rcsD, and micF genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 43. In the nucleotide sequence shown as SEQ ID NO: 43, the rcsB, rcsD, and micF genes correspond to the sequence of the positions 3,312 to 3,962, the sequence of the positions 623 to 3,295, and the sequence of the positions 219 to 311, respectively. The amino acid sequences of RcsB and RcsD proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 44 and 45, respectively.
(32) The ybiX, ybiI, ybiJ, ybiC, and ybiB genes are genes of unknown functions. The ybiX, ybiI, ybiJ, ybiC, and ybiB genes of the Escherichia coli K-12 MG1655 strain correspond to the complementary sequence of the sequence of the positions 837,753 to 838,430, the complementary sequence of the sequence of the positions 837,413 to 837,679, the complementary sequence of the sequence of the positions 836,888 to 837,148, the sequence of the positions 835,574 to 836,659, and the sequence of the positions 834,471 to 835,433 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The YbiX, Ybil, YbiJ, YbiC, and YbiB proteins of the MG1655 strain are registered as GenBank accession NP_415325 (version NP_415325.4 GI: 90111170), GenBank accession NP_415324 (version NP_415324.1 GI: 16128771), GenBank accession NP_415323 (version NP_415323.1 GI: 16128770), GenBank accession NP_415322 (version NP_415322.1 GI: 16128769), and GenBank accession NP_415321 (version NP_415321.1 GI: 16128768), respectively.
(33) The nucleotide sequence of a region containing the ybiX, ybiI, ybiJ, ybiC, and ybiB genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 37. In the nucleotide sequence shown as SEQ ID NO: 37, the ybiX, ybiI, and ybiJ genes correspond to the sequence of the positions 718 to 1,395, the sequence of the positions 1,469 to 1,735, and the sequence of the positions 2,000 to 2,260, respectively. In the nucleotide sequence shown as SEQ ID NO: 37, the ybiC and ybiB genes correspond to the complementary sequence of the sequence of the positions 2,488 to 3,574, and the complementary sequence of the sequence of the positions 3,715 to 4,677. The amino acid sequences of the YbiX, Ybil, YbiJ, YbiC, and YbiB proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 38 to 42, respectively.
(34) The rfaH and nusG genes encode a transcription factor. The rfaH and nusG genes of the Escherichia coli K-12 MG1655 strain correspond to the complementary sequence of the sequence of the positions 4,022,356 to 4,022,844, and the sequence of the positions 4,175,766 to 4,176,311 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The RfaH and NusG proteins of the MG1655 strain are registered as GenBank accession NP_418284 (version NP_418284.1 GI:16131688) and GenBank accession NP_418409 (version NP_418409.1 GI: 16131812), respectively.
(35) The nucleotide sequence of the rfaH gene of the Escherichia coli BL21(DE3) strain, and the amino acid sequence of the RfaH protein encoded by this gene are shown as SEQ ID NOS: 46 and 47, respectively. The nucleotide sequence of the nusG gene of the Escherichia coli BL21(DE3) strain, and the amino acid sequence of the NusG protein encoded by this gene are shown as SEQ ID NOS: 48 and 49, respectively.
(36) The pcoR, pcoS, and pcoE genes are genes encoding a factor that relates to copper resistance. The pcoR gene encodes a protein homologous to the activator of the pco operon. The pcoS gene encodes a protein homologous to the sensor protein of a two-component control system. The pcoE gene encodes a copper binding protein. These genes are not annotated in the genome of the Escherichia coli K-12 MG1655 strain.
(37) The nucleotide sequence of a region containing the pcoR, pcoS, and pcoE genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 50. In the nucleotide sequence shown as SEQ ID NO: 50, the pcoR, pcoS, and pcoE genes correspond to the sequence of the positions 128 to 808, the sequence of the positions 805 to 2,205, and the sequence of the positions 2,423 to 2,857, respectively. The amino acid sequences of the PcoR, PcoS, and PcoE proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 51 to 53, respectively.
(38) The yhcN gene encodes a factor that participates in stress response. The yhcN gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 3,383,560 to 3,383,823 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YhcN protein of the MG1655 strain is registered as GenBank accession NP_417705 (version NP_417705.2 GI: 90111561).
(39) The yhcO gene encodes a protein homologous to an inhibitor for RNase. The yhcO gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the sequence of the positions 3,383,879 to 3,384,151 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YhcO protein of the MG1655 strain is registered as GenBank accession NP_417706 (version NP_417706.1 GI: 16131129).
(40) The aaeB and aaeA genes encode a subunit of an efflux carrier of 4-hydroxybenzoic acid. The aaeX gene encodes a protein presumed to be an efflux carrier. The aaeB, aaeA, and aaeX genes of the Escherichia coli K-12 MG1655 strain correspond to the complementary sequence of the sequence of the positions 3,384,243 to 3,386,210, the complementary sequence of the sequence of the positions 3,386,216 to 3,387,148, and the complementary sequence of the sequence of the positions 3,387,156 to 3,387,359 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The AaeB, AaeA, and AaeX proteins of the MG1655 strain are registered as GenBank accession NP_417707 (version NP_417707.1 GI: 16131130), GenBank accession NP_417708 (version NP_417708.1 GI: 16131131), and GenBank accession NP_417709 (version NP_417709.2 GI: 90111562), respectively.
(41) The nucleotide sequence of a region containing the yhcN, yhcO, aaeB, aaeA, and aaeX genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 54. In the nucleotide sequence shown as SEQ ID NO: 54, the yhcN, yhcO, aaeB, aaeA, and aaeX genes correspond to the sequence of the positions 63 to 326, the complementary sequence of the sequence of the positions 382 to 654, the complementary sequence of the sequence of the positions 746 to 2,713, the complementary sequence of the sequence of the positions 2,719 to 3,651, and the complementary sequence of the sequence of the positions 3,659 to 3,931, respectively. The amino acid sequences of the YhcN, YhcO, AaeB, AaeA, and AaeX proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 55 to 59, respectively.
(42) The g1455 and g1453 genes are genes of unknown functions. These genes are not annotated in the genome of the Escherichia coli K-12 MG1655 strain.
(43) The alpA gene encodes an expression control factor of the intA gene. The alpA gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 2,756,666 to 2,756,878 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The AlpA protein of the MG1655 strain is registered as GenBank accession NP_417113 (version NP_417113.1 GI: 16130542).
(44) The nucleotide sequence of a region containing the g1455, alpA, and g1453 genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 60. In the nucleotide sequence shown as SEQ ID NO: 60, the g1455, alpA, and g1453 genes correspond to the complementary sequence of the sequence of the positions 568 to 1,140, the complementary sequence of the sequence of the positions 1,226 to 1,486, and the sequence of the positions 2,389 to 2,529, respectively. The amino acid sequences of G1455, AlpA, and G1453 proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 61 to 63, respectively.
(45) The yrbA gene (synonym is ibaG) encodes a protein presumed to be a DNA-binding type transcription factor. The yrbA gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the sequence of the positions 3,334,571 to 3,334,825 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YrbA protein of the MG1655 strain is registered as GenBank accession NP_417657 (version NP_417657.2 GI: 90111555).
(46) The mlaB, mlaC, mlaD, mlaE, and mlaF genes encode a constituent factor of a phospholipid ABC transporter. The mlaB, mlaC, mlaD, mlaE, and mlaF genes of the Escherichia coli K-12 MG1655 strain correspond to the complementary sequence of the sequence of the positions 3,334,985 to 3,335,278, the complementary sequence of the sequence of the positions 3,335,278 to 3,335,913, the complementary sequence of the sequence of the positions 3,335,932 to 3,336,483, the complementary sequence of the sequence of the positions 3,336,488 to 3,337,270, and the complementary sequence of the sequence of the positions 3,337,278 to 3,338,087 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The MlaB, MlaC, MlaD, MlaE, and MlaF proteins of the MG1655 strain are registered as GenBank accession NP_417658 (version NP_417658.4 GI: 90111556), GenBank accession NP_417659 (version NP_417659.1 GI: 16131082), GenBank accession NP_417660 (version NP_417660.1 GI: 16131083), GenBank accession NP_417661 (version NP_417661.1 GI: 16131084), and GenBank accession NP_417662 (version NP_417662.1 GI: 16131085), respectively.
(47) The yrbG gene encodes a protein presumed to be an Na.sup.+/Ca.sup.2+ antiporter. The yrbG gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 3,338,297 to 3,339,274 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YrbG protein of the MG1655 strain is registered as GenBank accession NP_417663 (version NP_417663.1 GI: 16131086).
(48) The nucleotide sequence of a region containing the yrbA, mlaB, mlaC, mlaD, mlaE, mlaF, and yrbG genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 64. In the nucleotide sequence shown as SEQ ID NO: 64, the yrbA, mlaB, mlaC, mlaD, mlaE, mlaF, and yrbG genes correspond to the complementary sequence of the sequence of the positions 977 to 1,246, the complementary sequence of the sequence of the positions 1,391 to 1,780, the complementary sequence of the sequence of the positions 1,684 to 2,319, the complementary sequence of the sequence of the positions 2,338 to 2,889, the complementary sequence of the sequence of the positions 2,894 to 3,676, the complementary sequence of the sequence of the positions 3,684 to 4,493, and the sequence of the positions 4,703 to 5,680, respectively. The amino acid sequences of YrbA, MlaB, MlaC, MlaD, MlaE, MlaF, and YrbG proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 65 to 71, respectively.
(49) The norW gene encodes an NO reductase. The norW gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 2,831,934 to 2,833,067 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The NorW protein of the MG1655 strain is registered as GenBank accession NP_417191 (version NP_417191.1 GI: 16130618).
(50) The nucleotide sequence of a region containing the norW gene of the Escherichia coli K5 strain is shown as SEQ ID NO: 72. In the nucleotide sequence shown as SEQ ID NO: 72, the norW gene corresponds to the sequence of the positions 1,201 to 2,334. The amino acid sequence of the NorW protein of the Escherichia coli K5 strain is shown as SEQ ID NO: 73.
(51) The ybjI gene encodes a flavin mononucleotide (FMN) phosphorylating enzyme. The ybjI gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the sequence of the positions 884,539 to 885,354 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YbjI protein of the MG1655 strain is registered as GenBank accession NP_415365 (version NP_415365.4 GI: 90111176).
(52) The ybjJ and ybjK genes are genes of unknown function. The ybjJ and ybjK genes of the Escherichia coli K-12 MG1655 strain correspond to the complementary sequence of the sequence of the positions 885,354 to 886,562, and the sequence of the positions 886,646 to 887,182 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The YbjJ and YbjK proteins of the MG1655 strain are registered as GenBank accession NP_415366 (version NP_415366.1 GI:16128813) and GenBank accession NP_415367 (version NP_415367.1 GI:16128814), respectively.
(53) The rybB gene encodes a low molecular weight RNA that participates in expression inhibition of OmpC and OmpW. The rybB gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the sequence of the positions 887,199 to 887,277 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990).
(54) The nucleotide sequence of a region containing the ybjI, ybjJ, ybjK, and rybB genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 74. In the nucleotide sequence shown as SEQ ID NO: 74, the ybjI, ybjJ, ybjK, and rybB genes correspond to the complementary sequence of the sequence of the positions 117 to 932, the complementary sequence of the sequence of the positions 932 to 2,140, the sequence of the positions 2,224 to 2,760, and the complementary sequence of the sequence of the positions 2,777 to 2,855, respectively. The amino acid sequences of the YbjI, YbjJ, and YbjK proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 75 to 77, respectively.
(55) The yjjY gene is a gene of unknown function. The yjjY gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 4,638,425 to 4,638,565 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YjjY protein of the MG1655 strain is registered as GenBank accession NP_418819 (version NP_418819.1 GI: 16132219).
(56) The yjtD gene encodes a protein presumed to be one of RNA methyltransferases. The yjtD gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 4,638,965 to 4,639,651 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YjtD protein of the MG1655 strain is registered as GenBank accession NP_418820 (version NP_418820.1 GI: 16132220).
(57) The thrL, thrA, and thrB genes encode an enzyme of the threonine biosynthesis pathway. The thrB gene encodes a homoserine kinase. The thrA gene encodes an enzyme having two functions of aspartate kinase I and homoserine dehydrogenase I. The thrL gene encodes a leader peptide of the thrLABC operon. The thrL, thrA, and thrB genes of the Escherichia coli K-12 MG1655 strain correspond to the sequence of the positions 190 to 255, the sequence of the positions 337 to 2,799, and the sequence of the positions 2,801 to 3,733 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The ThrL, ThrA, and ThrB proteins of the MG1655 strain are registered as GenBank accession NP_414542 (version NP_414542.1 GI: 16127995), GenBank accession NP_414543 (version NP_414543.1 GI: 16127996), and GenBank accession NP_414544 (version NP_414544.1 GI: 16127997), respectively.
(58) The nucleotide sequence of a region containing the yjjY, yjtD, thrL, thrA, and thrB genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 78. In the nucleotide sequence shown as SEQ ID NO: 78, the yjjY, yjtD, thrL, thrA, and thrB genes correspond to the sequence of the positions 124 to 264, the sequence of the positions 664 to 1,350, the sequence of the positions 1,564 to 1,629, the sequence of the positions 1,711 to 4,173, and the sequence of the positions 4,175 to 5,107, respectively. The amino acid sequences of the YjjY, YjtD, ThrL, ThrA, and ThrB proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 79 to 83, respectively.
(59) The fruA gene encodes a fructose PTS permease. The fruA gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the sequence of the positions 2,257,741 to 2,259,432 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The FruA protein of the MG1655 strain is registered as GenBank accession NP_416672 (version NP_416672.1 GI: 16130105).
(60) The psuK gene encodes a pseudouridine kinase. The psuK gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the sequence of the positions 2,256,377 to 2,257,318 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The PsuK protein of the MG1655 strain is registered as GenBank accession NP_416671 (version NP_416671.1 GI: 16130104).
(61) The nucleotide sequence of a region containing the fruA and psuK genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 84. In the nucleotide sequence shown as SEQ ID NO: 84, the fruA and psuK genes correspond to the sequence of the positions 897 to 2588, and the sequence of the positions 3,165 to 3,953, respectively. The amino acid sequences of the FruA and PsuK proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 85 and 86, respectively.
(62) The ytfT and yjfF genes are genes encoding a protein presumed to be a membrane constituent component of the galactose ABC transport carrier. The ytfT and yjfF genes of the Escherichia coli K-12 MG1655 strain correspond to the sequence of the positions 4,450,594 to 4,451,619 and the sequence of the positions 4,451,606 to 4,452,601 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The YtfT and YjfF proteins of the MG1655 strain are registered as GenBank accession NP_418651 (version NP_418651.3 GI:145698343) and GenBank accession NP_418652 (version NP_418652.2 GI: 90111710), respectively.
(63) The fbp gene encodes a fructose-1,6-diphosphate phosphatase (fructose-1,6-bisphosphatase). The fbp gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the sequence of the positions 4,452,634 to 4,453,632 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The Fbp protein of the MG1655 strain is registered as GenBank accession NP_418653 (version NP_418653.1 GI: 16132054).
(64) The nucleotide sequence of a region containing the ytfT, yjfF, and fbp genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 87. In the nucleotide sequence shown as SEQ ID NO: 87, the ytfT, yjfF, and fbp genes correspond to the sequence of the positions 252 to 1,277, the sequence of the positions 1,264 to 2,259, and the complementary sequence of the sequence of the positions 2,292 to 3,290, respectively. The amino acid sequences of the YtfT, YjfF, and Fbp proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 88 to 90, respectively.
(65) The yagU gene encodes a protein presumed to be an inner membrane protein. The yagU gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 302,215 to 302,829 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YagU protein of the MG1655 strain is registered as GenBank accession NP_414821 (version NP_414821.1 GI: 16128272).
(66) The paoA gene (also called yagT) and paoB gene (also called yagS) are genes encoding a constituent factor of an aldehyde oxidoreductase. The paoA and paoB genes of the Escherichia coli K-12 MG1655 strain correspond to the complementary sequence of the sequence of the positions 301,108 to 301,797, and the complementary sequence of the sequence of the positions 300,155 to 301,111 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The PaoA and PaoB proteins of the MG1655 strain are registered as GenBank accession NP_414820 (version NP_414820.1 GI:16128271) and GenBank accession NP_414819 (version NP_414819.1 GI: 16128270), respectively.
(67) The nucleotide sequence of a region containing the yagU, paoA, and paoB genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 91. In the nucleotide sequence shown as SEQ ID NO: 91, the yagU, paoA, and paoB genes correspond to the complementary sequence of the sequence of the positions 117 to 731, the sequence of the positions 1,149 to 1,838, and the sequence of the positions 1,835 to 2,791, respectively. The amino acid sequences of the YagU, PaoA, and PaoB proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 92 to 94, respectively.
(68) The gsiC and gsiD genes are genes encoding a constituent factor of a glutathione ABC transport carrier. The gsiC and gsiD genes of the Escherichia coli K-12 MG1655 strain correspond to the sequence of the positions 870,190 to 871,110, and the sequence of the positions 871,113 to 872,024 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The GsiC and GsiD proteins of the MG1655 strain are registered as GenBank accession NP_415352 (version NP_415352.1 GI:16128799) and GenBank accession NP_415353 (version NP_415353.1 GI: 16128800), respectively.
