Production of human milk oligosaccharides in microbial hosts with engineered import / export
12410454 ยท 2025-09-09
Assignee
Inventors
Cpc classification
C12P19/18
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
International classification
C12P19/18
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
Abstract
The present invention relates to methods for the production of oligosaccharides in genetically modified bacterial host cells, as well as to the genetically modified host cells used in the methods. The genetically modified host cell comprises at least one recombinant glycosyltransferase, and at least one nucleic acid sequence coding for a protein enabling the export of the oligosaccharide.
Claims
1. A method for production of an oligosaccharide by a genetically modified microbial host cell, wherein the oligosaccharide is a lacto-N-tetraose comprising a lacto-N-triose II (LNT-II; GlcNAc(b1-3)Gal(b1-4)Gluc) as a core trisaccharide, the method comprising: providing a genetically modified microbial host cell that comprises: at least one recombinant glycosyltransferase selected from the group consisting of a -1,3-N-acetylglucosaminyltransferase and a -1,3-galactosyltransferase; increased expression or activity of at least one homologous or heterologous sugar export protein capable of exporting the oligosaccharide; and decreased expression or inactivation of at least one export protein that exports LNT-II from the host cell; cultivating the host cell in a medium under conditions permissive for the production of the oligosaccharide, whereby the oligosaccharide is exported into the medium at an increased level compared to an unmodified host cell, and obtaining the oligosaccharide from the medium.
2. The method of claim 1, wherein the host cell further comprises: overexpression of at least one homologous or heterologous nucleic acid sequence coding for the sugar export protein capable of exporting the oligosaccharide into the culture medium; and/or deletion, disruption, diminishment or inactivation of at least one endogenous nucleic acid sequence coding for an export protein that exports precursors of the oligosaccharide outside the host cell; and/or overexpression of at least one homologous or heterologous sequence coding for a protein mediating import of a precursor of the oligosaccharide into said host cell, wherein the precursor is larger than a disaccharide.
3. The method of claim 2, wherein said sugar export protein capable of exporting the oligosaccharide belongs to the class of secondary active transporters.
4. The method of claim 2, wherein the nucleic acid sequence coding for the homologous or heterologous sugar export protein capable of exporting the oligosaccharide is of bacterial, archeal, plant, yeast or animal origin.
5. The method of claim 2, wherein said at least one nucleic acid sequence coding for the sugar export protein that exports precursors of the oligosaccharide outside the host cell is a gene selected from the group consisting of yebQ from Escherichia coli, yjhB from Escherichia coli, proP from Mannheimia succiniciproducens, and setA from Cedecea neteri.
6. The method of claim 1, wherein said -1,3-N-acetylglucosaminyltransferase belongs to the class of lgtA of Neisseria meningitides or PmnagT of Pasteurella multocida.
7. The method of claim 1, wherein the -1,3-galactosyltransferase is encoded by a WbdO gene.
8. The method of claim 1, wherein-ii the genetically modified microbial host cell further comprises an endogenous -galactosidase gene and glucosamine-6-phosphate deaminase gene that are inactivated or deleted, and wherein said genetically modified microbial host cell further comprises a nucleic acid sequence coding for a functional lactose permease protein.
9. The method of claim 1, wherein the genetically modified microbial host cell further comprises an increased UDP-N-acetylglucosamine and a UDP-galactose, or GDP-fucose, or CMP-N-acetylneuraminic acid production, as compared to a genetically unmodified host cell, wherein optionally said increased UDP-N-acetylglucosamine and UDP-galactose production capability is by an overexpression of one or more genes encoding a protein selected from the group consisting of L-glutamine:D-fructose-6-phosphate aminotransferase, N-acetyl glucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyl transferase, phosphoglucosamine mutase, UDP-galactose-4-epimerase, phosphoglucomutase, and glucose-1-phosphate uridylyltransferase.
10. The method of claim 8, wherein said genetically modified microbial host cell is cultivated in the presence of glucose, sucrose, glycerol or a combination thereof.
11. The method of claim 10, wherein the microbial host cell is cultured in the absence of N-acetylglucosamine and galactose.
12. A genetically modified microbial host cell for the production of lacto-N-tetraose comprising a lacto-N-triose II (LNT-II; GlcNAc(1-3)Gal(1-4)Gluc) as a core trisaccharide, wherein the host cell comprises at least one recombinant glycosyltransferase, selected from the group consisting of a -1,3-N-acetylglucosaminyltransferase and a -1,3-galactosyltransferase; at least one homologous or heterologous nucleic acid sequence coding for a sugar export protein capable exporting of the acto-N-tetraose into a culture medium the host cell is cultivated in wherein said sugar export protein capable of exporting the lacto-N-tetraose belongs to the class of secondary active transporters, and wherein said homologous or heterologous nucleic acid sequence coding for the sugar export protein capable of exporting the lacto-N-tetraose is overexpressed or under control of a promoter sequence enabling the overexpression of said nucleic acid sequence; and inactivated expression or activity of at least one endogenous sugar export protein that exports LNT-II from the host cell.
13. The microbial host cell of claim 12, further comprising an overexpression of at least one homologous or heterologous nucleic acid sequence coding for a protein mediating the import of a precursor of the acto-N-tetraose into said host cell through genetic modification of the microbial host cell, wherein the precursor is larger than a disaccharide.
14. The microbial host cell of claim 12, wherein said nucleic acid sequence coding for the sugar export protein capable of exporting the acto-N-tetraose is of bacterial, archeal, plant, yeast or animal origin.
15. The microbial host cell of claim 12, wherein said at least one nucleic acid sequence coding for the export protein that exports LNT-II from the host cell is a gene selected from the group consisting of yebQ from Escherichia coli, yjhB from Escherichia coli, proP from Mannheimia succiniciproducens, and setA from Cedecea neteri.
16. The microbial host cell of claim 14, wherein the sugar export protein capable of exporting the lacto-N-tetraose is selected from the group consisting of YebQ from Escherichia coli BL21(DE3), SpoVB of Bacillus amyloliquefaciens, YabM of Erwinia pyrilfolia, Bcr of E. coli MG1655, YdeA of E. coli MG1655, ProP2 of Haemophilus parainfluenzae, SetA of Pectobacterium carotovorum, FucP of E. coli MG1655, MdeA of Staphylococcus aureus Bmb9393, ImrA of Lactococcus lactis, SetA of Pseudomonas sp. MT-1 and SetA of Beauveria bassiana Dl-5.
17. The method of claim 1, wherein the sugar export protein capable of exporting the lacto-N-tetraose is selected from the group consisting of YebQ from Escherichia coli BL21(DE3), SpoVB of Bacillus amyloliquefaciens, YabM of Erwinia pyrilfolia, Bcr of E. coli MG1655, YdeA of E. coli MG1655, ProP2 of Haemophilus parainfluenzae, SetA of Pectobacterium carotovorum, FucP of E. coli MG1655, MdeA of Staphylococcus aureus Bmb9393, ImrA of Lactococcus lactis, SetA of Pseudomonas sp. MT-1 and SetA of Beauveria bassiana D1-5.
18. The method of claim 1, wherein the microbial host cell is an Escherichia coli cell, a Corynebacterium glutamicum cell or a Saccharomyces sp. cell.
19. The microbial host cell of claim 12, wherein the microbial host cell is an Escherichia coli cell, a Corynebacterium glutamicum cell or a Saccharomyces sp. cell.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
(10) In the method according to the invention, it is preferred if the desired oligosaccharide is a human milk oligosaccharide comprising a lacto-N-triose II (LNT-II; GlcNAc(1-3)Gal(1-4)Gluc) as a core trisaccharide. In this connection, an oligosaccharide having a core trisaccharide is meant to comprise the specific trisaccharide representing the reducing end of a desired oligosaccharide, and comprising, as the case may be, additional saccharide moieties, with the specific trisaccharide representing the major moiety.
(11) Accordingly, in an embodiment of the method and the host cell according to the invention, the desired oligosaccharide is selected from the group consisting of: lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose Ill, lacto-N-fucopentaose V, lacto-N-difucosylhexose I, lacto-N-difucosylhexaose II, lacto-N-sialylpentaose LSTa, LSTb, LSTc, disialyllacto-N-tetraose, disialyllacto-N-neotetraose.
(12) In order to overcome the above mentioned drawbacks of limited oligosaccharide export the object is further solved by a method according to the invention, wherein the host cell comprises: at least one homologous or heterologous nucleic acid sequence coding for a protein enabling the export of a desired oligosaccharide into the culture medium, wherein said host cell has been modified such, that the expression of the homologous or heterologous nucleic acid sequence is overexpressed or under control of a promoter enabling the overexpression of the nucleic acid sequence; and/or the deletion, disruption, diminishment or inactivation of at least one endogenous nucleic acid sequence coding for an exporter protein that exports precursors of the desired oligosaccharide outside the host cell; and/or at least one homologous or heterologous nuclei acid sequence coding for a protein mediating the import of a precursor of a desired oligosaccharide into said host cell, wherein preferably the nucleic acid sequence is overexpressed, and wherein preferably the precursor is larger than a disaccharide.
(13) The genetically modified microbial host cell comprising the characteristics as set forth herein are cultured in the presence of glucose, sucrose, glycerin or a combination thereofusing these substrates as carbon- and energy sourcesas well as in the presence of lactose or oligosaccharides larger than disaccharides, e.g., LNT-II.
(14) In a preferred embodiment of this method and host cell, said protein enabling the export of a desired oligosaccharide belongs to the class of secondary active transporters, and more preferably effects the export of an oligosaccharide comprising at least three moieties.
(15) According to preferred embodiments, for the export of desired oligosaccharides a suitable exporter is expressed in addition to the genes that are responsible for intracellular oligosaccharide biosynthesis.
(16) According to one aspect of the method and host cell of the invention, the at least one nucleic acid sequence coding for a protein enabling the export of a desired oligosaccharide is an endogenous or a recombinant nucleic acid.
(17) In a preferred embodiment of the method and the host cell of the invention, the nucleic acid sequence coding for a protein enabling the export of a desired oligosaccharide is of bacterial, archeal, plant, yeast or animal origin; preferably, the at least one nucleic acid sequence coding for a protein enabling the export of a desired oligosaccharide is a gene selected from the group consisting of yebQ and yjhB from Escherichia coli, proP from Mannheimia succiniciproducens and setA from Cedecea neteri or functional fragments thereof.
(18) Preferably, the oligosaccharide exporter is a protein selected from at least one of the following: SetA, SetB, SetC, YdeA, Cmr, YnfM, MdtD, YfcJ, YhhS, EmrD, YdhC, YbdA, YdeE, MhpT, YebQ, YjhB, Bcr and YdeA of E. coli, or ProP from Mannheimia succiniciproducens and SetA from Cedecea neteri or variants or homologs thereof.
(19) In yet another preferred embodiment, the recombinant glycosyltransferase is selected from at least one of the following: a galactosyltransferase, a sialyltransferase, an N-acetylglucosaminyltransferase and a fucosyltransferase, and is preferably selected from at least one of the following: -1,3-N-acetylglucosaminyltransferase, -1,3-galactosyltransferase, -1,4-galactosyltransferase, -1,6-galactosyltransferase, -2,3-sialyltransferase, -2,6-sialyltansferase, -1,2-fucosyltransferase, or -1,3-fucosyltransferase.