(69) The yliE gene encodes a protein presumed to be a c-di-GMP-specific phosphodiesterase. The yliE gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 872,202 to 874,550 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YliE protein of the MG1655 strain is registered as GenBank accession NP_415354 (version NP_415354.1 GI: 16128801).
(70) The nucleotide sequence of a region containing the gsiC, gsiD, and yliE genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 95. In the nucleotide sequence shown as SEQ ID NO: 95, the gsiC, gsiD, and yliE genes correspond to the sequence of the positions 264 to 1,184, the sequence of the positions 1,187 to 2,098, and the sequence of the positions 2,276 to 4,624, respectively. The amino acid sequences of the GsiC, GsiD, and YliE proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 96 to 98, respectively.
(71) The irp2 and irp1 genes encode a non-ribosormal peptide synthetase. The irp2 and irp1 genes are not annotated in the genome of the Escherichia coli K-12 MG1655 strain. In the present invention, the irp2 and irp1 gene may be generically called irp gene.
(72) The nucleotide sequence of a region containing a part of the irp gene of the Escherichia coli K5 strain is shown as SEQ ID NO: 99. This region contains the second half moiety of the irp2 gene (moiety of the positions 2,781 to 6,108 in the full length of 6108 bp, equivalent to about 54% of the full length), and the first half moiety of the irp1 gene (moiety of the positions 1 to 2,530 in the full length of 9492 bp, equivalent to about 27% of the full length). The nucleotide sequence of the irp2 gene of the Escherichia coli K5 strain and the amino acid sequence of the Irp2 protein encoded by that gene are shown as SEQ ID NOS: 100 and 101, respectively. The nucleotide sequence of the irp1 gene of the Escherichia coli K5 strain and the amino acid sequence of the Irp1 protein encoded by that gene are shown as SEQ ID NOS: 102 and 103, respectively.
(73) The bhsA gene (synonym is ycfR) encodes a protein presumed to be an outer membrane protein. The bhsA gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 1,168,296 to 1,168,553 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The BhsA protein of the MG1655 strain is registered as GenBank accession NP_415630 (version NP_415630.1 GI: 16129075).
(74) The ycJS gene encodes one of L,D-transpeptidases. The ycJS gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the sequence of the positions 1,168,635 to 1,169,597 in the genome sequence registered at the NCBI database as GenBank accessionNC_000913 (VERSION NC_000913.2 GI: 49175990). The YcfS protein of the MG1655 strain is registered as GenBank accession NP_415631 (version NP_415631.1 GI: 16129076).
(75) The nucleotide sequence of a region containing the bhsA and ycfS genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 104. In the nucleotide sequence shown as SEQ ID NO: 104, the bhsA and ycJS genes correspond to the sequence of the positions 440 to 697, and the complementary sequence of the sequence of the positions 779 to 1,741, respectively. The amino acid sequences of the BhsA and YcfS proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 105 and 106, respectively.
(76) The lepB gene encodes a signal peptidase. The lepB gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the sequence of the positions 2,702,357 to 2,703,331 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The LepB protein of the MG1655 strain is registered as GenBank accession NP_417063 (version NP_417063.1 GI: 16130493).
(77) The rnc gene encodes an RNaseIII. The rnc gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the sequence of the positions 2,701,405 to 2,702,085 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The Rnc protein of the MG1655 strain is registered as GenBank accession NP_417062 (version NP_417062.1 GI: 16130492).
(78) The era gene encodes a factor indispensable to survival. The era gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the sequence of the positions 2,700,503 to 2,701,408 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The Era protein of the MG1655 strain is registered as GenBank accession NP_417061 (version NP_417061.1 GI: 16130491).
(79) The nucleotide sequence of a region containing the lepB, rnc, and era genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 107. In the nucleotide sequence shown as SEQ ID NO: 107, the lepB, rnc, and era genes correspond to the sequence of the positions 1,344 to 2,318, the sequence of the positions 2,590 to 3,270, and the sequence of the positions 3,267 to 4,172, respectively. The amino acid sequences of the LepB, Rnc, and Era proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 108 to 110, respectively.
(80) The dapA gene encodes a 4-hydroxy-tetrahydrodipicolinate synthase. The dapA gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the sequence of the positions 2,596,904 to 2,597,782 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The DapA protein of the MG1655 strain is registered as GenBank accession NP_416973 (version NP_416973.1 GI: 16130403).
(81) The gcvR gene encodes a protein presumed to be a transcription control factor. The gcvR gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 2,597,928 to 2,598,500 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The GcvR protein of the MG1655 strain is registered as GenBank accession NP_416974 (version NP_416974.4 GI: 90111443).
(82) The bcp gene encodes a thiol peroxidase. The bcp gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 2,598,500 to 2,598,970 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The Bcp protein of the MG1655 strain is registered as GenBank accessionNP_416975 (version NP_416975.1 GI: 16130405).
(83) The hyfA gene encodes a protein presumed to participate in the electron transportation. The hyfA gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 2,599,223 to 2,599,840 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The HyfA protein of the MG1655 strain is registered as GenBank accession NP_416976 (version NP_416976.4 GI: 90111444).
(84) The nucleotide sequence of a region containing the dapA, gcvR, bcp, and hyfA genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 111. In the nucleotide sequence shown as SEQ ID NO: 111, the dapA, gcvR, bcp, and hyfA genes correspond to the complementary sequence of the sequence of the positions 858 to 1,736, the sequence of the positions 1,882 to 2,454, the sequence of the positions 2,454 to 2,924, and the sequence of the positions 3,177 to 3,794, respectively. The amino acid sequences of the DapA, GcvR, Bcp, and HyfA proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 112 to 115, respectively.
(85) The rpoE gene encodes SigmaE (.sup.E). The rpoE gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the sequence of the positions 2,707,459 to 2,708,034 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The RpoE protein of the MG1655 strain is registered as GenBank accession NP_417068 (version NP_417068.1 GI: 16130498).
(86) The nadB gene encodes an L-aspartic acid oxidase. The nadB gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 2,708,442 to 2,710,064 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The NadB protein of the MG1655 strain is registered as GenBank accession NP_417069 (version NP_417069.1 GI: 16130499).
(87) The yfiC gene encodes a methyltransferase that methylates N at the position 6 of A37 (adenine at the position 37) of valine tRNA. The yfiC gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the sequence of the positions 2,710,049 to 2,710,786 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YfiC protein of the MG1655 strain is registered as GenBank accession NP_417070 (version NP_417070.2 GI: 90111461).
(88) The srmB gene encodes a DEAD-box type RNA helicase. The srmB gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 2,710,918 to 2,712,252 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The SrmB protein of the MG1655 strain is registered as GenBank accession NP_417071 (version NP_417071.1 GI: 16130501).
(89) The nucleotide sequence of a region containing the rpoE, nadB, yfiC, and srmB genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 116. In the nucleotide sequence shown as SEQ ID NO: 116, the rpoE, nadB, yfiC, and srmB genes correspond to the complementary sequence of the sequence of the positions 355 to 930, the sequence of the positions 1,338 to 2,960, the complementary sequence of the sequence of the positions 2,945 to 3,682, and the sequence of the positions 3,814 to 5,148, respectively. Among these, the nucleotide sequence of the rpoE gene of the Escherichia coli K5 strain is especially shown as SEQ ID NO: 174. The amino acid sequences of the RpoE, NadB, YfiC, and SrmB proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 117 to 120, respectively.
(90) The g1414 and g1413 genes are genes of unknown function. These genes are not annotated in the genome of the Escherichia coli K-12 MG1655 strain.
(91) The nucleotide sequence of a region containing the g1414 and g1413 genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 121. In the nucleotide sequence shown as SEQ ID NO: 121, the g1414 and g1413 genes correspond to the sequence of the positions 28 to 699, and the sequence of the positions 831 to 1,157, respectively. The amino acid sequences of the G1414 and G1413 proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 122 and 123, respectively.
(92) The nuoE, nuoF, and nuoG genes encode a soluble fragment of NADH dehydrogenase I. The nuoE, nuoF, and nuoG genes of the Escherichia coli K-12 MG1655 strain correspond to the complementary sequence of the sequence of the positions 2,399,574 to 2,400,074, the complementary sequence of the sequence of the positions 2,398,240 to 2,399,577, and the complementary sequence of the sequence of the positions 2,395,461 to 2,398,187 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The NuoE, NuoF, and NuoG proteins of the MG1655 strain are registered as GenBank accession NP_416788 (version NP_416788.1 GI: 16130220), GenBank accession NP_416787 (version NP_416787.1 GI: 16130219), and GenBank accession NP_416786 (version NP_416786.4 GI: 145698290), respectively.
(93) The nucleotide sequence of a region containing the nuoE, nuoF, and nuoG genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 124. In the nucleotide sequence shown as SEQ ID NO: 124, the nuoE, nuoF, and nuoG genes correspond to the complementary sequence of the sequence of the positions 796 to 1,296, the complementary sequence of the sequence of the positions 1,293 to 2,630, and the complementary sequence of the sequence of the positions 2,683 to 5,409, respectively. The amino acid sequences of the NuoE, NuoF, and NuoG proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 125 to 127, respectively.
(94) The glmZ gene encodes a low molecular weight RNA. The glmZ gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 3,984,455 to 3,984,626 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990).
(95) The hemY, hemX, and hemD genes encode enzymes of the biosynthesis pathways of heme and choline. The hemY gene encodes a protoporphyrinogen oxidase. The hemX gene encodes a protein presumed to be a uroporphyrinogen III methylase. The hemD gene encodes a uroporphyrinogen III synthase. The hemY, hemX, and hemD genes of the K-12 MG1655 strain correspond to the complementary sequence of the sequence of the positions 3,984,709 to 3,985,905, the complementary sequence of the sequence of the positions 3,985,908 to 3,987,089, and the complementary sequence of the sequence of the positions 3,987,111 to 3,987,851 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The HemY, HemX, and HemD proteins of the MG1655 strain are registered as GenBank accession NP_418246 (version NP_418246.1 GI: 16131654), GenBank accession NP_418247 (version NP_418247.1 GI: 16131655), and GenBank accession NP_418248 (version NP_418248.1 GI: 16131656), respectively.
(96) The nucleotide sequence of a region containing the glmZ, hemY, hemX, and hemD genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 128. In the nucleotide sequence shown as SEQ ID NO: 128, the glmZ, hemY, hemX, and hemD genes correspond to the sequence of the positions 357 to 563, the sequence of the positions 611 to 1,807, the sequence of the positions 1,810 to 2,991, and the sequence of the positions 3,013 to 3,753, respectively. The amino acid sequences of the HemY, HemX, and HemD proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 129 to 131, respectively.
(97) The rlmL gene (synonym is rlmKL) encodes a methyltransferase that methylates G2445 and G2069 of 23S rRNA. The rlmL gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 1,007,067 to 1,009,175 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The RlmL protein of the MG1655 strain is registered as GenBank accession NP_415468 (version NP_415468.1 GI: 16128915).
(98) The nucleotide sequence of a region containing the rlmL gene of the Escherichia coli K5 strain is shown as SEQ ID NO: 132. In the nucleotide sequence shown as SEQ ID NO: 132, the rlmL gene corresponds to the sequence of the positions 571 to 2,679. The amino acid sequence of the RlmL protein of the Escherichia coli K5 strain is shown as SEQ ID NO: 133.
(99) The artQ, artM, and artJ genes encode subunits of an arginine ABC transporter. The artQ, artM, and artJ genes of the Escherichia coli K-12 MG1655 strain correspond to the complementary sequence of the sequence of the positions 900,757 to 901,473, the complementary sequence of the sequence of the positions 900,089 to 900,757, and the complementary sequence of the sequence of the positions 899,067 to 899,798 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The ArtQ, ArtM, and ArtJ proteins of the MG1655 strain are registered as GenBank accession NP_415383 (version NP_415383.1 GI: 16128830), GenBank accessionNP_415382 (version NP_415382.1 GI: 16128829), and GenBank accession NP_415381 (version NP_415381.1 GI: 16128828), respectively.
(100) The rlmC gene (synonym is rumB) encodes a methyltransferase that methylates U747 of 23S rRNA. The rlmC gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 897,741 to 898,868 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The RlmC protein of the MG1655 strain is registered as GenBank accession NP_415380 (version NP_415380.1 GI: 16128827).
(101) The ybjO gene encodes a protein presumed to be an inner membrane protein. The ybjO gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 897,212 to 897,700 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YbjO protein of the MG1655 strain is registered as GenBank accession NP_415379 (version NP_415379.1 GI: 16128826).
(102) The nucleotide sequence of a region containing the artQ, artM, artJ, rlmC, and ybjO genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 134. In the nucleotide sequence shown as SEQ ID NO: 134, the artQ, artM, artJ, rlmC, and ybjO genes correspond to the sequence of the positions 386 to 1,102, the sequence of the positions 1,102 to 1,770, the sequence of the positions 2,061 to 2,792, the complementary sequence of the sequence of the positions 2,991 to 4,118, and the complementary sequence of the sequence of the positions 4,159 to 4,647, respectively. The amino acid sequences of the ArtQ, ArtM, ArtJ, RlmC, and YbjO proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 135 to 139, respectively.
(103) The yejO gene encodes an outer membrane protein. The yejO gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the fused sequence consisting of the sequence of the positions 2,284,412 to 2,286,936 and the sequence of the positions 2,288,136 to 2,288,202 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The yejO gene of the MG1655 strain is considered to be a pseudogene.
(104) The yejM gene is a gene encoding a protein presumed to be one of hydrolases. The yejM gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 2,282,398 to 2,284,158 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YejM protein of the MG1655 strain is registered as GenBank accession NP_416693 (version NP_416693.1 GI: 16130126).
(105) The yejL gene is a gene of unknown function. The yejL gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 2,282,151 to 2,282,378 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YejL protein of the MG1655 strain is registered as GenBank accession NP_416692 (version NP_416692.1 GI: 16130125).
(106) The nucleotide sequence of a region containing the yejO, yejM, and yejL genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 140. In the nucleotide sequence shown as SEQ ID NO: 140, the yejO, yejM, and yejL genes correspond to the sequence of the positions 216 to 2,807, the complementary sequence of the sequence of the positions 3,061 to 4,821, and the complementary sequence of the sequence of the positions 4,841 to 5,068, respectively. The amino acid sequences of the YejO, YejM, and YejL proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 141 to 143, respectively.
(107) The rpoS gene encodes SigmaS (.sup.s). The rpoS gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the sequence of the positions 2,864,581 to 2,865,573 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The RpoS protein of the MG1655 strain is registered as GenBank accession NP_417221 (version NP_417221.1 GI: 16130648).
(108) The ygbN gene encodes a protein presumed to be a transporter belonging to the Gnt family. The ygbN gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 2,863,123 to 2,864,487 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YgbN protein of the MG1655 strain is registered as GenBank accession NP_417220 (version NP_417220.1 GI: 16130647).
(109) The ygbM gene is a gene of unknown function. The ygbM gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 2,862,258 to 2,863,034 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YgbM protein of the MG1655 strain is registered as GenBank accession NP_417219 (version NP_417219.1 GI: 16130646).
(110) The ygbL gene encodes a protein presumed to be one of aldolases. The ygbL gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 2,861,615 to 2,862,253 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YgbL protein of the MG1655 strain is registered as GenBank accession NP_417218 (version NP_417218.1 GI: 16130645).
(111) The nucleotide sequence of a region containing the rpoS, ygbN, ygbM, and ygbL genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 144. In the nucleotide sequence shown as SEQ ID NO: 144, the rpoS, ygbN, ygbM, and ygbL genes correspond to the sequence of the positions 318 to 1,310, the complementary sequence of the sequence of the positions 1,404 to 2,768, the complementary sequence of the sequence of the positions 2,857 to 3,633, and the complementary sequence of the sequence of the positions 3,638 to 4,276, respectively. The amino acid sequences of the RpoS, YgbN, YgbM, and YgbL proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 145 to 148, respectively.
(112) The g3798 gene encodes a protein presumed to be an SOS-response transcriptional repressor (RecA-mediated autopeptidase). The g3794 gene encodes a protein presumed to be a superinfection exclusion protein B. The g3793 gene encodes a protein presumed to be a restriction inhibitor protein ral (antirestriction protein). The g3797, g3796, g3795, and g3792 genes are genes of unknown functions. These genes are not annotated in the genome of the Escherichia coli K-12 MG1655 strain.
(113) The nucleotide sequence of a region containing the g3798, g3797, g3796, g3795, g3794, g3793, and g3792 genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 149. In the nucleotide sequence shown as SEQ ID NO: 149, the g3798, g3797, g3796, g3795, g3794, g3793, and g3792 genes correspond to the sequence of the positions 615 to 1,268, the sequence of the positions 1,368 to 2,219, the sequence of the positions 2,257 to 2,748, the sequence of the positions 3,021 to 3,203, the complementary sequence of the sequence of the positions 3,470 to 4,051, the sequence of the positions 4,280 to 4,480, and the sequence of the positions 4,520 to 4,717, respectively. The amino acid sequences of the G3798, G3797, G3796, G3795, G3794, G3793, and G3792 proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 150 to 156, respectively.