(20) A preferred embodiment of the method and the host cell of the invention, concerns the a host cell or its provision, wherein the host cell comprises (i) a -1,3-N-acetylglucosaminyltransferase, and (ii) a -1,3-galactosyltransferase or a -1,4-galactosyltransferase as glycosyltransferases. In this connection it is preferred, if said -1,3-N-acetylglucosaminyltransferase has the activity of ligating N-acetylglucosamine to lactose generating lacto-N-triose II, and if said -1,3-galactosyltransferase or said -1,4-galactosyltransferase, respectively, have the activity to galactosylate lacto-N-triose II thus generating lacto-N-tetraose or lacto-N-neotetraose, respectively. The here developed system is easily adaptable to even more complex oligosaccharides by the expression of further glycosyltransferases.
(21) With the microbial cell and the method according to the invention, it is possible to ferment a desired oligosaccharide in large quantities, especially an oligosaccharide comprising LNT-II as core structure, and to recover it from the culture broth.
(22) In a preferred embodiment, said -1,3-N-acetylglucosaminyltransferase belongs to the class of lgtA of Neisseria meningitides or PmnagT of Pasteurella multocida, or variants thereof.
(23) Preferably, the glycosyltransferase is selected from a galactosyltransferase, a sialyltransferase, an N-acetylglucosaminyltransferase and a fucosyltransferase.
(24) In yet another preferred embodiment, the lacto-N-tetraose generating -1,3-galactosyltransferase is WbdO or a functional variant thereof. According to an aspect of the invention, the -1,3-galactosyltransferase is a -1,3-galactosyltransferase derived from Salmonella enterica (wbdO, acc. no. AY730594), and is preferably encoded by a gene selected from the group consisting of wbgO from Escherichia coli O55:H7 or furA from Lutiella nitroferrum, or a functional fragments thereof.
(25) The invention also concerns a genetically modified microbial host cell, preferably a bacterial host cell, as described above in which the endogenous -galactosidase gene is inactivated or deleted and in which a functional lactose permease gene is present.
(26) Accordingly, in a preferred embodiment of the method and the host cell of the invention, a genetically modified host cell is provided, in which, where applicable, an endogenous -galactosidase gene and a glucosamine-6-phosphate deaminase gene are inactivated or deleted, and wherein said genetically modified host cell comprises a nucleic acid sequence coding for a functional lactose permease protein, preferably LacY.
(27) In a preferred embodiment, the genetically modified host cell comprises an increased UDP-N-acetylglucosamine and UDP-galactose, GDP-fucose or CMP-N-acetylneuraminic acid production capability as compared to a genetically unmodified host cell.
(28) In a refinement of this embodiment of the method of and of the host cell of the invention, said increased UDP-N-acetylglucosamine and UDP-galactose production capability comprises the overexpression of one or more genes encoding for proteins comprising the following activities for a: L-glutamine: D-fructose-6-phosphate aminotransferase, N-acetyl glucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyl transferase, phosphoglucosamine mutase, UDP-galactose-4-epimerase, phosphoglucomutase, glucose-1-phosphate uridylyltransferase.
(29) For the synthesis of, e.g. LNT, UDP-galactose and UDP-N-acetylglucosamine are required. UDP-galactose can be obtained by feeding galactose to the HMO producing bacterial host cell via the fermentation medium. The galactose is then taken up by the cell, phosphorylated to galactose-1-phosphate and then converted to UDP-galactose. Genes encoding these enzymatic activities are well known in the literature (Grossiord et al., J. Bacteriol 2003 185(3) 870-878). The supply for UDP-galactose can be also obtained from the cells own metabolism, and the metabolism can be improved by further genetic modification, such as the overexpression of the UDP-galactose-4-epimerase, or the UDP-galactose-4-epimerase in combination with the glucose-1-phosphate-1-uridinyltransferase. UDP-N-acetlyglucosamine can be also obtained from the bacterial host cell's own UDP-N-acetylglucosamine metabolism. The provision of UDP-N-acetylglucosamine for the synthesis of N-aectylglucosamine containing oligosaccharides can be improved by the inactivation of the N-acetylglucosamine catabolism within the producing cell.
(30) According to one aspect of the invention, the genetically modified host cell is cultivated in the presence of glucose, sucrose, glycerol or a combination thereof, but neither by addition or in the presence of N-acetylglucosamine or galactose nor in a combination thereof.
(31) In a preferred embodiment of the method and of the host cell of the invention, the desired oligosaccharide is lacto-N-triose II, which is produced by total fermentation from a simple carbon source in the host cell by the action of the heterologous expressed glycosyltransferases -1,4-galactosyltransferase and -1,3-N-acetylglucosaminyltransferase.
(32) The present invention, as already mentioned above, also concerns a genetically modified host cell for the production of a desired oligosaccharide, the oligosaccharide comprising a lacto-N-triose II (LNT-II; GlcNAc(1-3)Gal(1-4)Gluc) as a core trisaccharid, wherein the host cell comprises at least one recombinant glycosyltransferase, the glycosyltransferase being preferably selected from a galactosyltransferase, a sialyltransferase, and an N-acetylglucosaminyltransferase, and has the expression or activity of at least endogenous sugar transport protein modified such, that the expression or activity of the endogenous sugar transport protein is functionally inactivated for the export of a precursor of the desired oligosaccharide.
(33) A preferred embodiment concerns a host cell as described above, comprising (i) a heterologous expressed -1,3-N-acetylglucosaminyltransferase, and (ii) a heterologous expressed -1,3-galactosyltransferase or a heterologous expressed -1,4-galactosyltransferase as glycosyltransferases, wherein the host cell further preferably comprises at least one homologous or heterologous nucleic acid sequence coding for a protein enabling the export of the oligosaccharide into a culture medium the host cell is cultivated in, wherein said protein enabling the export of the desired oligosaccharide belongs to the class of secondary active transporters, wherein said host cell has been modified such, that the expression of the homologous or heterologous nucleic acid sequence is overexpressed or under control of a promoter enabling the overexpression of the nucleic acid sequence. In preferred embodiments of the host cell, said at least one nucleic acid sequence coding for a protein enabling the export of the desired oligosaccharide is an endogenous or a recombinant nucleic acid sequence.
(34) As already outlined for the method according to the invention, it is also preferred in the host cell of the invention, if said nucleic acid sequence coding for a protein enabling the export of a desired oligosaccharide is of bacterial, archeal, plant, yeast or animal origin.
(35) According to another aspect of the invention, the host cell as described above further comprises: the deletion, disruption, diminishment or inactivation of at least one endogenous nucleic acid sequence coding for an exporter protein that exports precursors of the desired oligosaccharide outside the host cell; and/or at least one homologous or heterologous nucleic acid sequence coding for a protein enabling the import of a precursor of a desired oligosaccharide into said host cell, wherein preferably the nucleic acid sequence is overexpressed, and wherein preferably the precursor is larger than a disaccharide.
(36) With the overexpression of at least one homologous or heterologous nucleic acid sequence coding for a protein enabling the import of a precursor of a desired oligosaccharide into said host cell, it is possible to feed precursors of a desired oligosaccharide to the culture medium, which get imported into the host cell, such as, e.g., LNT-II.
(37) According to one aspect of the invention, in the host cell said at least one nucleic acid sequence coding for a protein enabling the export of a desired oligosaccharide is a gene selected from the group consisting of yebQ and yjhB from Escherichia coli, proP from Mannheimia succiniciproducens and setA from Cedecea neteri or functional fragments thereof.
(38) According to yet another preferred embodiment, the desired oligosaccharide is lacto-N-triose II, and the protein enabling the export of the oligosaccharide into a culture medium the host cell is cultivated in, is YjhB from Escherichia coli, ProP from Mannheimia succiniciproducens and SetA from Cedecea neteri or functional fragments thereof.
(39) According to a preferred embodiment, the microbial host according to the invention is further modified not to express proteins exporting precursors of a desired oligosaccharide.
(40) In a preferred embodiment of the host cell, the desired oligosaccharide is lacto-N-tetraose, the precursor is lacto-N-triose II, and the host cell has deleted, disrupted or inactivated at least one nucleic acid sequence coding for an exporter protein that is able to export lacto-N-triose II outside the host cell.
(41) In this connection it is preferred, if the protein enabling the export of lacto-N-tetraose is selected from YebQ from Escherichia coli BL21(DE3), SpoVB of Bacillus amyloliquefaciens, YabM of Erwinia pyrilfolia, Bcr of E. coli MG1655, YdeA of E. coli MG1655, ProP2 of Haemophilus parainfluenzae, SetA of Pectobacterium carotovorum, FucP of E. coli MG1655, MdeA of Staphylococcus aureus Bmb9393, ImrA of Lactococcus lactis, SetA of Pseudomonas sp. MT-1 and SetA of Beauveria bassiana D1-5.
(42) Preferably, the oligosaccharide exporter is a protein selected from at least one of the following: SetA, SetB, SetC, YdeA, Cmr, YnfM, MdtD, YfcJ, YhhS, EmrD, YdhC, YbdA, YdeE, MhpT, YebQ, YjhB, Bcr and YdeA of E. coli, or ProP from Mannheimia succiniciproducens and SetA from Cedecea neteri or variants or homologs thereof.
(43) Presently, the term nucleic acid refers to a single- or double-stranded deoxyribonucleotide or ribonucleotide macromolecule and encompasses known analogues or natural or synthetically produced nucleotides that hybridize with the desired nucleic acid and that encode a certain polypeptide.
(44) The term recombinant or genetically modified, as used herein with reference to a microbial host cell indicates that the microbial host cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid (i.e., a sequence foreign to said cell). Recombinant cells can contain genes that are not found within the native (non-recombinant) form of the cell. Recombinant cells can also contain genes found in the native form of the cell wherein the genes are modified and re-introduced into the cell by artificial means. The term also encompasses cells that contain a nucleic acid endogenous to the cell that has been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, site-specific mutation, and related techniques. Accordingly, a recombinant polypeptide is one which has been produced by a recombinant cell. A heterologous sequence or a heterologous nucleic acid, as used herein, is one that originates from a source foreign to the particular host cell (e.g. from a different species), or, if from the same source, is modified from its original form. Thus, a heterologous nucleic acid operably linked to a promoter is from a source different from that from which the promoter was derived, or, if from the same source, is modified from its original form. The heterologous sequence may be stably introduced, e.g. by transfection, transformation, conjugation or transduction, into the genome of the host microbial host cell, thus representing a genetically modified host cell. Techniques may be applied which will depend on the host cell the sequence is to be introduced. Various techniques are known to a person skilled in the art and are, e.g., disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
(45) Accordingly, a microbial host cell is presently understood as a microbial, preferably bacterial, cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence.
(46) Thus, the nucleic acid sequences as used in the present invention, may, e.g., be comprised in a vector which is to be stably transformed/transfected or otherwise introduced into host microorganism cells.
(47) Presently, the term operably linked as used herein, shall mean a functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence affects transcription and/or translation of the nucleic acid corresponding to the second sequence. Accordingly, the term Promoter designates DNA sequences which usually precede a gene in a DNA polymer and provide a site for initiation of the transcription into mRNA. Regulator DNA sequences, also usually upstream of (i.e., preceding) a gene in a given DNA polymer, bind proteins that determine the frequency (or rate) of transcriptional initiation. Collectively referred to as promoter/regulator or control DNA sequence, these sequences which precede a selected gene (or series of genes) in a functional DNA polymer cooperate to determine whether the transcription (and eventual expression) of a gene will occur. DNA sequences which follow a gene in a DNA polymer and provide a signal for termination of the transcription into mRNA are referred to as transcription terminator sequences.