(114) The ryjA gene encodes a low molecular weight RNA. The ryjA gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the sequence of the positions 4,275,950 to 4,276,089 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990).
(115) The soxR and soxS genes are genes encoding a transcriptional control factor. The soxR and soxS genes of the Escherichia coli K-12 MG1655 strain correspond to the sequence of the positions 4,275,492 to 4,275,956, and the complementary sequence of the sequence of the positions 4,275,083 to 4,275,406 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The SoxR and SoxS proteins of the MG1655 strain are registered as GenBank accession NP_418487 (version NP_418487.1 GI:16131889) and GenBank accession NP_418486 (version NP_418486.1 GI: 16131888), respectively.
(116) The yjcC gene encodes a c-di-GMP-specific phosphodiesterase. The yjcC gene of the Escherichia coli K-12 MG1655 strain corresponds to the sequence of the positions 4,273,494 to 4,275,080 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YjcC protein of the MG1655 strain is registered as GenBank accession NP_418485 (version NP_418485.1 GI: 16131887).
(117) The yjcB gene is a gene of unknown function. The yjcB gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the sequence of the positions 4,272,783 to 4,273,064 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YjcB protein of the MG1655 strain is registered as GenBank accession NP_418484 (version NP_418484.4 GI: 90111681).
(118) The nucleotide sequence of a region containing the ryjA, soxR, soxS, yjcC, and yjcB genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 157. In the nucleotide sequence shown as SEQ ID NO: 157, the ryjA, soxR, soxS, yjcC, and yjcB genes correspond to the sequence of the positions 657 to 796, the complementary sequence of the sequence of the positions 790 to 1,254, the sequence of the positions 1,340 to 1,663, the complementary sequence of the sequence of the positions 1,666 to 3,252, and the sequence of the positions 3,682 to 3,963, respectively. The amino acid sequences of the SoxR, SoxS, YjcC, and YjcB proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 158 to 161, respectively.
(119) The efeU and efeO genes are genes encoding a component of a divalent iron ion transport carrier. The efeU and efeO genes of the Escherichia coli K-12 MG1655 strain correspond to the sequence of the positions 1,080,579 to 1,081,408, and the sequence of the positions 1,081,466 to 1,082,593 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The efeU gene of the MG1655 strain is considered to be a pseudogene. The EfeO protein of the MG1655 strain is registered as GenBank accession NP_415537 (version NP_415537.1 GI: 16128982).
(120) The nucleotide sequence of a region containing the efeU and efeO genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 162. In the nucleotide sequence shown as SEQ ID NO: 162, the efeU and efeO genes correspond to the sequence of the positions 753 to 1,583, and the sequence of the positions 1,641 to 2,768, respectively. The amino acid sequences of EfeU and EfeO proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 163 and 164, respectively.
(121) The bacterium of the present invention may have been modified so that, for example, expression of at least the rfaH gene among the genes of Tables 1 to 3 is increased, or expression of at least one or more genes among the genes of Tables 1 to 3 other than the rfaH gene is increased. The bacterium of the present invention may have been also modified so that expression of the rfaH gene and expression of one or more kinds of the genes of Tables 1 to 3 other than the rfaH gene are increased. Specifically, the bacterium of the present invention may have been modified so that, for example, expression of the rfaH gene and expression is increased of one or more genes such as rbsR, rbsK, rbsB, hsrA, glgB, glgX, micF, rcsD, rcsB, ybiX, ybiJ ybiC, ybiB, nusG, pcoR, pcoS, pcoE, yhcN, yhcO, aaeB, aaeA, aaeX, g1455, alpA, g1453, yrbA, mlaB, mlaC, mlaD, mlaE, mlaF, yrbG, norW, ybjI, ybjJ, ybjK, rybB, yjjY, yjtD, thrL, thrA, thrB, fruA, psuK, ytfT, yjfF, fbp, yagU, paoA, paoB, gsiC, gsiD, yliE, irp2, irp1, bhsA, and ycfS. The bacterium of the present invention may have been also modified so that, for example, expression of at least the rpoE gene among the genes of Tables 1 to 3 is increased. The combination of the genes of Tables 1 to 3 of which expression is to be increased is not particularly limited. Examples of the combination include, for example, the combinations described in Examples depicted herein.
(122) The methods for increasing gene expression will be described later. Expression of the gene(s) of Tables 1 to 3 may be increased by, for example, increasing the copy number of a DNA containing the gene(s) of Tables 1 to 3, such as a DNA having the nucleotide sequence shown as SEQ ID NO: 29, 34, 37, 43, 50, 54, 60, 64, 72, 74, 78, 84, 87, 91, 95, 99, 104, 107, 111, 116, 121, 124, 128, 132, 134, 140, 144, 149, 157, or 162. As for the irp gene, the copy number of a DNA containing a part of the irp gene, such as a DNA having the nucleotide sequence shown as SEQ ID NO: 99, may also be increased. Such DNA as mentioned above of which the copy number is to be increased may be a variant of a DNA having the nucleotide sequence shown as SEQ ID NO: 29, 34, 37, 43, 50, 54, 60, 64, 72, 74, 78, 84, 87, 91, 95, 99, 104, 107, 111, 116, 121, 124, 128, 132, 134, 140, 144, 149, 157, or 162. For variants of DNA, the descriptions about conservative variants of the genes mentioned in Tables 1 to 3 can be similarly applied. Namely, for example, the copy number of a DNA showing a homology of 90% or more to the nucleotide sequence shown as SEQ ID NOS: 29, 34, 37, 43, 50, 54, 60, 64, 72, 74, 78, 84, 87, 91, 95, 99, 104, 107, 111, 116, 121, 124, 128, 132, 134, 140, 144, 149, 157, or 162 may be increased.
(123) These genes can be obtained by PCR using a chromosome of a strain having any of these genes as the template, and oligonucleotides produced on the basis of any of these known gene sequences as the primers.
(124) The genes of Tables 1 to 3 each may be a variant of the genes exemplified above, so long as the variant maintains the original function. Similarly, the proteins encoded by the genes of Tables 1 to 3 each may be a variant of the proteins exemplified above, so long as the variant maintains the original function. Such a variant that maintains the original function may be referred to as a conservative variant. In the present invention, the genes specified with the aforementioned gene names and the proteins specified with names corresponding to the gene names include the genes and proteins exemplified above, respectively, and in addition, conservative variants thereof. Namely, for example, the term rpoE gene includes the rpoE genes exemplified above (i.e. rpoE genes of the Escherichia coli K-12 MG1655 strain and the Escherichia coli K5 strain), and in addition, conservative variants thereof. Similarly, for example, the term RpoE protein includes the RpoE proteins exemplified above (i.e. RpoE proteins of the Escherichia coli K-12 MG1655 strain and the Escherichia coli K5 strain), and in addition, conservative variants thereof. Examples of the conservative variants include, for example, homologues and artificially modified variants of the genes and proteins exemplified above.
(125) The expression variant maintains the original function means that the variant of a gene or protein has a function (such as activity or property) corresponding to the function (such as activity or property) of the original gene or protein.
(126) That is, the expression variant maintains the original function means that, in the case of the genes of Tables 1 to 3, a variant of any of the genes has a property of increasing heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount of the variant is increased in the bacterium. Furthermore, the expression variant maintains the original function may also mean that, in the case of the genes of Tables 1 to 3, a variant of any of the genes encodes a protein that maintains the original function. That is, the genes of Tables 1 to 3 may encode a conservative variant of the proteins exemplified above.
(127) Similarly, the expression variant maintains the original function means that, in the case of the proteins encoded by the genes of Tables 1 to 3, a variant of any of the proteins has a property of increasing heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount of the variant is increased in the bacterium. Further, the expression variant maintains the original function may also mean that, in the case of the proteins encoded by the genes of Tables 1 to 3, a variant of any of the proteins has the above-mentioned function of the corresponding protein, for example, the function of the sigmaE (.sup.E) in the case of the RpoE protein.
(128) Whether a variant of a gene or protein has the property of increasing heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount thereof is increased in the bacterium can be confirmed by introducing the gene or a gene encoding the protein into the Escherichia bacterium having heparosan-producing ability, and confirming whether the heparosan-producing ability is improved or not.
(129) Homologues of the genes of Tables 1 to 3 can be easily obtained from public databases by, for example, BLAST search or FASTA search using any of the nucleotide sequences of the genes exemplified above as a query sequence. Further, homologues of the genes of Tables 1 to 3 can also be obtained by, for example, PCR using a chromosome of a microorganism such as bacterium as the template, and oligonucleotides prepared on the basis of any of these known gene sequences as the primers.
(130) The genes of Tables 1 to 3 each may encode a protein having any of the aforementioned amino acid sequences including substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions, so long as the protein maintains the original function. For example, the N-terminus and/or C-terminus of the encoded protein may be extended or shortened. Although the number of one or several may differ depending on the positions in the three-dimensional structure of the protein or the types of amino acid residues, specifically, it is, for example, 1 to 50, 1 to 40, or 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3.
(131) The aforementioned substitution, deletion, insertion, or addition of one or several amino acid residues is a conservative mutation that maintains normal function of the protein. Typical examples of the conservative mutation are conservative substitutions. The conservative substitution is a mutation wherein substitution takes place mutually among Phe, Trp, and Tyr, if the substitution site is an aromatic amino acid; among Leu, Ile, and Val, if it is a hydrophobic amino acid; between Gln and Asn, if it is a polar amino acid; among Lys, Arg, and His, if it is a basic amino acid; between Asp and Glu, if it is an acidic amino acid; and between Ser and Thr, if it is an amino acid having a hydroxyl group. Examples of substitutions considered as conservative substitutions include, specifically, substitution of Ser or Thr for Ala, substitution of Gln, His, or Lys for Arg, substitution of Glu, Gln, Lys, His, or Asp for Asn, substitution of Asn, Glu, or Gln for Asp, substitution of Ser or Ala for Cys, substitution of Asn, Glu, Lys, His, Asp, or Arg for Gln, substitution of Gly, Asn, Gln, Lys, or Asp for Glu, substitution of Pro for Gly, substitution of Asn, Lys, Gln, Arg, or Tyr for His, substitution of Leu, Met, Val, or Phe for Ile, substitution of Ile, Met, Val, or Phe for Leu, substitution of Asn, Glu, Gln, His, or Arg for Lys, substitution of Ile, Leu, Val, or Phe for Met, substitution of Trp, Tyr, Met, Ile, or Leu for Phe, substitution of Thr or Ala for Ser, substitution of Ser or Ala for Thr, substitution of Phe or Tyr for Trp, substitution of His, Phe, or Trp for Tyr, and substitution of Met, Ile, or Leu for Val. Further, such substitution, deletion, insertion, addition, inversion, or the like of amino acid residues as mentioned above includes a naturally occurring mutation due to an individual difference, or a difference of species of the organism from which the gene is derived (mutant or variant).
(132) The genes of Tables 1 to 3 each may be a gene encoding a protein showing a homology of, for example, 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more, to the total amino acid sequence of any of the amino acid sequences mentioned above, so long as the protein maintains the original function. In this description, homology may mean identity.
(133) The genes of Tables 1 to 3 each may also be a DNA that is able to hybridize under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to a partial or entire sequence of any of the aforementioned nucleotide sequences. The stringent conditions refer to conditions under which a so-called specific hybrid is formed, and a non-specific hybrid is not formed. Examples of the stringent conditions include those under which highly homologous DNAs hybridize to each other, for example, DNAs not less than 80% homologous, not less than 90% homologous, not less than 95% homologous, not less than 97% homologous, not less than 99% homologous, hybridize to each other, and DNAs less homologous than the above do not hybridize to each other, or conditions of washing of typical Southern hybridization, i.e., conditions of washing once, preferably 2 or 3 times, at a salt concentration and temperature corresponding to 1SSC, 0.1% SDS at 60 C., or 0.1SSC, 0.1% SDS at 60 C., or 0.1SSC, 0.1% SDS at 68 C.
(134) As described above, the probe used for the aforementioned hybridization may be a part of a sequence that is complementary to a gene. Such a probe can be prepared by PCR using oligonucleotides prepared on the basis of a known gene sequence as the primers and a DNA fragment containing any of the genes of Tables 1 to 3 as the template. As the probe, for example, a DNA fragment having a length of about 300 bp can be used. When a DNA fragment having a length of about 300 bp is used as the probe, the washing conditions of the hybridization may be, for example, 50 C., 2SSC and 0.1% SDS.
(135) Furthermore, since the degeneracy of codons differs depending on the host, the genes of Tables 1 to 3 each may be a gene in which an arbitrary codon is replaced with an equivalent codon, so long as the original function is maintained. For example, the genes of Tables 1 to 3 each may be modified so that they have optimal codons according to codon usage of the host.
(136) A variant of the genes of Tables 1 to 3 can be obtained by, for example, modifying a coding region of the genes by site-specific mutagenesis so that a specific site of the encoded protein include substitution, deletion, insertion, or addition of amino acid residues. Further, a variant of the genes of Tables 1 to 3 can also be obtained by the conventionally known mutagenesis. Examples of the mutagenesis include such methods as treating a DNA molecule having a nucleotide sequence of any of the genes of Tables 1 to 3 in vitro with hydroxylamine or the like, irradiating X-ray or ultraviolet ray on a microorganism such as a microorganism belonging to Enterobacteriaceae containing any of the genes of Tables 1 to 3, treating such a microorganism with a mutagen such as N-methyl-N-nitro-N-nitrosoguanidine (NTG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS), performing error prone PCR (Cadwell, R. C., PCR Meth. Appl., 2, 28 (1992)), DNA shuffling (Stemmer, W. P., Nature, 370, 389 (1994)), and StEP-PCR (Zhao, H., Nature Biotechnol., 16, 258 (1998)), and so forth.
(137) <1-3> Method for Increasing Expression of Gene
(138) Hereafter, methods for increasing (rising) expression of a gene will be explained.
(139) The expression of a gene may be increased 1.5 times or more, 2 times or more, or 3 times or more, as compared with that of a non-modified strain. Further, the state that the expression of a gene is increased includes not only a state that the expression amount of an objective gene is increased in a strain that inherently expresses the objective gene, but also a state that the gene is introduced into a strain that does not inherently express the objective gene, and expressed therein. That is, the phrase the expression of a gene is increased may also mean, for example, that an objective gene is introduced into a strain that does not possess the gene, and is expressed therein. The state that the expression of a gene is increased may also be referred to as the expression of a gene is enhanced.
(140) The expression of a gene can be increased by, for example, increasing the copy number of the gene.
(141) The copy number of a gene can be increased by introducing the gene into the chromosome of a host. A gene can be introduced into a chromosome by, for example, using homologous recombination (Miller, J. H., Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Only one copy, or two or more copies of a gene may be introduced. For example, by performing homologous recombination using a sequence which is present in multiple copies on a chromosome as a target, multiple copies of a gene can be introduced into the chromosome. Examples of such a sequence which is present in multiple copies on a chromosome include repetitive DNAs, and inverted repeats located at the both ends of a transposon. Alternatively, homologous recombination may be performed by using an appropriate sequence on a chromosome such as a gene unnecessary for the production of an objective substance as a target. Homologous recombination can be performed by, for example, a method using a linear DNA such as Red-driven integration (Datsenko, K. A., and Wanner, B. L., Proc. Natl. Acad. Sci. USA, 97:6640-6645 (2000)), a method of using a plasmid containing a temperature sensitive replication origin, a method of using a plasmid capable of conjugative transfer, a method of using a suicide vector not having a replication origin that functions in a host, or a transduction method using a phage. Furthermore, a gene can also be randomly introduced into a chromosome by using a transposon or Mini-Mu (Japanese Patent Laid-open (Kokai) No. 2-109985, U.S. Pat. No. 5,882,888, EP 805867 B1).
(142) Introduction of a target gene into a chromosome can be confirmed by Southern hybridization using a probe having a sequence complementary to the whole gene or a part thereof, PCR using primers prepared on the basis of the sequence of the gene, or the like.
(143) Furthermore, the copy number of a gene can also be increased by introducing a vector containing the gene into a host. For example, the copy number of a target gene can be increased by ligating a DNA fragment containing the target gene with a vector that functions in a host to construct an expression vector of the gene, and transforming the host with the expression vector. The DNA fragment containing the target gene can be obtained by, for example, PCR using the genomic DNA of a microorganism having the target gene as the template. As the vector, a vector autonomously replicable in the cell of the host can be used. The vector is preferably a multi-copy vector. Furthermore, the vector preferably has a marker such as an antibiotic resistance gene for selection of transformant. Furthermore, the vector may have a promoter and/or terminator for expressing the introduced gene. The vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, cosmid, phagemid, or the like. Specific examples of vector autonomously replicable in Enterobacteriaceae bacteria such as Escherichia coli include, for example, pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, pSTV29 (all of these are available from Takara Bio), pACYC184, pMW219 (NIPPON GENE), pTrc99A (Pharmacia), pPROK series vectors (Clontech), pKK233-2 (Clontech), pET series vectors (Novagen), pQE series vectors (QIAGEN), and the broad host spectrum vector RSF1010.