(48) A great variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression system constructs may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and to synthesize a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., supra.
(49) The art is rich in patent and literature publications relating to recombinant DNA methodologies for the isolation, synthesis, purification and amplification of genetic materials for use in the transformation of selected host organisms. Thus, it is common knowledge to transform host organisms with hybrid viral or circular plasmid DNA which includes selected exogenous (i.e. foreign or heterologous) DNA sequences. The procedures known in the art first involve generation of a transformation vector by enzymatically cleaving circular viral or plasmid DNA to form linear DNA strands. Selected foreign DNA strands usually including sequences coding for desired protein product are prepared in linear form through use of the same/similar enzymes. The linear viral or plasmid DNA is incubated with the foreign DNA in the presence of ligating enzymes capable of effecting a restoration process and hybrid vectors are formed which include the selected exogenous DNA segment spliced into the viral or circular DNA plasmid.
(50) As used herein, the term cultivating means growing a bacterial cell in a medium and under conditions permissive and suitable for the production of the desired oligosaccharide(s). A couple of suitable bacterial host cells as well as mediums and conditions for their cultivation will be readily available for one skilled in the art upon reading the disclosure of this invention in connection with the skilled person's technical and expert background.
(51) As used herein, the term recovering or obtaining means isolating, harvesting, purifying, collecting or otherwise separating from the host cell culture the oligosaccharide produced by the host cell according to the invention.
(52) A microbial host cell according to the invention, and as generally understood, means any microorganism, including bacteria, fungi and archaea, which is generally suitable for cultivation in large amounts, and which can be genetically modified according to the invention in order to produce a desired oligosaccharide. Preferred microorganisms are bacteria, e.g. Escherichia coli, Corynebacterium glutamicum and the yeast Saccharomyces sp., which have the advantage that these microorganisms can be grown easily and inexpensively in laboratory settings, and the bacteria and yeast have been intensively investigated for over many years
(53) Generally, and throughout the present invention, the term glycosyltransferase activity or glycosyltransferase designates and encompasses enzymes that are responsible for the biosynthesis of disaccharides, oligosaccharides and polysaccharides, and they catalyze the transfer of monosaccharide moieties from an activated nucleotide monosaccharide/sugar (the glycosyl donor) to a glycosyl acceptor molecule.
(54) Generally, and throughout the present invention, the terms exporter or exporter protein or protein enabling the export of a desired oligosaccharide, which terms are presently being used synonymously, designates one or more polypeptides that solely or as part of a multi-protein complex transfers an oligosaccharide from the intracellular milieu of a bacterial cell into the periplasm of said cell or the culture supernatant, thus, enabling the oligosaccharide to pass the cellular membrane and/or the cell wall of said cell.
(55) Within the scope of the present invention, also nucleic acid/polynucleotide and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs are comprised by those terms, that have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acids, to a polypeptide encoded by a wild type glycosyltransferase activity or oligosaccharide export displaying protein.
(56) Variant(s) as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a poly-peptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to the persons skilled in the art.
(57) Accordingly, a functional fragment of any of the genes/proteins disclosed therein, is meant to designate sequence variants of the genes/proteins still retaining the same or somewhat lesser activity of the gene or protein the respective fragment is derived from.
(58) In this connection, the term nucleic acid sequence encoding . . . generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA, and generally represents a gene which encodes a certain polypeptide or protein.
(59) In this context, the term polypeptide(s) refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. Polypeptide(s) refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids. Polypeptide(s) include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide, without essentially altering the activity of the polypeptide. Also, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini.
(60) Further, with the expression precursor compounds are encompassed which are involved in the biosynthetic pathway of the oligosaccharide according to the invention or which are produced and naturally present in the host cell.
(61) A precursor that is larger than a disaccharide is presently understood as a sugar moiety that comprises more than two monosaccharide residues.
(62) The term desired oligosaccharide refers to a sugar polymer consisting of at least three moieties, thus, comprising trisaccharides, tetrasaccharides, pentasaccharides etc., preferably an oligosaccharide selected from at least one of the following: lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-difucosylhexose I, lacto-N-difucosylhexaose II, lacto-N-sialylpentaose LSTa, LSTb, LSTc, disialyllacto-N-tetraose, disialyllacto-N-neotetraose.
(63) Presently, and as generally understood in the relevant field, the expression homologous refers to a nucleic acid sequence that encodes for a specific product or products and is derived from the same species, in which said nucleic acid sequence is inserted. Accordingly, the term heterologous refers to a nucleic acid sequence encoding for a specific product or products and being derived from a species other than those in which said nucleic acid sequence is inserted.
(64) The term endogenous herein and generally within the field means that the nucleic acid encoding for an enzyme of interest is originating from the bacterial host cell and has not been introduced into said host cell, whereas a recombinant nucleic acid has been introduced into said host cell and does not originates from said host cell.
(65) The expression overexpressed, or overexpressing or under control of a promoter sequence enabling the overexpression of said nucleic acid sequence presently, and generally in the art, means the expression of a gene in greater-than-normal amounts, i.e. in increased quantity thus leading to an increased amount of the protein the nucleic acid sequence is coding for.
(66) In some embodiments, the nucleic acid sequence is placed under the control of an inducible promoter, which is a promoter that directs expression of a gene where the level of expression is alterable by environmental or developmental factors such as, for example, temperature, pH, anaerobic or aerobic conditions, light, transcription factors and chemicals. Such promoters are referred to herein as inducible promoters, which allow one to control the timing of expression of the proteins used in the present invention. For E. coli, and other microbial host cells, inducible promoters are known to those of skill in the art.
(67) Further advantages are evident from the description and the drawings.
(68) It is understood that the features mentioned above and those yet to be explained below can be used not only in the respective combinations indicated, but also in other combinations or in isolation, without leaving the context of the present invention.