(144) When a gene is introduced, it is sufficient that the gene is expressibly harbored by the bacterium of the present invention. Specifically, it is sufficient that the gene is introduced so that it is expressed under control by a promoter sequence that functions in the bacterium of the present invention. The promoter may be a promoter derived from the host, or a heterogenous promoter. The promoter may be the native promoter of the gene to be introduced, or a promoter of another gene. As the promoter, for example, such a stronger promoter as mentioned later may also be used.
(145) A terminator for termination of gene transcription may be located downstream of the gene. The terminator is not particularly limited so long as it functions in the bacterium of the present invention. The terminator may be a terminator derived from the host, or a heterogenous terminator. The terminator may be the native terminator of the gene to be introduced, or a terminator of another gene. Specific examples of the terminator include, for example, T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator.
(146) Vectors, promoters, and terminators available in various microorganisms are disclosed in detail in Fundamental Microbiology Vol. 8, Genetic Engineering, KYORITSU SHUPPAN CO., LTD, 1987, and those can be used.
(147) Furthermore, when two or more of genes are introduced, it is sufficient that the genes each are expressibly harbored by the bacterium of the present invention. For example, all the genes may be carried by a single expression vector or a chromosome. Furthermore, the genes may be separately carried by two or more expression vectors, or separately carried by a single or two or more expression vectors and a chromosome. An operon constituted by two or more genes may also be introduced. The case of introducing two or more genes include, for example, introducing respective genes coding for two or more kinds of enzymes, introducing respective genes coding for two or more subunits constituting a single enzyme, and a combination of the foregoing cases.
(148) The gene to be introduced is not particularly limited so long as it codes for a protein that functions in the host. The gene to be introduced may be a gene derived from the host, or may be a heterogenous gene. The gene to be introduced can be obtained by, for example, PCR using primers designed on the basis of the nucleotide sequence of the gene, and using the genomic DNA of an organism having the gene, a plasmid carrying the gene, or the like as a template. The gene to be introduced may also be totally synthesized, for example, on the basis of the nucleotide sequence of the gene (Gene, 60(1), 115-127 (1987)).
(149) In addition, when a protein functions as a complex consisting of a plurality of subunits, a part or all of the plurality of subunits may be modified, so long as the activity of the protein is eventually increased. That is, for example, when the activity of a protein is increased by increasing the expression of a gene, the expression of a part or all of the plurality of genes that code for the subunits may be enhanced. It is usually preferable to enhance the expression of all of the plurality of genes coding for the subunits. Furthermore, the subunits constituting the complex may be derived from a single kind of organism or two or more kinds of organisms, so long as the complex has a function of the objective protein. That is, for example, genes of the same organism coding for a plurality of subunits may be introduced into a host, or genes of different organisms coding for a plurality of subunits may be introduced into a host.
(150) Further, the expression of a gene can be increased by improving the transcription efficiency of the gene. The transcription efficiency of a gene can be improved by, for example, replacing the promoter of the gene on a chromosome with a stronger promoter. The stronger promoter means a promoter providing an improved transcription of a gene compared with an inherently existing wild-type promoter of the gene. Examples of stronger promoters include, for example, the known high expression promoters such as T7 promoter, trp promoter, lac promoter, thr promoter, tac promoter, trc promoter, tet promoter, araBAD promoter, rpoH promoter, PR promoter, and PL promoter. Furthermore, as the stronger promoter, a highly-active type of an existing promoter may also be obtained by using various reporter genes. For example, by making the 35 and 10 regions in a promoter region closer to the consensus sequence, the activity of the promoter can be enhanced (WO00/18935). Examples of highly active-type promoter include various tac-like promoters (Katashkina J I et al., Russian Federation Patent Application No. 2006134574) and pnlp8 promoter (WO2010/027045). Methods for evaluating the strength of promoters and examples of strong promoters are described in the paper of Goldstein et al. (Prokaryotic Promoters in Biotechnology, Biotechnol. Annu. Rev., 1, 105-128 (1995)), and so forth.
(151) Furthermore, the expression of a gene can also be increased by improving the translation efficiency of the gene. The translation efficiency of a gene can be improved by, for example, replacing the Shine-Dalgarno (SD) sequence (also referred to as ribosome binding site (RBS)) for the gene on a chromosome with a stronger SD sequence. The stronger SD sequence means a SD sequence that provides an improved translation of mRNA compared with the inherently existing wild-type SD sequence of the gene. Examples of stronger SD sequences include, for example, RBS of the gene 10 derived from phage T7 (Olins P. O. et al, Gene, 1988, 73, 227-235). Furthermore, it is known that substitution, insertion, or deletion of several nucleotides in a spacer region between RBS and the start codon, especially in a sequence immediately upstream of the start codon (5-UTR), significantly affects the stability and translation efficiency of mRNA, and hence, the translation efficiency of a gene can also be improved by modifying them.
(152) In the present invention, sites that affect the expression of a gene, such as promoter, SD sequence, and spacer region between RBS and the start codon, may also be collectively referred to as expression control region. Expression control regions can be identified by using a promoter search vector or gene analysis software such as GENETYX. These expression control regions can be modified by, for example, a method of using a temperature sensitive vector, or the Red driven integration method (WO2005/010175).
(153) The translation efficiency of a gene can also be improved by, for example, modifying codons. In Escherichia coli etc., a clear codon bias exists among the 61 amino acid codons found within the population of mRNA molecules, and the level of cognate tRNA appears directly proportional to the frequency of codon usage (Kane, J. F., Curr. Opin. Biotechnol., 6 (5), 494-500 (1995)). That is, if there is a large amount of mRNA containing an excess amount of rare codons, a translational problem may arise. According to recent research, it is suggested that clusters of AGG/AGA, CUA, AUA, CGA, or CCC codons may especially reduce both the quantity and quality of a synthesized protein. Such a problem occurs especially at the time of expression of a heterologous gene. Therefore, in the case of heterogenous expression of a gene or the like, the translation efficiency of the gene can be improved by replacing a rare codon present in the gene with a synonymous codon more frequently used. Codons can be replaced by, for example, the site-specific mutation method for introducing an objective mutation into an objective site of DNA. Examples of the site-specific mutation method include the method utilizing PCR (Higuchi, R., 61, in PCR Technology, Erlich, H. A. Eds., Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)), and the method utilizing phage (Kramer, W. and Frits, H. J., Meth. in Enzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth. in Enzymol., 154, 367 (1987)). Alternatively, a gene fragment in which objective codons are replaced may be totally synthesized. Frequencies of codons in various organisms are disclosed in the Codon Usage Database (www.kazusa.or.jp/codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292 (2000)).
(154) Furthermore, the expression of a gene can also be increased by amplifying a regulator that increases the expression of the gene, or deleting or attenuating a regulator that reduces the expression of the gene.
(155) Such methods for increasing the gene expression as mentioned above may be used independently or in any arbitrary combination.
(156) The method for the transformation is not particularly limited, and conventionally known methods can be used. There can be used, for example, a method of treating recipient cells with calcium chloride so as to increase the permeability thereof for DNA, which has been reported for the Escherichia coli K-12 strain (Mandel, M. and Higa, A., J. Mol. Biol., 1970, 53, 159-162), and a method of preparing competent cells from cells which are in the growth phase, followed by transformation with DNA, which has been reported for Bacillus subtilis (Duncan, C. H., Wilson, G A. and Young, F. E., Gene, 1977, 1:153-167). Alternatively, there can also be used a method of making DNA-recipient cells into protoplasts or spheroplasts, which can easily take up recombinant DNA, followed by introducing a recombinant DNA into the DNA-recipient cells, which is known to be applicable to Bacillus subtilis, actinomycetes, and yeasts (Chang, S. and Choen, S. N., 1979, Mol. Gen. Genet., 168:111-115; Bibb, M. J., Ward, J. M. and Hopwood, O. A., 1978, Nature, 274:398-400; Hinnen, A., Hicks, J. B. and Fink, G R., 1978, Proc. Natl. Acad. Sci. USA, 75:1929-1933). Further, the electric pulse method reported for coryneform bacteria (Japanese Patent Laid-open (Kokai) No. 2-207791) can also be used.
(157) An increase in the expression of a gene can be confirmed by confirming an increase in the transcription amount of the gene, or by confirming an increase in the amount of a protein expressed from the gene. An increase in the expression of a gene can also be confirmed by confirming an increase in the activity of a protein expressed from the gene.
(158) An increase of the transcription amount of a gene can be confirmed by comparing the amount of mRNA transcribed from the gene with that of a non-modified strain such as a wild-type strain or parent strain. Examples of the method for evaluating the amount of mRNA include Northern hybridization, RT-PCR, and so forth (Sambrook, J., et al., Molecular Cloning A Laboratory Manual/Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The amount of mRNA may increase, for example, 1.5 times or more, 2 times or more, or 3 times or more, as compared with that of a non-modified strain.
(159) An increase in the amount of a protein can be confirmed by Western blotting using antibodies (Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The amount of the protein may increase, for example, 1.5 times or more, 2 times or more, or 3 times or more, as compared with that of a non-modified strain.
(160) An increase in the activity of a protein can be confirmed by measuring the activity of the protein. The activity of the protein may increase, for example, 1.5 times or more, 2 times or more, or 3 times or more, as compared with that of a non-modified strain.
(161) The aforementioned methods for increasing the expression of a gene can be used for enhancement of the expression of arbitrary genes such as the genes of Tables 1 to 3 and genes encoding a protein that participates in heparosan production.
(162) <2> Method for Producing Heparosan
(163) The method for producing heparosan of the present invention includes steps, for example, of culturing the bacterium of the present invention in a medium to produce and accumulate heparosan in the medium, and collecting heparosan from the medium.
(164) The medium to be used is not particularly limited, so long as the bacterium of the present invention can proliferate in the medium, and heparosan is produced and accumulated. As the medium, for example, a usual medium used for culture of bacteria can be used. Specific examples of the medium include, for example, the LB medium (Luria-Bertani medium, containing 10.0 g of Bacto tryptone, 5.0 g of Bacto yeast extract, and 5.0 g of NaCl in 1 litter), but are not limited thereto. As the medium, for example, a medium containing carbon source, nitrogen source, phosphorus source, and sulfur source, as well as components selected from other various organic components and inorganic components as required can be used. Types and concentrations of the medium components may be arbitrarily determined by those skilled in the art.
(165) The carbon source is not particularly limited, so long as the bacterium of the present invention can utilize it to generate heparosan. Specific examples of the carbon source include, for example, saccharides such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, blackstrap molasses, starch hydrolysates, and hydrolysates of biomass, organic acids such as acetic acid, fumaric acid, citric acid, succinic acid, and malic acid, alcohols such as glycerol, crude glycerol, and ethanol, and aliphatic acids. As the carbon source, a single kind of carbon source may be used, or two or more kinds of carbon sources may be used in combination.
(166) Specific examples of the nitrogen source include, for example, ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen sources such as peptone, yeast extract, meat extract, and soybean protein decomposition products, ammonia, and urea. As the nitrogen source, a single kind of nitrogen source may be used, or two or more kinds of nitrogen sources may be used in combination.
(167) Specific examples of the phosphate source include, for example, phosphoric acid salts such as potassium dihydrogenphosphate and dipotassium hydrogenphosphate, and phosphoric acid polymers such as pyrophosphoric acid. As the phosphate source, a single kind of phosphate source may be used, or two or more kinds of phosphate sources may be used in combination.
(168) Specific examples of the sulfur source include, for example, inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. As the sulfur source, a single kind of sulfur source may be used, or two or more kinds of sulfur sources may be used in combination.
(169) Specific examples of other various organic components and inorganic components include, for example, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components containing those such as peptone, casamino acid, yeast extract, and soybean protein decomposition product. As other various organic components and inorganic components, a single kind of component may be used, or two or more kinds of components may be used in combination.
(170) Further, when an auxotrophic mutant that requires an amino acid or the like for growth thereof is used, it is preferable to supplement a required nutrient to the medium. Furthermore, when a gene is introduced by using a vector carrying an antibiotic resistance gene, it is preferable to add the corresponding antibiotic to the medium.
(171) Culture conditions are not particularly limited, so long as the bacterium of the present invention can proliferate, and heparosan is produced and accumulated. The culture can be performed with, for example, usual conditions used for culture of bacteria. The culture conditions may be appropriately chosen by those skilled in the art.
(172) The culture can be performed, for example, aerobically as aeration culture or shaking culture by using a liquid medium. The culture temperature may be, for example, 30 to 37 C. The culture period may be, for example, 16 to 72 hours. The culture can be performed as batch culture, fed-batch culture, continuous culture, or a combination of these. The culture may be performed as preculture and main culture. The preculture may be performed by using, for example, a plate medium or liquid medium.
(173) As a result of culture of the bacterium of the present invention as described above, heparosan is accumulated in the medium.
(174) The method for collecting heparosan from the culture broth is not particularly limited, so long as heparosan can be collected. Examples of the method for collecting heparosan from the culture broth include, for example, the method described in Examples. Specifically, for example, culture supernatant can be separated from the culture broth, and then heparosan contained in the supernatant can be precipitated by ethanol precipitation. The volume of ethanol to be added may be, for example, 2.5 to 3.5 times the volume of the supernatant. The solvent used for precipitating heparosan is not limited to ethanol, and organic solvents miscible with water in an arbitrary ratio can be used. Examples of such organic solvents include methanol, n-propanol, isopropanol, n-butanol, t-butanol, sec-butanol, propylene glycol, acetonitrile, acetone, DMF, DMSO, N-methylpyrrolidone, pyridine, 1,2-dimethoxyethane, 1,4-dioxane, and THF, as well as ethanol. Precipitated heparosan can be dissolved with, for example, water in a volume of 2 times the volume of the original supernatant. The collected heparosan may contain such components as bacterial cells, medium components, moisture, and by-product metabolites of the bacterium, in addition to heparosan. Heparosan may be purified in a desired degree. Purity of heparosan may be, for example, 30% (w/w) or higher, 50% (w/w) or higher, 70% (w/w) or higher, 80% (w/w) or higher, 90% (w/w) or higher, or 95% (w/w) or higher.
(175) Detection and quantification of heparosan can be performed by a known method. Specifically, for example, heparosan can be detected and quantified by the carbazole method. The carbazole method is a technique widely used as a quantification method for uronic acid, in which a thermal reaction of heparosan and carbazole can be carried out in the presence of sulfuric acid, and absorption at 530 nm of the reaction mixture provided by the generated color substance can be measured to detect and quantify heparosan (Bitter T. and Muir H. M. (1962) A modified uronic acid carbazole reaction, Analytical Biochemistry, 4(4):330-334). Heparosan can also be detected and quantified by, for example, treating heparosan with heparinase III, which is a heparosan decomposition enzyme, and performing disaccharide composition analysis.
(176) <3> Method for Producing Heparin
(177) Heparin can be produced by using heparosan produced by the bacterium of the present invention. That is, the method for producing heparin of the present invention is a method for producing heparin comprising culturing the bacterium of the present invention in a medium to produce and accumulate heparosan in the medium, chemically and/or enzymatically treating the heparosan to produce heparin, and collecting the heparin. Heparin has an anticoagulant activity, and can be used as an ingredient in drug formulations.
(178) The method for producing heparin from heparosan has already been reported. Specifically, for example, by subjecting heparosan as a starting material to the steps of (1) N-deacetylation, (2) N-sulfation, (3) C5-epimerization, (4) 2-O-sulfation, (5) 6-O-sulfation, and (6) 3-O-sulfation, heparin having an anticoagulant activity can be produced (Zhang Z. et al. (2008) Solution Structures of Chemoenzymatically Synthesized Heparin and Its Precursors, J. Am. Chem. Soc., 130(39):12998-13007). The method for producing heparin may further include a depolymerization step. Such steps as mentioned above for producing heparin from heparosan are also collectively referred to as heparin production process. The implementation order of the steps in the heparin production process is not particularly limited, so long as heparin having desired properties can be obtained.
(179) When heparosan is present in the medium, the medium may be subjected to the heparin production process, or heparosan collected from the medium may be subjected to the heparin production process. Furthermore, heparosan may be subjected to an arbitrary pretreatment, and then may be subjected to the heparin production process. Examples of the pretreatment include, for example, purification, dilution, concentration, drying, dissolution, and so forth. These pretreatments may also be performed in an appropriate combination. For example, a culture broth containing heparosan as it is, or heparosan purified from such a culture broth to a desired extent may be subjected to the heparin production process.
(180) The N-deacetylation can be chemically performed by using, for example, sodium hydroxide. The reaction conditions can be appropriately determined by those skilled in the art. For example, conditions mentioned in the published reference (Kuberan B. et al. (2003) Chemoenzymatic Synthesis of Classical and Non-classical Anticoagulant Heparan Sulfate Polysaccharides, J. Biol. Chem., 278(52):52613-52621) can be referred to.
(181) The N-sulfation can be chemically performed by using, for example, sulfur trioxide/trimethylamine complex. The reaction conditions can be appropriately determined by those skilled in the art. For example, conditions mentioned in the published reference (Kuberan B. et al. (2003) Chemoenzymatic Synthesis of Classical and Non-classical Anticoagulant Heparan Sulfate Polysaccharides, J. Biol. Chem., 278(52):52613-52621) can be referred to.
(182) The C5-epimerization can be enzymatically performed by using, for example, C5-epimerase. The C5-epimerase is not particularly limited so long as a C5-epimerase that can catalyze the epimerization of the glucuronic acid (GlcUA) residue into the iduronic acid (IdoA) residue is chosen. Depending on the order of the C5-epimerization, N-deacetylation, and/or 0-sulfation, a C5-epimerase showing suitable substrate specificity can be chosen and used. The C5-epimerase may be derived from any origin such as animal, plant, and microorganism. As the C5-epimerase, for example, human C5-epimerase can be used. The reaction conditions can be appropriately determined by those skilled in the art. For example, conditions mentioned in the published reference (Chen J., et al., Enzymatic redesigning of biologically active heparan sulfate, J. Biol. Chem., 2005 December, 30; 280(52):42817-25) can be referred to.
(183) The 2-O-sulfation can be enzymatically performed by using, for example, a 2-O-sulfation enzyme (2-OST). 2-OST is not particularly limited, so long as the chosen 2-OST can catalyze sulfation of the 0-2 position of the IdoA residue. Depending on the order of the 2-O-sulfation, N-deacetylation, C5-epimerization, 6-O-sulfation, and/or 3-O-sulfation, 2-OST showing suitable substrate specificity can be chosen and used. 2-OST may be derived from any origin such as animal, plant, and microorganism. As 2-OST, for example, hamster 2-OST can be used. The reaction conditions can be appropriately determined by those skilled in the art. For example, conditions mentioned in the published reference (Chen J., et al., Enzymatic redesigning of biologically active heparan sulfate, J. Biol. Chem., 2005 December, 30; 280(52):42817-25) can be referred to.
(184) The 6-O-sulfation can be enzymatically performed by using, for example, a 6-O-sulfation enzyme (6-OST). 6-OST is not particularly limited so long as the chosen 6-OST can catalyze sulfation of the 0-6 position of N-sulfated glucosamine (GlcNS) residue. Depending on the order of the 6-O-sulfation, N-deacetylation, C5-epimerization, 2-O-sulfation, and/or 3-O-sulfation, 6-OST showing suitable substrate specificity can be chosen and used. 6-OST may be derived from any origin such as animal, plant, and microorganism. As 6-OST, for example, hamster 6-OST-1 or mouse 6-OST-3 can be used. The reaction conditions can be appropriately determined by those skilled in the art. For example, conditions mentioned in the published reference (Chen J., et al., Enzymatic redesigning of biologically active heparan sulfate, J. Biol. Chem., 2005 December, 30; 280(52):42817-25) can be referred to.
(185) The 3-O-sulfation can be enzymatically performed by using, for example, a 3-O-sulfation enzyme (3-OST). 3-OST is not particularly limited so long as 3-OST that can catalyze sulfation of the O-3 position of N-sulfated and 6-O-sulfated glucosamine residue is chosen. Depending on the order of the 3-O-sulfation, N-deacetylation, C5-epimerization, 2-O-sulfation, and/or 6-O-sulfation, 3-OST showing suitable substrate specificity can be chosen and used. 3-OST may be derived from any origin such as animal, plant, and microorganism. As 3-OST, for example, mouse 3-OST-1 can be used. The reaction conditions can be appropriately determined by those skilled in the art. For example, conditions mentioned in the published reference (Chen J., et al., Enzymatic redesigning of biologically active heparan sulfate, J. Biol. Chem., 2005 December, 30; 280(52):42817-25) can be referred to.
(186) The depolymerization can be performed, for example, by using nitrous acid or by the photolysis method. Degree of the depolymerization is not particularly limited. The depolymerization may be performed so that heparin having a molecular weight of, for example, 1000 to 35000 Da is produced.
(187) The produced heparin can be collected by known methods used for separation and purification of compounds. Examples of such methods include, for example, ion-exchange resin method, membrane treatment, precipitation, and crystallization. These methods can be used in an appropriate combination. The collected heparin may contain components such as those used for the heparin production process, and moisture, in addition to heparin. Heparin may be purified in a desired degree. Purity of heparin may be, for example, 30% (w/w) or higher, 50% (w/w) or higher, 70% (w/w) or higher, 80% (w/w) or higher, 90% (w/w) or higher, or 95% (w/w) or higher.
(188) The obtained heparin can be further fractionated to obtain a low molecular weight heparin. Low molecular weight heparin means, for example, a fraction of a molecular weight of 1000 to 10000 Da (average molecular weight, 4000 to 6000 Da). Low molecular weight heparin has an advantage that it shows less adverse reaction of hemorrhage compared with a non-fractionated heparin.
EXAMPLES
(189) Hereafter, the present invention will be more specifically explained with reference to Examples.
Example 1: Construction of Heparosan-Producing Strain from Escherichia coli BL21(DE3) Strain
(190) Construction of Expression Plasmid for kfiABCD Genes of Escherichia coli K5 Strain
(191) From the Escherichia coli K5 strain (ATCC 23506), the kfiABCD genes (kfiABCD operon) were cloned into the pVK9 vector (SEQ ID NO: 1, U.S. Published Patent Application No. 20050196846) to construct a kfiABCD gene expression plasmid, pVK9-kfiABCD.
(192) The details of the construction of the expression plasmid are described below. By PCR using the chromosomal DNA of the Escherichia coli K5 strain as the template, as well as the primer KfiABCD-kpnF (SEQ ID NO: 2) and primer KfiABCD-xbaR (SEQ ID NO: 3), a DNA fragment containing the kfiABCD genes and an upstream sequence thereof of about 450 bp was obtained. PrimeStar Polymerase (TaKaRa) was used for PCR, and PCR was performed in the reaction composition described in the attached protocol. The PCR cycles consisted of 94 C. for 5 minutes, following 30 cycles of 98 C. for 5 seconds, 55 C. for 10 seconds, and 72 C. for 8 minutes, and final maintenance at 4 C. Further, by PCR using pVK9 as the template DNA and the oligonucleotides of SEQ ID NOS: 4 and 5 as the primers, a DNA fragment of pVK9 was obtained. PrimeStar Polymerase (TaKaRa) was used for PCR, and PCR was performed in the reaction composition described in the attached protocol. The PCR cycles consisted of 94 C. for 5 minutes, following 30 cycles of 98 C. for 5 seconds, 55 C. for 10 seconds, and 72 C. for 6 minutes, and final maintenance at 4 C. Both the obtained DNA fragments were ligated by using In-Fusion (registered trademark) HD Cloning Kit (Clontech) to construct a kfiABCD gene expression plasmid, pVK9-kfiABCD. A nucleotide sequence containing the cloned kfiABCD genes and the upstream sequence thereof of about 450 bp is shown as SEQ ID NO: 24.
(193) Construction of kfiABCD Gene-Expressing Strain of Escherichia coli BL21(DE3)
(194) The kfiABCD gene expression plasmid, pVK9-kfiABCD, was introduced into the Escherichia coli BL21(DE3) strain (Life Technologies) by electroporation (cell 80 L, 200 , 25 F, 1.8 kV, cuvette 0.1 mL) to obtain Escherichia coli BL21(DE3)/pVK9-kfiABCD strain. This strain was spread on the LB agar medium containing 25 g/mL kanamycin, and precultured overnight at 37 C. Then, the cells on the plate were scraped and inoculated into 2 mL of a production medium contained in a test tube. Shaking culture was performed at 37 C. for 40 hours, and the culture was finished when glycerol contained in the medium was completely consumed.
(195) The composition of the production medium is shown below.
(196) Production Medium: (Concentrations of Components are Final Concentrations)
(197) Component 1:
(198) TABLE-US-00004 Glycerol 10 g/L
(199) Component 2:
(200) TABLE-US-00005 MOPS (3-N-morpholino-propanesulphonic acid) 41.9 g/L
(201) Components 3:
(202) TABLE-US-00006 Tryptone 8.8 g/L Yeast extract 4.4 g/L Sodium chloride 8.8 g/L
(203) Components 1 and 3 were separately sterilized by autoclaving at 120 C. for 20 minutes, and component 2 was sterilized by filter sterilization. After cooling to room temperature, three of the components were mixed.
(204) Quantification of Polysaccharides by Carbazole Method
(205) The produced polysaccharides were quantified by the carbazole method (Bitter, T. and Murir H. M., Anal. Biochem., 1962, 4:330-334). The procedures are shown below.
(206) The culture supernatant was collected from the culture broth (fermentation broth) by centrifugation. To 150 L of the culture supernatant, 500 L of 100% ethanol was added, and the polysaccharide components were precipitated by centrifugation. The obtained precipitates were air-dried, and dissolved in 300 L of 0.2 N aqueous sodium hydroxide solution. The obtained sample (solution, 30 L) was calmly added to 150 L of sulfuric acid containing 0.025 M tetraboronic acid, and the resulting mixture was heated at 100 C. for 10 minutes. After the mixture was cooled to room temperature, 30 L of a 0.025% carbazole solution (obtained by dissolving 0.125 g of carbazole in 100 mL of 100% ethanol) was added. The resulting mixture was heated at 100 C. for 15 minutes, and then cooled to room temperature, and absorbance was measured at 530 nm. As a result of quantification performed by using a standard curve prepared with D-glucuronic acid, the concentration of the polysaccharides contained in the sample (solution) was calculated to be 140.5 mg/L in terms of glucuronic acid concentration.
Example 2: Structural Analysis of Produced Polysaccharides
(207) (2-1) Nuclear Magnetic Resonance (NMR) Spectrum Analysis
(208) The fermentation broth obtained in Example 1 was subjected to bactofugation, and the supernatant was filtered through a 0.45 m MF membrane. The obtained filtrate (31 g) was concentrated to 1.1 g by using a UF membrane of 100 KDa (Amicon-15K, 5000 rpm). The concentrate was further washed twice with 40 mL of water. The washed concentrate was further concentrated under reduced pressure in an evaporator, 600 L of heavy water was added to the residue to prepare a solution, and then 41-NMR measurement was performed.
(209) The analysis conditions are shown below.
(210) (A) Apparatus: AVANCE400 produced by Bruker; 1H, 400 MHz
(211) (B) Solvent: Heavy water
(212) (C) Temperature: Room temperature
(213) (D) Number of times of measurement: 16 times
(214) As a result, there was observed a spectrum of .sup.1H-NMR (D.sub.2O) including peaks of a: 1.9 (methyl proton of N-acetyl group), 3.3-4.5 (methylene and methine protons of C2 to C6), and 5.3 (methine proton of C1). This spectrum was the same as the .sup.1H-NMR spectrum of heparosan produced by Iduron (lot number, B.N.4).
(215) (2-2) Disaccharide Composition Analysis by Liquid Chromatography-Mass Spectrometry (LC-MS)
(216) The fermentation broth obtained in Example 1 was subjected to bactofugation, and the supernatant was filtered through a 0.45 m MF membrane. The obtained filtrate (40 mL) was concentrated to 4 mL by using a UF membrane of 100 KDa (Amicon-15K, 5000 rpm). The concentrate was further washed twice with 40 mL of water. To 50 L of the washed concentrate, 10 L of Tris-buffer (200 mM Tris-HCl, 1 M NaCl, 15 mM CaCl.sub.2, adjusted to pH 7 (25 C.) with 35% hydrochloric acid), 10 L of heparinase III (0.005 unit/mL, produced by Iduron), and 30 L of water were added, and enzyme treatment was performed at 37 C. for 16 hours. To the obtained enzyme-treated mixture, 900 L of water was added, and used for LC-MS analysis.
(217) The analysis conditions are shown below:
(218) (A) Apparatus: LC-MS 2010 produced by Shimadzu
(219) (B) Column: UG80 (SCX, Shiseido), 2.0 mm250 mm, particle size 5 m
(220) (C) Mobile phase: CH.sub.3CN/10 mM formic acid=8/2
(221) (D) Flow rate: 0.2 mL/minute
(222) (E) Column temperature: 40 C.
(223) (F) Injection volume: 10
(224) (G) UV (PDA): 200 to 600 nm
(225) (H) MS (ESI): 100 to 2000 (positive and negative)
(226) As a result, fragment ions of [m/z]=362 (M+H-H.sub.2O), 380 (M+H), and 418 (M+K) were detected at a retention time of 6 minutes. The retention time and fragment pattern of the enzyme-treated mixture agreed with the retention time and fragment pattern of a GlcUA-GlcNAc standard sample (Heparin disaccharide IV-A sodium salt, Sigma-Aldrich), which is a heparinase decomposition product of heparin and heparan sulfate. The structural formula of the GlcUA-GlcNAc standard sample is shown below as the formula (I).
(227) ##STR00001##
(228) On the basis of the aforementioned results of NMR and LC-MS, it was identified that the polymer component obtained from the culture broth of the BL21(DE3)/pVK9-kfiABCD strain was objective heparosan. Therefore, the glucuronic acid concentration multiplied by a coefficient 2.067 was used as heparosan concentration measured by the carbazole method.
(229) (2-3) Gel Filtration Chromatography (GPC) Analysis
(230) The fermentation broth obtained in Example 1 was subjected to bactofugation, and the supernatant was filtered through a 0.45 m MF membrane. The obtained filtrate (31 g) was concentrated to 1.1 g by using a UF membrane of 100 KDa (Amicon-15K, 5000 rpm). The concentrate was further washed twice with 40 mL of water. GPC measurement of the washed concentrate was performed.
(231) Analysis conditions are shown below.
(232) (A) Apparatus: HPLC produced by Shimadzu
(233) (B) Column: Asahipak GS520HQ, 7.5 mm300 mm
(234) (C) Mobile phase: 100 mM KH.sub.2PO.sub.4
(235) (D) Flow rate: 0.6 mL/minute
(236) (E) Column temperature: 40 C.
(237) (F) Injection volume: 20
(238) (G) UV: 200 nm
(239) (H) Molecular weight standard sample: Pullulan (P-82, Showa Denko)
(240) As a result, it was confirmed that retention time (peak top) was 8.3 minutes, number average molecular weight (Mn) was 240,000, weight average molecular weight (Mw) was 320,000, and Mw/Mn was 1.3.
Example 3: Screening for Factors that Improve Heparosan-Producing Ability
(241) In this example, screening was performed for factors that improve heparosan-producing ability by introducing a genomic library of the Escherichia coli K5 strain into a heparosan-producing strain.
(242) (3-1) Construction of Escherichia coli BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD Strain
(243) As a heparosan-producing strain to be introduced with the genomic library, Escherichia coli BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strain introduced with the kfiABCD gene and showing enhanced expression of the rfaH gene was constructed in accordance with the following procedures.
(244) A rfaH gene expression-enhanced strain was obtained by replacing the native promoter region of the rfaH gene on the chromosome with a potent tac promoter (Amann E. et al. (1983) Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli, Gene, 25(2-3):167-78). The rfaH promoter was replaced with the tac promoter by using the method called Red-driven integration, which was originally developed by Datsenko and Wanner (One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645). According to this technique, a strain in which a DNA fragment amplified by PCR is inserted into the genomic DNA can be obtained.
(245) First, by PCR using the genomic DNA of the Pantoea ananatis NA1c1129 strain (WO2010/027022A1) as the template, as well as the primer rfaH-attL Fw (SEQ ID NO: 6) and primer rfaH-Ptac Rv (SEQ ID NO: 7), a DNA fragment for promoter substitution was amplified. PrimeStar Polymerase was used for PCR, and the PCR cycles consisted of 94 C. for 5 minutes, following 30 cycles of 98 C. for 5 seconds, 55 C. for 10 seconds, and 72 C. for 3 minutes, and final maintenance at 4 C. The primer rfaH-attL Fw (SEQ ID NO: 6) shows homology to both a region locating upstream from the rfaH gene, and a region adjacent to the gene that imparts kanamycin (km) resistance existing in the genomic DNA of the NA1c1129 strain. The Km resistance gene kan existing in the genomic DNA of the NA1c1129 strain is inserted between the attL and attR genes, which are the attachment sites of phage, and the tac promoter (Ptac, SEQ ID NO: 8) is inserted further downstream therefrom in the order of attL-kan-attR-Ptac. The primer rfaH-Ptac Rv (SEQ ID NO: 7) shows homology to both the rfaH region and a region locating downstream from the tac promoter in the genomic DNA of the NA1c1129 strain.
(246) Then, into Escherichia coli BL21(DE3)/pKD46 strain obtained by introducing the plasmid pKD46 having a temperature sensitive replication origin (Datsenko and Wanner, Proc. Natl. Acad. Sci. USA, 2000, 97:12:6640-45) into Escherichia coli BL21(DE3) strain (C6000-03, Life Technologies), the PCR product obtained above was introduced by electroporation to attain substitution of the promoter region. The plasmid pKD46 contains the genes of the Red homologous recombination system (, , and exo genes), i.e. a 2,154 nucleotide DNA fragment of phage (GenBank/EMBL Accession No. J02459, nucleotide positions 31088 to 33241), under the control of the arabinose-inducible P.sub.araB promoter. The plasmid pKD46 is necessary for integration of the PCR product into the chromosome of the BL21(DE3) strain. The Escherichia coli BL21(DE3)/pKD46 strain was grown overnight at 30 C. in the LB medium containing ampicillin (100 mg/L). This culture was diluted 100 times with the LB medium (100 mL) containing ampicillin and L-arabinose (1 mM). The cells were grown at 30 C. with aeration until OD600 became about 0.3, then concentrated 100 times, washed 3 times with ice-cooled aqueous glycerol solution (10%), and thereby made into electrocompetent cells. Electroporation was performed by using 70 l of the competent cells and about 100 ng of the PCR product. After the electroporation, the cells were incubated in 1 mL of the SOC medium (Molecular Cloning A Laboratory Manual, 2nd edition, Sambrook, J. et al., Cold Spring Harbor Laboratory Press (1989)) at 37 C. for 2.5 hours, applied to the LB agar medium, and grown at 37 C. to select Km resistant strains.