(69) The invention will be described in more detail in the examples and the attached figures, in which
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EXAMPLES
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(80) The exporters are membrane-bound, and their expression can be either overexpressed, whichin case of overexpression of the LNT-II exporter leads to an increased LNT-II export and to a decreased LNT export, whereas when the LNT-II exporting exporter protein is deleted or otherwise inactivated, this leads to an improved LNT-export. The LNT-II exporter preferably is an endogenous exporter protein, whereas the LNT-exporter protein preferably is a heterologous exporter protein.
Example 1
(81) Development of an E. coli Lacto-N-Triose II Production Strain
(82) Escherichia coli BL21(DE3) was used to construct a lacto-N-triose II (LNT-2) producing strain. Metabolic engineering included mutagenesis and deletions of specific genes, respectively, and genomic integrations of heterologous genes. The genes lacZ and araA were inactivated by mutagenesis using mismatch-oligonucleotides as described by Ellis et al., High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides, Proc. Natl. Acad. Sci. USA 98: 6742-6746 (2001).
(83) Genomic deletions were performed according to the method of Datsenko and Warner (Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)). To prevent intracellular degradation of N-acetylglucosamine, genes encoding N-acetylglucosamine-6-phosphate deacetylase (nagA) and glucosamine-6-phosphate deaminase (nagB) were deleted from the genome of the E. coli strain BL21 (DE3) strain. Also genes wzxC-wcaJ were deleted. WcaJ encodes an UDP-glucose:undecaprenyl phosphate glucose-1-phosphate transferase catalysing the first step in colanic acid synthesis (Stevenson et al., J. Bacteriol. 1996, 178:4885-4893). In addition the genes fucI and fucK, coding for L-fucose isomerase and L-fuculose kinase, respectively, were removed.
(84) Genomic integration of heterologous genes was performed by transposition. Either the EZ-Tn5 transposase (Epicentre, USA) was used to integrate linear DNA-fragments or the hyperactive C9-mutant of the mariner transposase Himarl (Lampe et al., Proc. Natl. Acad. Sci. 1999, USA 96:11428-11433) was employed for transposition. To produce EZ-Tn5 transposomes the gene of interest together with a FRT-site flanked antibiotic resistance marker was amplified with primer 1119 and 1120 (all primer used are listed in table 3 below); the resulting PCR-product carried on both sites the 19-bp Mosaic End recognition sites for the EZ-Tn5 transposase. For integration using Himarl transposase expression constructs (operons) of interest were similarly cloned together with a FRT-site flanked antibiotic resistance marker into the pEcomar vector. The pEcomar vector encodes the hyperactive C9-mutant of the mariner transposase Himarl under the control of the arabinose inducible promoter P.sub.araB. The expression fragment <P.sub.tet-lacY-FRT-aadA-FRT> (SeqID1) was integrated by using the EZ-Tn5 transposase. After successful integration of the gene for the lactose importer LacY from E. coli K12 TG1 (acc. no. ABN72583) the resistance gene was eliminated from streptomycin resistant clones by the FLP recombinase encoded on plasmid pCP20 (Datsenko and Warner, Proc. Natl. Acad. Sci. 2000, USA 97:6640-6645). The N-acetylglucosaminyltransferase gene lgtA from Neisseria meningitidis MC58 (acc. no. NP_274923) was codon-optimized for expression in E. coli and prepared synthetically by gene synthesis. Together with the gene galT, encoding a galactose-1-phosphate uridylyltransferase from E. coli K-12 substr. MG1655 (acc. no. NP_415279) that was similarly obtained by gene synthesis, lgtA was inserted by transposition (SeqID2) using plasmid pEcomar-lgtA-galT. To enhance de novo synthesis of UDP-N-acetylglucosamine, genes encoding L-glutamine:D-fuctose-6-phosphate aminotransferase (glmS), phosphoglucosamine mutase from E. coli K-12 substr. MG1655 (glmM) and N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase (glmU) from E. coli K-12 substr. MG1655 (acc. no. NP_418185, NP_417643, NP_418186, respectively) were codon-optimized and obtained by gene synthesis. The operon glmUM was cloned under the control of constitutive tetracyclin promoter P.sub.tet while glmS was cloned under the constitutive P.sub.T5 promoter. The transposon cassette <P.sub.tet-glmUM-P.sub.T5-glmS-FRT-dhfr-FRT> (SeqID3), flanked by the inverted terminal repeats specifically recognized by the mariner-like element Himarl transposase was inserted from pEcomar-glmUM-glmS revealing a lacto-N-triose II production strain. Additionally, the expression fragment <P.sub.tet-lacY(6H/S)-FRT-aadA-FRT> (SeqID4) was integrated by using the EZ-Tn5 transposase.
(85) The gal-operon (galETKM) was amplified from E. coli K12 TG1 (SeqID6) using primer 605 and 606 and inserted into the galM ybhJ locus of E. coli BL21 (DE3) strain by homologous recombination facilitated by using the red recombinase helper plasmid pKD46 (Datsenko and Warner, Proc. Natl. Acad. Sci. 2000, USA 97:6640-6645). Sequences of the heterologous genes and gene clusters are deposit in appendix 1.
Example 2
(86) Batch Fermentation of E. coli BL21 (DE3) 707 screening various -1,3-N-acetyl-glycosaminyltransferases
(87) The gene for the -1,3-N-acetyl-glucosaminyltransferase PmnagT from Pasteurella multocida subsp. multocida str. HN06 (acc. no. PMCN06_0022) was codon-optimized and synthetically synthesized by GenScript Cooperation (Piscataway, USA). Cloning of the gene occurred by sequence and ligation-independent cloning into the plasmid pET-DUET (Merck KGaA, Darmstadt, Germany). All primer used for cloning are listed in table 3 below.