(247) The substitution of the tac promoter for the rfaH promoter was confirmed by PCR using the primer rfaH CF (SEQ ID NO: 9) and primer rfaH CR (SEQ ID NO: 10), which are specific to the nucleotide sequence after the promoter substitution. PrimeStar Polymerase was used for PCR. The PCR cycles consisted of 94 C. for 5 minutes, following 30 cycles of 98 C. for 5 seconds, 55 C. for 10 seconds, and 72 C. for 2 minutes, and final maintenance at 4 C. A strain in which amplification of a DNA fragment of 1.6 kbp could be confirmed was designated as BL21(DE3)-Ptac-rfaH(KmR) strain.
(248) In order to remove the Km resistance marker from the BL21(DE3)-Ptac-rfaH(KmR) strain, plasmid pMW118-int-xis (ampicillin resistant (AmpR)) was introduced (WO2005/010175) to the strain. AmpR clones were grown at 30 C. on an LB agar plate containing 150 mg/L of ampicillin. Several tens of AmpR clones were picked up, and a Km-sensitive strain was selected. By incubating the Km sensitive strain at 42 C. on an LB agar plate, the plasmid pMW118-int-xis was removed from the Km-sensitive strain. An obtained Amp sensitive strain was designated as BL21(DE3)-Ptac-rfaH strain. The plasmid pVK9-kfiABCD produced in Example 1 was introduced into the BL21(DE3)-Ptac-rfaH strain by electroporation to obtain BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strain. Culture was performed in test tubes by using the same medium and culture method as those of Example 1, and heparosan production amount was determined by the carbazole method. The heparosan production amounts of the BL21(DE3)/pVK9-kfiABCD strain of which expression of the rfaH gene was not enhanced, and the BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strain of which expression of the rfaH gene was enhanced are shown in Table 4.
(249) TABLE-US-00007 TABLE 4 Heparosan production amount of BL21(DE3)- Ptac-rfaH/pVK9-kfiABCD strain Strain Heparosan (mg/L) BL21(DE3)/pVK9-kfiABCD 290.4 32.7 BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD 506.2 69.9
(250) (3-2) Construction of Genomic Library of Escherichia coli K5 Strain
(251) Fragments of the genomic DNA of the Escherichia coli K5 strain were cloned into the pSTV28 vector (SEQ ID NO: 11, TaKaRa) to constructed genomic library.
(252) The details of the construction of the genomic library are shown below. The genomic DNA of the Escherichia coli K5 strain (3 g) was randomly fragmented by using a DNA fragmentation apparatus (Hydro-Shear, Gene Machine), and fractionated by agarose electrophoresis. A portion containing DNAs of about 3 to 5 kb was cut out from the agarose gel, and DNAs were extracted, purified, and then blunt-ended. Then, the genomic DNA fragments were ligated with 50 ng of the plasmid vector pSTV28 (TaKaRa) digested with HincII and dephosphorylated with Alkaline Phosphatase (E. coli C75) (TaKaRa). The Escherichia coli HST08 strain (TaKaRa) was transformed with the ligation product by electroporation. Seventy percent or more of the obtained transformants contained inserts of about 3 to 5 kb. The transformants were cultured, and the plasmids were extracted to obtain a genomic library.
(253) (3-3) Selection of Strains Showing Heparosan-Producing Ability Improved by Introduction of Genomic Library
(254) The genomic library or pSTV28 as a control was introduced into the BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strain by electroporation. One clone was selected from each of the obtained genomic library transformants, and used to perform fermentative production culture. Media of the following compositions were used for the culture.
(255) Seed Medium: (Concentrations of Components are Final Concentrations)
(256) TABLE-US-00008 Tryptone 10 g/L Yeast extract 5 g/L Sodium chloride 10 g/L
(257) The seed medium was sterilized by autoclaving at 120 C. for 20 minutes.
(258) Production Medium: (Concentrations of Components are Final Concentrations)
(259) Component 1:
(260) TABLE-US-00009 Glycerol 10 g/L
(261) Component 2:
(262) TABLE-US-00010 MOPS (3-N-morpholino-propanesulphonic acid) 41.9 g/L
(263) Components 3:
(264) TABLE-US-00011 Tryptone 8.8 g/L Yeast extract 4.4 g/L Sodium chloride 8.8 g/L
(265) The components 1 and 3 were separately sterilized by autoclaving at 120 C. for 20 minutes, and the component 2 was sterilized by filter sterilization. After cooling to room temperature, three of the components were mixed.
(266) The heparosan production culture was performed according to the following procedures. First, one colony of each transformant was inoculated into each well of a 96-well plate (MEDISCAN), which contained 750 L of the seed medium, and shaking culture was performed overnight at 37 C. on a shaking machine (Tietech). Then, 20 L of the seed culture broth was inoculated into 2 mL of the production medium contained in a test tube, shaking culture was performed at 37 C. for 30 hours, and the culture was terminated when the glycerol in the medium was completely consumed. In order to make the cells harbor the plasmids, kanamycin (25 mg/L) and chloramphenicol (25 mg/L) were added to the medium over the whole culture period. Heparosan produced in the medium was quantified by the carbazole method (Bitter, T. and Murir H. M., Anal. Biochem., 1962, 4:330-334). There were isolated clones that showed increased heparosan accumulation amounts as compared with the simultaneously cultured control vector (pSTV28)-introduced strain. In order to identify the genes inserted into the plasmids contained in the isolated clones, the nucleotide sequences of the inserted DNA fragments were determined by using the primer pSTV Fw (SEQ ID NO: 12) and primer pSTV Rv (SEQ ID NO: 13). As a result, it was revealed that the respective plasmids contained rbsBKR-hsrA, glgBX, ybiXIJCB, rcsBD-micF, pcoESR, yhcNO-aaeBAX, g1455-alpA-g1453, yrbA-mlaBCDEF-yrbG, norW, ybjIJK-rybB, thrBAL-yjtD-yjjY, fruA-psuK, ytfT-yjfF-fbp, yagU-paoAB, gsiCD-yliE, irp (part), bhsA-ycfS, lepB-rnc-era, dapA-gcvR-bcp-hyfA, rpoE-nadB-yfiC-srmB, g1414-g1413, nuoEFG, glmZ-hemYXD, rlmL, artQMJ-rlmC-ybjO, yejOML, rpoS-ygbNML, g3798-g3797-g3796-g3795-g3794-g3793-g3792, ryjA-soxRS-yjcCB, and efeUO. The irp (part) means a part of the irp2 gene and a part of the irp1 gene. The nucleotide sequences of the inserted fragments containing rbsBKR-hsrA, glgBX, ybiXIJCB, rcsBD-micF, pcoESR, yhcNO-aaeBAX, g1455-alpA-g1453, yrbA-mlaBCDEF-yrbG, norW, ybjIJK-rybB, thrBAL-yjtD-yjjY, fruA-psuK, ytfT-yjfF-fbp, yagU-paoAB, gsiCD-yliE, irp (part), bhsA-ycfS, lepB-rnc-era, dapA-gcvR-bcp-hyfA, rpoE-nadB-yfiC-srmB, g1414-g1413, nuoEFG, glmZ-hemYXD, rlmL, artQMJ-rlmC-ybjO, yejOML, rpoS-ygbNML, g3798-g3797-g3796-g3795-g3794-g3793-g3792, ryjA-soxRS-yjcCB, and efeUO are shown as SEQ ID NOS: 29, 34, 37, 43, 50, 54, 60, 64, 72, 74, 78, 84, 87, 91, 95, 99, 104, 107, 111, 116, 121, 124, 128, 132, 134, 140, 144, 149, 157, and 162, respectively. From the respective isolated clones, plasmids pSTV28-rbsBKR-hsrA, pSTV28-glgBX, pSTV28-ybiXIJCB, pSTV28-rcsBD-micF, pSTV28-pcoESR, pSTV28-yhcNO-aaeBAX, pSTV28-g1455-alpA-g1453, pSTV28-yrbA-mlaBCDEF-yrbG, pSTV28-norW, pSTV28-ybjIJK-rybB, pSTV28-thrBAL-yjtD-yjjY, pSTV28-fruA-psuK, pSTV28-ytfT-yjfF-fbp, pSTV28-yagU-paoAB, pSTV28-gsiCD-yliE, pSTV28-irp, pSTV28-bhsA-ycfS, pSTV28-lepB-rnc-era, pSTV28-dapA-gcvR-bcp-hyfA, pSTV28-rpoE-nadB-yfiC-srmB, pSTV28-g1414-g1413, pSTV28-nuoEFG, pSTV28-glmZ-hemYXD, pSTV28-rlmL, pSTV28-artQMJ-rlmC-ybjO, pSTV28-yejOML, pSTV28-rpoS-ygbNML, pSTV28-g3798-g3797-g3796-g3795-g3794-g3793-g3792, pSTV28-ryjA-soxRS-yjcCB, and pSTV28-efeUO were extracted.
Example 4: Heparosan Production Using rbsBKR-hsrA, glgBX, ybiXIJCB, and rcsBD-micF Gene Expression-Enhanced Strains (1)
(267) There were constructed respective strains from the Escherichia coli BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strain constructed in Example 1 by introducing thereto pSTV28-rbsBKR-hsrA, pSTV28-glgBX, pSTV28-ybiXIJCB, and pSTV28-rcsBD-micF isolated in Example 3, and pSTV28 as a control. Fermentative production culture was performed with these strains, and amounts of the produced heparosan were compared. The strains were cultured in test tubes in quadruplicate by using the same medium and culture method as those shown in Example 1, and heparosan was quantified by the carbazole method. Averages and standard deviations of the measured heparosan concentrations are shown in Table 5.
(268) TABLE-US-00012 TABLE 5 Effect of enhancement of rbsBKR-hsrA, glgBX, ybiXIJCB, or micF-rcsDB gene expression in BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strain Strain Heparosan (mg/L) BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28 529.6 46.3 BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 766.4 156.7 rbsBKR-hsrA BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 679.8 9.7 glgBX BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 753.4 129.2 ybiXIJCB BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 656.7 57.7 micF-rcsDB
Example 5: Heparosan Production Using rbsBKR-hsrA, glgBX, ybiXIJCB, and rcsBD-micF Gene Expression-Enhanced Strains (2)
(269) There were constructed respective strains from the Escherichia coli BL21(DE3)/pVK9-kfiABCD strain constructed in Example 1 by introducing thereto pSTV28-rbsBKR-hsrA, pSTV28-glgBX, pSTV28-ybiXIJCB, and pSTV28-rcsBD-micF isolated in Example 3, and pSTV28 as a control. Fermentative production culture was performed with these strains, and amounts of the produced heparosan were compared. The strains were cultured in test tubes in quadruplicate by using the same medium and culture method as those shown in Example 1, and heparosan was quantified by the carbazole method. Averages and standard deviations of the measured heparosan concentrations are shown in Table 6.
(270) TABLE-US-00013 TABLE 6 Effect of enhancement of rbsBKR-hsrA, glgBX, ybiXIJCB, or micF-rcsDB gene expression in BL21(DE3)/pVK9-kfiABCD strain Strain Heparosan (mg/L) BL21(DE3)/pVK9-kfiABCD/pSTV28 145.3 14.3 BL21(DE3)/pVK9-kfiABCD/pSTV28-rbsBKR-hsrA 286.9 53.3 BL21(DE3)/pVK9-kfiABCD/pSTV28-glgBX 238.7 44.2 BL21(DE3)/pVK9-kfiABCD/pSTV28-ybiXIJCB 238.1 55.4 BL21(DE3)/pVK9-kfiABCD/pSTV28-micF-rcsDB 167.0 7.0
Example 6: Heparosan Production Using rfaH Gene Expression-Enhanced Strain
(271) (6-1) Construction of Expression Plasmid for rfaH Gene of Escherichia coli B Strain
(272) The rfaH gene was cloned from the Escherichia coli BL21(DE3) strain into pMIV-Pn1p0-ter to construct a rfaH gene expression plasmid, pMIV-Pn1p0-rfaH. pMIV-Pn1p0-ter contains the potent n1p0 promoter (Pn1p0) and the rrnB terminator, and the promoter and the terminator can function as an expression unit of a target gene when the target gene is inserted therebetween. Pn1p0 means the wild-type promoter of the nlpD gene of the Escherichia coli K-12 strain.
(273) The details of the construction of the expression plasmid are shown below. By PCR using the chromosomal DNA of Escherichia coli MG1655 as the template, as well as the primer P1 (SEQ ID NO: 14) and primer P2 (SEQ ID NO: 15), there was obtained a DNA fragment containing the promoter region of the nlpD gene of about 300 bp (wild-type nlpD gene promoter is henceforth referred to as Pn1p0). The sites for the restriction enzymes SalI and PaeI were designed in the 5 end regions of the respective primers. The PCR cycles consisted of 95 C. for 3 minutes, following 2 cycles of 95 C. for 60 seconds, 50 C. for 30 seconds, and 72 C. for 40 seconds, 25 cycles of 94 C. for 20 seconds, 55 C. for 20 seconds, and 72 C. for 15 seconds, and 72 C. for 5 minutes as the final cycle. The obtained fragment was treated with SalI and PaeI, and inserted into pMIV-5JS (Japanese Patent Laid-open (Kokai) No. 2008-99668) at the SalI-PaeI site to obtain plasmid pMIV-Pn1p0. The nucleotide sequence of the PaeI-SalI fragment of the Pn1p0 promoter inserted into this pMIV-Pn1p0 plasmid is as shown as SEQ ID NO: 16.
(274) Then, by PCR using the chromosomal DNA of MG1655 as the template, as well as the primer P3 (SEQ ID NO: 17) and primer P4 (SEQ ID NO: 18), a DNA fragment (SEQ ID NO: 19) containing about 300 bp of the terminator region of the rrnB gene was obtained. The sites for the restriction enzymes XbaI and BamHI were designed in the 5 end regions of the respective primers. The PCR cycles consisted of 95 C. for 3 minutes, following 2 cycles of 95 C. for 60 seconds, 50 C. for 30 seconds, and 72 C. for 40 seconds, 25 cycles of 94 C. for 20 seconds, 59 C. for 20 seconds, and 72 C. for 15 seconds, and 72 C. for 5 minutes as the final cycle. The obtained fragment was treated with XbaI and BamHI, and inserted into pMIV-Pn1p0 at the XbaI-BamHI site to obtain plasmid pMIV-Pn1p0-ter.
(275) Then, by PCR using the chromosomal DNA of the Escherichia coli BL21(DE3) strain as the template, as well as the primer rfaH Fw (SEQ ID NO: 20) and primer rfaH Rv (SEQ ID NO: 21), a rfaH gene fragment was obtained. The sites for the restriction enzymes SalI and XbaI were designed in the 5 end regions of the respective primers. PrimeStar Polymerase was used for PCR, and the PCR cycles consisted of 94 C. for 5 minutes, following 30 cycles of 98 C. for 5 seconds, 55 C. for 10 seconds, and 72 C. for 4 minutes, and final maintenance at 4 C. The obtained fragment was treated with SalI and XbaI, and inserted into pMIV-Pn1p0-ter at the SalI-XbaI site to obtain plasmid pMIV-Pn1p0-rfaH. As described above, there was constructed an rfaH expression unit comprising the nlpD promoter, rfaH gene, and rrnB terminator connected in this order in the pMIV-5JS vector. The nucleotide sequence of the rfaH gene of the Escherichia coli BL21(DE3) strain cloned in this experiment is shown as SEQ ID NO: 46.
(276) (6-2) Heparosan Production Using rfaH Gene Expression-Enhanced Strain
(277) There were constructed respective strains from the Escherichia coli BL21(DE3)/pVK9-kfiABCD strain constructed in Example 1 by introducing thereto pMIV-Pn1p0-rfaH and pMIV-5JS as a control. Fermentative production culture was performed with these strains, and amounts of the produced heparosan were compared. The medium, culture method, and quantification method for heparosan were the same as those described above. Averages and standard deviations of the measured heparosan concentrations are shown in Table 7.
(278) TABLE-US-00014 TABLE 7 Effect of enhancement of rfaH gene expression in BL21(DE3)/pVK9-kfiABCD strain Strain Heparosan (mg/L) BL21(DE3)/pVK9-kfiABCD/pMIV-5JS 376.9 48.3 BL21(DE3)/pVK9-kfiABCD/pMIV-Pnlp0-rfaH 857.9 219.1
Example 7: Heparosan Production Using nusG Gene Expression-Enhanced Strain
(279) (7-1) Construction of Expression Plasmid for nusG Gene of Escherichia coli B Strain
(280) By PCR using the chromosomal DNA of the Escherichia coli BL21(DE3) strain as the template, as well as the primer nusG Fw (SEQ ID NO: 22) and primer nusG Rv (SEQ ID NO: 23), a nusG gene fragment was obtained. The sites for the restriction enzymes SalI and XbaI were designed in the 5 end regions of the respective primers. PrimeStar Polymerase was used for PCR, and the PCR cycles consisted of 94 C. for 5 minutes, following 30 cycles of 98 C. for 5 seconds, 55 C. for 10 seconds, and 72 C. for 4 minutes, and final maintenance at 4 C. The obtained fragment was treated with SalI and XbaI, and inserted into pMIV-Pn1p0-ter treated with the same restriction enzymes at the SalI-XbaI site to obtain plasmid pMIV-Pn1p0-nusG in which the nusG gene was cloned. The nucleotide sequence of the nusG gene of the Escherichia coli BL21(DE3) strain cloned in this experiment is shown as SEQ ID NO: 48.
(281) (7-2) Heparosan Production Using nusG Gene Expression-Enhanced Strain
(282) There were constructed respective strains from the Escherichia coli BL21(DE3)/pVK9-kfiABCD strain constructed in Example 1 by introducing thereto pMIV-Pn1p0-nusG and pMIV-5JS as a control. Fermentative production culture was performed with these strains, and amounts of the produced heparosan were compared. The medium, culture method, and quantification method for heparosan were the same as those described above. Averages and standard deviations of the measured heparosan concentrations are shown in Table 8.
(283) TABLE-US-00015 TABLE 8 Effect of enhancement of nusG gene expression in BL21(DE3)/pVK9-kfiABCD strain Strain Heparosan (mg/L) BL21(DE3)/pVK9-kfiABCD/pMTV-5JS 376.9 48.3 BL21(DE3)/pVK9-kfiABCD/pMTV-Pnlp0-nusG 618.1 14.6
Example 8: Heparosan Production Using pcoESR, yhcNO-aaeBAX, g1455-alpA-g1453, yrbA-mlaBCDEF-yrbG, norW, ybjIJK-rybB, thrBAL-yjtD-yjjY, fruA-psuK, ytfT-yjfF-fbp, yagU-paoAB, gsiCD-yliE, Irp (Part), and bhsA-ycfS Gene Expression-Enhanced Strains (1)
(284) There were constructed respective strains from the Escherichia coli BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strain constructed in Example 1 by introducing thereto pSTV28-pcoESR, pSTV28-yhcNO-aaeBAX, pSTV28-g1455-alpA-g1453, pSTV28-yrbA-mlaBCDEF-yrbG, pSTV28-norW, pSTV28-ybjIJK-rybB, pSTV28-thrBAL-yjtD-yjjY, pSTV28-fruA-psuK, pSTV28-ytfT-yjfF-fbp, pSTV28-yagU-paoAB, pSTV28-gsiCD-yliE, pSTV28-irp, and pSTV28-bhsA-ycfS isolated in Example 3, and pSTV28 as a control. Fermentative production culture was performed with these strains, and amounts of the produced heparosan were compared. The strains were cultured in test tubes in quadruplicate by using the same medium and culture method as those shown in Example 1, and heparosan was quantified by the carbazole method. Averages and standard deviations of the measured heparosan concentrations are shown in Table 9.
(285) TABLE-US-00016 TABLE 9 Effect of enhancement of pcoESR, yhcNO-aaeBAX, g1455- alpA-g1453, yrbA-mlaBCDEF-yrbG, norW, ybjIJK-rybB, thrBAL-yjtD-yjjY, fruA-psuK, ytfT-yjfF-fbp, yagU-paoAB, gsiCD-yliE, irp (part), or bhsA-ycfS gene expression in BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strain Strain Heparosan (mg/L) BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28 559.8 72.6 BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 692.0 95.1 pcoESR BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 663.3 136.1 yhcNO-aaeBAX BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 843.1 81. 5 g1455-alpA-g1453 BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 877.8 137.8 yrbA-mlaBCDEF-yrbG BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 741.0 21.1 norW BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 796.0 235.8 ybjIJK-rybB BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 635.1 7.9 thrBAL-yjtD-yjjY BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 826.0 125.1 fruA-psuK BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 872.3 20.1 ytfT-yjfF-fbp BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 690.2 63.1 yagU-paoAB BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 804.1 92.9 gsiCD-yliE BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 691.5 92.4 irp BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 994.3 124.1 bhsA-ycfS
Example 9: Heparosan Production Using pcoESR, yhcNO-aaeBAX, g1455-alpA-g1453, yrbA-mlaBCDEF-yrbG, norW, ybjIJK-rybB, thrBAL-yjtD-yjjY, fruA-psuK, ytfT-yjfF-Fbp, yagU-paoAB, gsiCD-yliE, Irp (Part), and bhsA-ycfS Gene Expression-Enhanced Strains (2)
(286) There were constructed respective strains from the Escherichia coli BL21(DE3)/pVK9-kfiABCD strain constructed in Example 1 by introducing thereto pSTV28-pcoESR, pSTV28-yhcNO-aaeBAX, pSTV28-g1455-alpA-g1453, pSTV28-yrbA-mlaBCDEF-yrbG, pSTV28-norW, pSTV28-ybjIJK-rybB, pSTV28-thrBAL-yjtD-yjjY, pSTV28-fruA-psuK, pSTV28-ytfT-yjfF-fbp, pSTV28-yagU-paoAB, pSTV28-gsiCD-yliE, pSTV28-irp, and pSTV28-bhsA-ycfS isolated in Example 3, and pSTV28 as a control. Fermentative production culture was performed with these strains, and amounts of the produced heparosan were compared. The strains were cultured in test tubes in quadruplicate by using the same medium and culture method as those shown in Example 1, and heparosan was quantified by the carbazole method. Averages and standard deviations of the measured heparosan concentrations are shown in Table 10.
(287) TABLE-US-00017 TABLE 10 Effect of enhancement of pcoESR, yhcNO-aaeBAX, g1455-alpA-g1453, yrbA-mlaBCDEF-yrbG, norW, ybjIJK-rybB, thrBAL-yjtD-yjjY, fruA- psuK, ytfT-yjfF-fbp, yagU-paoAB, gsiCD-yliE, irp (part), or bhsA-ycfS gene expression in BL21(DE3)/pVK9-kfiABCD strain Strain Heparosan (mg/L) BL21(DE3)/pVK9-kfiABCD/pSTV28 106.4 12.4 BL21(DE3)/pVK9-kfiABCD/pSTV28-pcoESR 260.8 47.8 BL21(DE3)/pVK9-kfiABCD/pSTV28-yhcNO-aaeBAX 237.2 36.6 BL21(DE3)/pVK9-kfiABCD/pSTV28-g1455-alpA- 225.6 8.5 g1453 BL21(DE3)/pVK9-kfiABCD/pSTV28-yrbA- 215.2 14.9 mlaBCDEF-yrbG BL21(DE3)/pVK9-kfiABCD/pSTV28-norW 216.4 36.6 BL21(DE3)/pVK9-kfiABCD/pSTV28-ybjIJK-rybB 301.9 46.9 BL21(DE3)/pVK9-kfiABCD/pSTV28-thrBAL-yjtD- 327.2 39.8 yjjY BL21(DE3)/pVK9-kfiABCD/pSTV28-fruA-psuK 209.3 8.1 BL21(DE3)/pVK9-kfiABCD/pSTV28-ytfT-yjfF- 220.1 18.5 fbp BL21(DE3)/pVK9-kfiABCD/pSTV28-yagU-paoAB 258.0 26.4 BL21(DE3)/pVK9-kfiABCD/pSTV28-gsiCD-yliE 323.3 15.5 BL21(DE3)/pVK9-kfiABCD/pSTV28-irp 202.0 15.0 BL21(DE3)/pVK9-kfiABCD/pSTV28-bhsA-ycfS 225.5 37.4
Example 10: Heparosan Production Using lepB-Rnc-Era, dapA-gcvR-Bcp-hyfA, rpoE-nadB-yfiC-srmB, g1414-g1413, nuoEFG, glmZ-hemYXD, rlmL, artQMJ-rlmC-ybjO, yejOML, rpoS-ygbNML, g3798-g3797-g3796-g3795-g3794-g3793-g3792, ryjA-soxRS-yjcCB, and efeUO Gene Expression-Enhanced Strains
(288) There were constructed respective strains from the Escherichia coli BL21(DE3)/pVK9-kfiABCD strain constructed in Example 1 by introducing thereto pSTV28-lepB-rnc-era, pSTV28-dapA-gcvR-bcp-hyfA, pSTV28-rpoE-nadB-yfiC-srmB, pSTV28-g1414-g1413, pSTV28-nuoEFG, pSTV28-glmZ-hemYXD, pSTV28-rlmL, pSTV28-artQMJ-rlmC-ybjO, pSTV28-yejOML, pSTV28-rpoS-ygbNML, pSTV28-g3798-g3797-g3796-g3795-g3794-g3793-g3792, pSTV28-ryjA-soxRS-yjcCB, and pSTV28-efeUO isolated in Example 3, and pSTV28 as a control. Fermentative production culture was performed with these strains, and amounts of the produced heparosan were compared. The strains were cultured in test tubes in quadruplicate by using the same medium and culture method as those shown in Example 1, and heparosan was quantified by the carbazole method. Averages and standard deviations of the measured heparosan concentrations are shown in Tables 11 and 12.
(289) TABLE-US-00018 TABLE 11 Effect of enhancement of lepB-rnc-era, dapA-gcvR-bcp-hyfA, rpoE-nadB-yfiC-srmB, g1414-g1413, nuoEFG, or glmZ-hemYXD gene expression in BL21(DE3)/pVK9-kfiABCD strain Strain Heparosan (mg/L) BL21(DE3)/pVK9-kfiABCD/pSTV28 210.4 23.6 BL21(DE3)/pVK9-kfiABCD/pSTV28-lepB-rnc-era 488.7 83.5 BL21(DE3)/pVK9-kfiABCD/pSTV28-dapA-gcvR- 379.8 49.1 bcp-hyfA BL21(DE3)/pVK9-kfiABCD/pSTV28-rpoE-nadB- 282.9 54.1 yfiC-srmB BL21(DE3)/pVK9-kfiABCD/pSTV28-g1414-g1413 423.9 119.5 BL21(DE3)/pVK9-kfiABCD/pSTV28-nuoEFG 428.5 64.6 BL21(DE3)/pVK9-kfiABCD/pSTV28-glmZ-hemYXD 604.2 177.5
(290) TABLE-US-00019 TABLE 12 Effect of enhancement of rlmL, artQMJ-rlmC-ybjO, yejOML, rpoS-ygbNML, g3798-g3797-g3796-g3795-g3794- g3793-g3792, ryjA-soxRS-yjcCB, or efeUO gene expression in BL21(DE3)/pVK9-kfiABCD strain Strain Heparosan (mg/L) BL21(DE3)/pVK9-kfiABCD/pSTV28 147.4 16.3 BL21(DE3)/pVK9-kfiABCD/pSTV28-rlmL 242.7 45.8 BL21(DE3)/pVK9-kfiABCD/pSTV28-artQMJ-rlmC- 243.4 97.9 ybjO BL21(DE3)/pVK9-kfiABCD/pSTV28-yejOML 310.7 40.8 BL21(DE3)/pVK9-kfiABCD/pSTV28-rpoS-ygbNML 257.1 56.8 BL21(DE3)/pVK9-kfiABCD/pSTV28-g3798-g3797- 251.6 68.5 g3796-g3795-g3794-g3793-g3792 BL21(DE3)/pVK9-kfiABCD/pSTV28-ryjA-soxRS- 287.5 46.0 yjcCB BL21(DE3)/pVK9-kfiABCD/pSTV28-efeUO 385.7 88.5
Example 11: Heparosan Production Using rpoE Gene Expression-Enhanced Strain
(291) (11-1) Construction of Expression Plasmid for rpoE Gene of Escherichia coli K5 Strain
(292) The rpoE gene was cloned from the Escherichia coli K5 strain into pMIV-Pn1p8-ter to construct a rpoE gene expression plasmid, pMIV-Pn1p0-rpoE. pMIV-Pn1p0-ter contains the potent n1p8 promoter (Pn1p8), and the promoter and a terminator can function as an expression unit of a target gene when the target gene is inserted therebetween. Pn1p8 means a variant promoter of the nlpD gene of the Escherichia coli K-12 strain.
(293) The details of the construction of the expression vector pMIV-Pn1p8-ter are shown below. In order to make the wild-type nlpD promoter (Pn1p0) be a stronger promoter by modifying the 10 region thereof, the 10 region was randomized according to the following procedures. The wild-type nlpD promoter region (
(294) In the same manner, by PCR using the plasmid pMIV-Pn1p0-ter as the template, as well as the primer P2 (SEQ ID NO: 15) and primer P8 (SEQ ID NO: 167), there was obtained a DNA fragment of the wild-type nlpD promoter (Pn1p0) of which 10 region (10(Pn1p2)) contained on the 5 end side was randomized. The PCR cycle consisted of 95 C. for 3 minutes, following 2 cycles of 95 C. for 60 seconds, 50 C. for 30 seconds, and 72 C. for 40 seconds, 25 cycles of 94 C. for 20 seconds, 60 C. for 20 seconds, and 72 C. for 15 seconds, and 72 C. for 5 minutes as the final cycle.
(295) The obtained fragments for the 3 end side and 5 end side were ligated by using the BglII sites designed in the primers P7 and P8 to construct a DNA fragment containing a variant nlpD promoter in full length, of which two 10 regions were randomized. By PCR using this DNA fragment as the template, as well as the primer P1 and primer P2, the DNA fragment containing the full length of the variant nlpD promoter was amplified. The PCR cycles consisted of 95 C. for 3 minutes, following 2 cycles of 95 C. for 60 seconds, 50 C. for 30 seconds, and 72 C. for 40 seconds, 12 cycles of 94 C. for 20 seconds, 60 C. for 20 seconds, and 72 C. for 15 seconds, and 72 C. for 5 minutes as the final cycle.
(296) The amplified DNA fragment containing the full length of the variant nlpD promoter was treated with the restriction enzymes SalI and PaeI designed in the 5 end regions of the primers, and inserted into the plasmid pMIV-Pn1p0-ter similarly treated with SalI and PaeI to replace the wild-type nlpD promoter (Pn1p0) on the plasmid with the variant nlpD promoter. From plasmids obtained as described above, one having the promoter sequence shown in
(297) The details of the construction of the rpoE gene expression plasmid, pMIV-Pn1p8-rpoE, are described below. By PCR using the chromosomal DNA of the Escherichia coli K5 strain as the template, as well as the primer rpoE-SalI Fw (SEQ ID NO: 170) and primer rpoE-xba Rv (SEQ ID NO: 171), a DNA fragment of the rpoE gene was obtained. PrimeStar Polymerase (TaKaRa) was used for PCR, and PCR was performed in the reaction composition described in the attached protocol. The PCR cycles consisted of 94 C. for 5 minutes, following 30 cycles of 98 C. for 5 seconds, 55 C. for 10 seconds, and 72 C. for 2 minutes, and final maintenance at 4 C. Further, by PCR using pMIV-Pn1p8-ter as the template DNA, as well as the oligonucleotides of SEQ ID NOS: 172 and 173 as the primers, a DNA fragment of pMIV-Pn1p8-ter was obtained. PrimeStar Polymerase (TaKaRa) was used for PCR, and PCR was performed in the reaction composition described in the attached protocol. The PCR cycles consisted of 94 C. for 5 minutes, following 30 cycles of 98 C. for 5 seconds, 55 C. for 10 seconds, and 72 C. for 6 minutes, and final maintenance at 4 C. Both the obtained DNA fragments were ligated by using In-Fusion (registered trademark) HD Cloning Kit (Clontech) to construct an rpoE gene expression plasmid, pMIV-Pn1p8-rpoE. The nucleotide sequence of the cloned rpoE gene is shown as SEQ ID NO: 174.
(298) (11-2) Heparosan Production Using rpoE Gene Expression-Enhanced Strain
(299) There were constructed respective strains from the Escherichia coli BL21(DE3)/pVK9-kfiABCD strain constructed in Example 1 by introducing thereto pMIV-Pn1p8-rpoE and pMIV-5JS as a control. Fermentative production culture was performed with these strains, and amounts of the produced heparosan were compared. The medium, culture method, and quantification method for heparosan were the same as those described above. Averages and standard deviations of the measured heparosan concentrations are shown in Table 13.
(300) TABLE-US-00020 TABLE 13 Effect of enhancement of rpoE gene expression in BL21(DE3)/pVK9-kfiABCD strain Strain Heparosan (mg/L) BL21(DE3)/pVK9-kfiABCD/pMIV-5JS 96.1 5.8 BL21(DE3)/pVK9-kfiABCD/pMIV-Pnlp8-rpoE 183.6 7.8
(301) While the invention has been described in detail with reference to the preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. Each of the aforementioned documents is incorporated by reference herein in its entirety.
INDUSTRIAL APPLICABILITY
(302) According to the present invention, heparosan-producing ability of bacteria can be improved, and heparosan can be efficiently produced.