(88) E. coli BL21(DE3) 707 (table 2 below) harbouring plamid pET-PmnagT coding for a -1,3-N-acetyl glucosaminyltransferase was grown at 30 C. in mineral salts medium (Samain et al., J. Biotech. 1999, 72:33-47) supplemented with 2% (wt/vol) glucose and ampicillin 100 g ml.sup.1. When the cultures reached an OD660 nm of 0.1, gene expression was induced by addition of 0.3 mM IPTG. After four hours of incubation 1.5 mM lactose was added. After an additional incubation for 24 hours at 30 C. in shaking flasks cells were harvested. LNT-2 was detected by thin layer chromatography. Therefore, cells were mechanically disrupted in a defined volume using glass beads. Subsequently, samples were applied on TLC Silica Gel 60 F.sub.254 (Merck KGaA, Darmstadt, Germany). The mobile phase was composed of acetone:butanol:acetic acid:water (35:35:7:23).
(89) The result of the TLC analysis is shown in
Example 3
Generation of an E. coli Lacto-N-Triose II Production Strain Overexpressing a Homologous Sugar Efflux Transporter
(90) The export of oligosaccharides produced in E. coli was proven to be a limiting factor during the fermentation process. However, trisaccharides like 2-fucosyllactose and LNT-2 are translocated into the culture supernatant to some extent, thus probably encoding a working sugar efflux transporter. In order to improve the efflux of lacto-N-triose II (LNT-II; GluNAc(1-3)Gal(1-4)Glc), the E. coli BL21(DE3) strain 1326 (table 2 below) was used for the screening of a library of sugar efflux transporters (SET). Putative SET proteins from E. coli were amplified from genomic DNA of E. coli BL21 (DE3) and integrated into vector pINT by sequence and ligation-independent cloning. Using the example of the gene yjhB, the primer 2567, 2568, 2526 and 2443 were used, generating the plasmid pINT-yjhB.
(91) The primer sequences used for cloning are listed in table 3 below. E. coli BL21(DE3) 1326 harbouring plasmids encoding for 20 different E. coli transporters were grown at 30 C. in mineral salts medium (Samain et al., J. Biotech. 1999, 72:33-47) supplemented with 2% (wt/vol) glucose, ampicillin 100 g ml.sup.1 and zeocin 40 g ml.sup.1. When the cultures reached an OD660 nm of 0.1, gene expression of the genes was induced by addition of 200 ng/ml anhydrotetracycline. After four hours of incubation 2.5 mM lactose was added. After an additional incubation for 24 and 48 hours at 30C in shaking flasks the LNT-II concentration in the supernatant was determined by LC-MS.
(92) Mass analysis was performed by characteristic fragment ion detection using an LC Triple-Quadrupole MS detection system. Precursor ions are selected and analyzed in quadrupole 1, fragmentation takes place in the collision cell using nitrogen as CID gas, selection of fragment ions is performed in quadrupole 3.
(93) Lacto-N-tetraose (LNT (Gal(1-3)GlcNAc(1-3)Gal(1-4)Glc)), LNT-II and Maltotriose (internal standard for quantification) were analyzed in ESI positive ionization mode. LNT forms an ion of m/z 708.3 [M+H.sup.+], LNT-II an ion of m/z 546.1 [M+H.sup.+] and Maltotriose an ion of m/z 522.0 [M+NH.sub.4.sup.+]. Adduct formation of this carbohydrate [m/z 504.0] takes place with an ammonium ion (NH4.sup.+), resulting in mass shift of +18. Thus for Maltotriose a precursor ion of m/z 522.0 was selected. The precursor ion was further fragmented in the collision cell into the characteristic fragment ions m/z 487.1, m/z 325.0 and m/z 163.2. The molecular ion of LNT (m/z 708.3) was fragmented into m/z 546.3, m/z 528.3, m/z 366.2 and m/z 204.0. LNT-II (m/z 546.1) was fragmented into m/z 204.2, 186.0, 138.0 and 126.0 (see method description).
(94) Chromatographic separation of LNT and LNT-II was performed on a Luna NH.sub.2 HPLC column (Phenomenex, Aschaffenburg, Germany). This was necessary due to partial fragmentation of LNT during ionization resulting in LNT-II signals affecting quantification results of the individual carbohydrates.
(95) Only for the strain expressing the gene yjhB, an increased amount of LNT-2 in the culture supernatant was observed (see table 1 below).
(96) Table 1: Calculated concentrations of LNT-II in the culture supernatant of an E. coli BL21 (DE3) strain overexpressing yjhB and the reference strain.
(97) TABLE-US-00001 Calc. conc. after 24 h Calc. conc. after 48 h Analyte Sample of incubation [M] of incubation [M] RT 1326 751 1265 0.616 1326 pINT-yjhB 413 1975 0.609
Example 4
(98) Batch Fermentations of E. coli BL21(DE3) 724 Screening Various -1,4-Galactosyltransferases
(99) The genes for the -1,4-galactosyltransferases lex1 from Aggregatibacter aphrophilus NJ8700 (acc. no. YP_003008647), PmgalT7 from Pasteurella multocida subsp. multocida str. HN06 (acc. No. PMCN06_0021), MsgalT8 from Myxococcus stipitatus DSM14675 (acc. no. MYSTI_04346), KdgalT10 from Kingella denitrificans ATCC 33394 (acc. no. HMPREF9098_2407), gatD from Pasteurella multocida M1404 (acc. no. GQ444331), BfgalT2 from Bacterioidis fragilis NCTC9343 (acc. no. BF9343 0585), IsgD from Haemophilus influenza (acc. no. AAA24981) and HpgalT from Helicobacter pylori (acc. no. AB035971) were codon-optimized and synthetically synthesized by GenScript Cooperation (Piscataway, USA). Cloning of the genes occurred by sequence and ligation-independent cloning (Li and Elledge, Nat Methods. 2007 March; 4(3):251-6.). Therefore, the plasmid pINT, harbouring the malE gene under control of an anhydrotetracyline-inducible promoter, was used, enabling the generation of a N-terminal fusion of the -1,4-galactosyltransferase genes with ma/E. Solely, the -1,4-galactosyltransferase encoding gene waaX from Pectobacterium atrosepticum JG10-08 (acc. no. ECA0154) was cloned into plasmid pACYC-Duet (Merck KGaA, Darmstadt, Germany). All primer used for cloning are listed in table 3 below.