DESCRIPTION OF SEQUENCE LISTING
(303) SEQ ID NO: 1, Nucleotide sequence of pVK9
(304) SEQ ID NOS: 2 to 7, Primers
(305) SEQ ID NO: 8, Nucleotide sequence of tac promoter
(306) SEQ ID NOS: 9 and 10, Primers
(307) SEQ ID NO: 11, Nucleotide sequence of pSTV28
(308) SEQ ID NOS: 12 to 15, Primers
(309) SEQ ID NO: 16, Nucleotide sequence of PaeI-SalI fragment containing wild-type nlpD promoter (Pn1p0)
(310) SEQ ID NOS: 17 and 18, Primers
(311) SEQ ID NO: 19, Nucleotide sequence of rrnB terminator
(312) SEQ ID NOS: 20 to 23, Primers
(313) SEQ ID NO: 24, Nucleotide sequence of kfiABCD operon of Escherichia coli K5 strain
(314) SEQ ID NO: 25, Amino acid sequence of KfiA protein of Escherichia coli K5 strain
(315) SEQ ID NO: 26, Amino acid sequence of KfiB protein of Escherichia coli K5 strain
(316) SEQ ID NO: 27, Amino acid sequence of KfiC protein of Escherichia coli K5 strain
(317) SEQ ID NO: 28, Amino acid sequence of KfiD protein of Escherichia coli K5 strain
(318) SEQ ID NO: 29, Nucleotide sequence of region containing rbsBKR-hsrA genes of Escherichia coli K5 strain
(319) SEQ ID NO: 30, Amino acid sequence of RbsB protein of Escherichia coli K5 strain
(320) SEQ ID NO: 31, Amino acid sequence of RbsK protein of Escherichia coli K5 strain
(321) SEQ ID NO: 32, Amino acid sequence of RbsR protein of Escherichia coli K5 strain
(322) SEQ ID NO: 33, Amino acid sequence of HsrA protein of Escherichia coli K5 strain
(323) SEQ ID NO: 34, Nucleotide sequence of region containing glgBX genes of Escherichia coli K5 strain
(324) SEQ ID NO: 35, Amino acid sequence of GlgB protein of Escherichia coli K5 strain
(325) SEQ ID NO: 36, Amino acid sequence of GlgX protein of Escherichia coli K5 strain
(326) SEQ ID NO: 37, Nucleotide sequence of region containing ybiXIJCB genes of Escherichia coli K5 strain
(327) SEQ ID NO: 38, Amino acid sequence of YbiX protein of Escherichia coli K5 strain
(328) SEQ ID NO: 39, Amino acid sequence of Ybil protein of Escherichia coli K5 strain
(329) SEQ ID NO: 40, Amino acid sequence of YbiJ protein of Escherichia coli K5 strain
(330) SEQ ID NO: 41, Amino acid sequence of YbiC protein of Escherichia coli K5 strain
(331) SEQ ID NO: 42, Amino acid sequence of YbiB protein of Escherichia coli K5 strain
(332) SEQ ID NO: 43, Nucleotide sequence of region containing rcsBD-micF genes of Escherichia coli K5 strain
(333) SEQ ID NO: 44, Amino acid sequence of RcsB protein of Escherichia coli K5 strain
(334) SEQ ID NO: 45, Amino acid sequence of RcsD protein of Escherichia coli K5 strain
(335) SEQ ID NO: 46, Nucleotide sequence of rfaH gene of Escherichia coli BL21(DE3) strain
(336) SEQ ID NO: 47, Amino acid sequence of RfaH protein of Escherichia coli BL21(DE3) strain
(337) SEQ ID NO: 48, Nucleotide sequence of nusG gene of Escherichia coli BL21(DE3) strain
(338) SEQ ID NO: 49, Amino acid sequence of NusG protein of Escherichia coli BL21(DE3) strain
(339) SEQ ID NO: 50, Nucleotide sequence of region containing pcoRSE genes of Escherichia coli K5 strain
(340) SEQ ID NO: 51, Amino acid sequence of PcoR protein of Escherichia coli K5 strain
(341) SEQ ID NO: 52, Amino acid sequence of PcoS protein of Escherichia coli K5 strain
(342) SEQ ID NO: 53, Amino acid sequence of PcoE protein of Escherichia coli K5 strain
(343) SEQ ID NO: 54, Nucleotide sequence of region containing yhcNO-aaeBAX genes of Escherichia coli K5 strain
(344) SEQ ID NO: 55, Amino acid sequence of YchN protein of Escherichia coli K5 strain
(345) SEQ ID NO: 56, Amino acid sequence of YchO protein of Escherichia coli K5 strain
(346) SEQ ID NO: 57, Amino acid sequence of AaeB protein of Escherichia coli K5 strain
(347) SEQ ID NO: 58, Amino acid sequence of AaeA protein of Escherichia coli K5 strain
(348) SEQ ID NO: 59, Amino acid sequence of AaeX protein of Escherichia coli K5 strain
(349) SEQ ID NO: 60, Nucleotide sequence of region containing g1455-alpA-g1453 genes of Escherichia coli K5 strain
(350) SEQ ID NO: 61, Amino acid sequence of G1455 protein of Escherichia coli K5 strain
(351) SEQ ID NO: 62, Amino acid sequence of AlpA protein of Escherichia coli K5 strain
(352) SEQ ID NO: 63, Amino acid sequence of G1453 protein of Escherichia coli K5 strain
(353) SEQ ID NO: 64, Nucleotide sequence of region containing yrbA-mlaBCDEF-yrbG genes of Escherichia coli K5 strain
(354) SEQ ID NO: 65, Amino acid sequence of YrbA protein of Escherichia coli K5 strain
(355) SEQ ID NO: 66, Amino acid sequence of MlaB protein of Escherichia coli K5 strain
(356) SEQ ID NO: 67, Amino acid sequence of MlaC protein of Escherichia coli K5 strain
(357) SEQ ID NO: 68, Amino acid sequence of MlaD protein of Escherichia coli K5 strain
(358) SEQ ID NO: 69, Amino acid sequence of MlaE protein of Escherichia coli K5 strain
(359) SEQ ID NO: 70, Amino acid sequence of MlaF protein of Escherichia coli K5 strain
(360) SEQ ID NO: 71, Amino acid sequence of YrbG protein of Escherichia coli K5 strain
(361) SEQ ID NO: 72, Nucleotide sequence of region containing norW gene of Escherichia coli K5 strain
(362) SEQ ID NO: 73, Amino acid sequence of NorW protein of Escherichia coli K5 strain
(363) SEQ ID NO: 74, Nucleotide sequence of region containing ybjIJK-rybB genes of Escherichia coli K5 strain
(364) SEQ ID NO: 75, Amino acid sequence of YbjI protein of Escherichia coli K5 strain
(365) SEQ ID NO: 76, Amino acid sequence of YbjJ protein of Escherichia coli K5 strain
(366) SEQ ID NO: 77, Amino acid sequence of YbjK protein of Escherichia coli K5 strain
(367) SEQ ID NO: 78, Nucleotide sequence of region containing yjjY-yjtD-thrLAB genes of Escherichia coli K5 strain
(368) SEQ ID NO: 79, Amino acid sequence of YjjY protein of Escherichia coli K5 strain
(369) SEQ ID NO: 80, Amino acid sequence of YjtD protein of Escherichia coli K5 strain
(370) SEQ ID NO: 81, Amino acid sequence of ThrL protein of Escherichia coli K5 strain
(371) SEQ ID NO: 82, Amino acid sequence of ThrA protein of Escherichia coli K5 strain
(372) SEQ ID NO: 83, Amino acid sequence of ThrB protein of Escherichia coli K5 strain
(373) SEQ ID NO: 84, Nucleotide sequence of region containing fruA-psuK genes of Escherichia coli K5 strain
(374) SEQ ID NO: 85, Amino acid sequence of FruA protein of Escherichia coli K5 strain
(375) SEQ ID NO: 86, Amino acid sequence of PsuK protein of Escherichia coli K5 strain
(376) SEQ ID NO: 87, Nucleotide sequence of region containing ytfT-yjfF-fbp genes of Escherichia coli K5 strain
(377) SEQ ID NO: 88, Amino acid sequence of YtfT protein of Escherichia coli K5 strain
(378) SEQ ID NO: 89, Amino acid sequence of YjfF protein of Escherichia coli K5 strain
(379) SEQ ID NO: 90, Amino acid sequence of Fbp protein of Escherichia coli K5 strain
(380) SEQ ID NO: 91, Nucleotide sequence of region containing yagU-paoAB genes of Escherichia coli K5 strain
(381) SEQ ID NO: 92, Amino acid sequence of YagU protein of Escherichia coli K5 strain
(382) SEQ ID NO: 93, Amino acid sequence of PaoA protein of Escherichia coli K5 strain
(383) SEQ ID NO: 94, Amino acid sequence of PaoB protein of Escherichia coli K5 strain
(384) SEQ ID NO: 95, Nucleotide sequence of region containing gsiCD-yliE genes of Escherichia coli K5 strain
(385) SEQ ID NO: 96, Amino acid sequence of GsiC protein of Escherichia coli K5 strain
(386) SEQ ID NO: 97, Amino acid sequence of GsiD protein of Escherichia coli K5 strain
(387) SEQ ID NO: 98, Amino acid sequence of YliE protein of Escherichia coli K5 strain
(388) SEQ ID NO: 99, Nucleotide sequence of region containing a part of irp gene of Escherichia coli K5 strain
(389) SEQ ID NO: 100, Nucleotide sequence of irp2 gene of Escherichia coli K5 strain
(390) SEQ ID NO: 101, Amino acid sequence of Irp2 protein of Escherichia coli K5 strain
(391) SEQ ID NO: 102, Nucleotide sequence of irp1 gene of Escherichia coli K5 strain
(392) SEQ ID NO: 103, Amino acid sequence of Irp1 protein of Escherichia coli K5 strain
(393) SEQ ID NO: 104, Nucleotide sequence of region containing bhsA-ycfS genes of Escherichia coli K5 strain
(394) SEQ ID NO: 105, Amino acid sequence of BhsA protein of Escherichia coli K5 strain
(395) SEQ ID NO: 106, Amino acid sequence of YcfS protein of Escherichia coli K5 strain
(396) SEQ ID NO: 107, Nucleotide sequence of region containing lepB-rnc-era genes of Escherichia coli K5 strain
(397) SEQ ID NO: 108, Amino acid sequence of LepB protein of Escherichia coli K5 strain
(398) SEQ ID NO: 109, Amino acid sequence of Rnc protein of Escherichia coli K5 strain
(399) SEQ ID NO: 110, Amino acid sequence of Era protein of Escherichia coli K5 strain
(400) SEQ ID NO: 111, Nucleotide sequence of region containing dapA-gcvR-bcp-hyfA genes of Escherichia coli K5 strain
(401) SEQ ID NO: 112, Amino acid sequence of DapA protein of Escherichia coli K5 strain
(402) SEQ ID NO: 113, Amino acid sequence of GcvR protein of Escherichia coli
(403) K5 strain
(404) SEQ ID NO: 114, Amino acid sequence of Bcp protein of Escherichia coli K5 strain
(405) SEQ ID NO: 115, Amino acid sequence of HyfA protein of Escherichia coli K5 strain
(406) SEQ ID NO: 116, Nucleotide sequence of region containing rpoE-nadB-yfiC-srmB genes of Escherichia coli K5 strain
(407) SEQ ID NO: 117, Amino acid sequence of RpoE protein of Escherichia coli K5 strain
(408) SEQ ID NO: 118, Amino acid sequence of NadB protein of Escherichia coli K5 strain
(409) SEQ ID NO: 119, Amino acid sequence of YfiC protein of Escherichia coli K5 strain
(410) SEQ ID NO: 120, Amino acid sequence of SrmB protein of Escherichia coli K5 strain
(411) SEQ ID NO: 121, Nucleotide sequence of region containing g1414-g1413 genes of Escherichia coli K5 strain
(412) SEQ ID NO: 122, Amino acid sequence of G1414 protein of Escherichia coli K5 strain
(413) SEQ ID NO: 123, Amino acid sequence of G1413 protein of Escherichia coli K5 strain
(414) SEQ ID NO: 124, Nucleotide sequence of region containing nuoEFG genes of Escherichia coli K5 strain
(415) SEQ ID NO: 125, Amino acid sequence of NuoE protein of Escherichia coli K5 strain
(416) SEQ ID NO: 126, Amino acid sequence of NuoF protein of Escherichia coli K5 strain
(417) SEQ ID NO: 127, Amino acid sequence of NuoG protein of Escherichia coli K5 strain
(418) SEQ ID NO: 128, Nucleotide sequence of region containing glmZ-hemYXD genes of Escherichia coli K5 strain
(419) SEQ ID NO: 129, Amino acid sequence of HemY protein of Escherichia coli
(420) K5 strain
(421) SEQ ID NO: 130, Amino acid sequence of HemX protein of Escherichia coli K5 strain
(422) SEQ ID NO: 131, Amino acid sequence of HemD protein of Escherichia coli K5 strain
(423) SEQ ID NO: 132, Nucleotide sequence of region containing rlmL gene of Escherichia coli K5 strain
(424) SEQ ID NO: 133, Amino acid sequence of RlmL protein of Escherichia coli K5 strain
(425) SEQ ID NO: 134, Nucleotide sequence of region containing artQMJ-rlmC-ybjO genes of Escherichia coli K5 strain
(426) SEQ ID NO: 135, Amino acid sequence of ArtQ protein of Escherichia coli K5 strain
(427) SEQ ID NO: 136, Amino acid sequence of ArtM protein of Escherichia coli K5 strain
(428) SEQ ID NO: 137, Amino acid sequence of ArtJ protein of Escherichia coli K5 strain
(429) SEQ ID NO: 138, Amino acid sequence of RlmC protein of Escherichia coli K5 strain
(430) SEQ ID NO: 139, Amino acid sequence of YbjO protein of Escherichia coli K5 strain
(431) SEQ ID NO: 140, Nucleotide sequence of region containing yejOML genes of Escherichia coli K5 strain
(432) SEQ ID NO: 141, Amino acid sequence of YejO protein of Escherichia coli K5 strain
(433) SEQ ID NO: 142, Amino acid sequence of YejM protein of Escherichia coli K5 strain
(434) SEQ ID NO: 143, Amino acid sequence of YejL protein of Escherichia coli K5 strain
(435) SEQ ID NO: 144, Nucleotide sequence of region containing rpoS-ygbNML genes of Escherichia coli K5 strain
(436) SEQ ID NO: 145, Amino acid sequence of RpoS protein of Escherichia coli K5 strain
(437) SEQ ID NO: 146, Amino acid sequence of YgbN protein of Escherichia coli K5 strain
(438) SEQ ID NO: 147, Amino acid sequence of YgbM protein of Escherichia coli K5 strain
(439) SEQ ID NO: 148, Amino acid sequence of YgbL protein of Escherichia coli K5 strain
(440) SEQ ID NO: 149, Nucleotide sequence of region containing g3798-g3797-g3796-g3795-g3794-g3793-g3792 genes of Escherichia coli K5 strain
(441) SEQ ID NO: 150, Amino acid sequence of G3798 protein of Escherichia coli K5 strain
(442) SEQ ID NO: 151, Amino acid sequence of G3797 protein of Escherichia coli K5 strain
(443) SEQ ID NO: 152, Amino acid sequence of G3796 protein of Escherichia coli K5 strain
(444) SEQ ID NO: 153, Amino acid sequence of G3795 protein of Escherichia coli K5 strain
(445) SEQ ID NO: 154, Amino acid sequence of G3794 protein of Escherichia coli K5 strain
(446) SEQ ID NO: 155, Amino acid sequence of G3793 protein of Escherichia coli K5 strain
(447) SEQ ID NO: 156, Amino acid sequence of G3792 protein of Escherichia coli K5 strain
(448) SEQ ID NO: 157, Nucleotide sequence of region containing ryjA-soxRS-yjcCB genes of Escherichia coli K5 strain
(449) SEQ ID NO: 158, Amino acid sequence of SoxR protein of Escherichia coli K5 strain
(450) SEQ ID NO: 159, Amino acid sequence of SoxS protein of Escherichia coli K5 strain
(451) SEQ ID NO: 160, Amino acid sequence of YjcC protein of Escherichia coli K5 strain
(452) SEQ ID NO: 161, Amino acid sequence of YjcB protein of Escherichia coli K5 strain
(453) SEQ ID NO: 162, Nucleotide sequence of region containing efeUO genes of Escherichia coli K5 strain
(454) SEQ ID NO: 163, Amino acid sequence of EfeU protein of Escherichia coli K5 strain
(455) SEQ ID NO: 164, Amino acid sequence of EfeO protein of Escherichia coli K5 strain
(456) SEQ ID NO: 165, Nucleotide sequence of wild-type nlpD promoter (Pn1p0)
(457) SEQ ID NOS: 166 and 167, Primers
(458) SEQ ID NO: 168, Nucleotide sequence of variant nlpD promoter (Pn1p8)
(459) SEQ ID NO: 169, Nucleotide sequence of PaeI-SalI fragment containing variant nlpD promoter (Pn1p8)
(460) SEQ ID NOS: 170 to 173, Primers
(461) SEQ ID NO: 174, Nucleotide sequence of rpoE gene of Escherichia coli K5 strain