(100) E. coli BL21(DE3) 724 (table 2 below) harbouring plamid pCDF-galE and a plasmid coding for the gene fusion of malE with a -1,4-galactosyltransferase was grown at 30 C. in mineral salts medium (Samain et al., J. Biotech. 1999, 72:33-47) supplemented with 2% (wt/vol) glucose, ampicillin 100 g ml.sup.1 and zeocin 40 g ml.sup.1. When the cultures reached an OD660 nm of 0.1, gene expression of the galE gene and the -1,4-galactosyltransferase was induced by addition of 0.3 mM IPTG and 200 ng/ml anhydrotetracycline. E. coli BL21(DE3) 534 (table 2 below) harbouring plamids pET-lgtA, pCOLA-glmUM-glmS, pCDF-galT-galE and pACYC-waaX was grown at 30 C. in mineral salts medium supplemented with 2% (wt/vol) glucose, ampicillin 100 g ml.sup.1, chloramphenicol 34 g ml.sup.1, streptomycin 50 g ml.sup.1 and kanamycin 30 g ml.sup.1. When the cultures reached an OD660 nm of 0.1, gene expression was induced by addition of 0.3 mM IPTG. Four hours after induction of gene expression 2 mM lactose were added. After an additional incubation for 48 hours at 30 C. in shaking flasks, cells were harvested and mechanically disrupted. Lacto-N-neotetraose (LNnT (Gal(1-4)GlcNAc(1-3)Gal(1-4)Glc)) was detected by thin layer chromatography. Therefore, cells were mechanically disrupted using glass beads. Subsequently, samples were applied on TLC Silica Gel 60 F.sub.254 (Merck KGaA, Darmstadt, Germany). The mobile phase was composed of acetone:butanol:acetic acid:water (35:35:7:23).
(101) The results of the TLC analyses are shown in
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(104) The formation of a compound showing the same migration rate as the tetrasaccharide standard LNnT could be observed when the following genes were overexpressed: lex1, PmgalT7, MsgalT8, BfgalT2, gatD, IsgD, KdgalT10, HpgalT, wax.
(105) The -1,4-galactosyltransferases cpsIaJ and cpsI14J, known from literature to produce LNnT (Watanabe et al., J Biochem. 2002 February; 131(2):183-91; Kolkman et al., J Bacteriol. 1996 July; 178(13):3736-41), were also included in the activity screening and served as positive control. Using the described expression system, the formation of LNnT could be observed by CpsIaJ and CpsI14J (
Example 5
(106) Batch Fermentations of E. coli BL21(DE3) 534 Screening Different -1,3-Galactosyltransferases
(107) Using genomic DNA of E. coli K12 DH5a as template, galE was amplified using primer 1163 and 1162. The PCR product was purified, restricted with restriction endonucleases NdeI and XhoI and ligated into the second multiple cloning site of vector pCDFDuet (Merck KGaA, Darmstadt, Germany), which was cut with the same enzymes. GalE is expressed from the IPTG inducible T7 promoter. The E. coli K12 gene galT was amplified from genomic DNA and integrated into plasmid pCDF-galE by sequence and ligation-independent cloning using primer 991-994, producing the plasmid pCDF-galT-galE.
(108) Using the codon-optimized gene of lgtA as template, amplification occurred using primer 688 and 689. The PCR product was purified, restricted with restriction endonucleases NdeI and AatII and ligated into the multiple cloning site of vector pETDuet (Merck KGaA, Darmstadt, Germany), which was cut with the same enzymes, producing the plasmid pET-lgtA.
(109) Cloning of the codon-optimized gene construct of glmUM occurred by sequence and ligation-independent cloning into the plasmid pCOLA-Duet (Merck KGaA, Darmstadt, Germany) using primer 848-851. The codon-optimized form of glmS was amplified using primer 852 and 853. The PCR product was purified, restricted with restriction endonucleases NdeI and AatII and ligated into the second multiple cloning site of vector pCOLA-glmUM, which was cut with the same enzymes, producing the plasmid pCOLA-glmUM-glmS.
(110) The genes for the -1,3-galactosyltransferases wbdO from Salmonella enterica subsp. salamae serovar Greenside (acc. no. AY730594) and furA from Lutiella nitroferrum 2002 (FuraDRAFT_0419) were also codon-optimized and synthetically synthesized by GenScript Cooperation (Piscataway, USA). Cloning of the genes occurred by sequence and ligation-independent cloning into the plasmid pACYC-Duet (Merck KGaA, Darmstadt, Germany). All primer used for cloning are listed in table 3 below.
(111) E. coli BL21(DE3) 534 harbouring plasmids pET-lgtA, pCOLA-glmUM-glmS, pCDF-galT-galE and a plasmid coding for a -1,3-galactosyltransferase pACYC-furA or pACYC-wbdO was grown at 30 C. in mineral salts medium (Samain et al., J. Biotech. 1999, 72:33-47) supplemented with 2% (w/v) glucose, ampicillin 100 g ml.sup.1, chloramphenicol 34 g ml.sup.1, streptomycin 50 g ml.sup.1 and kanamycin 30 g ml.sup.1. When the cultures reached an OD660 nm of 0.1, gene expression was induced by addition of 0.3 mM IPTG. After four hours of incubation 2 mM lactose was added. After an additional incubation for 48 hours at 30 C. in shaking flasks, cells were harvested. LNT was detected by thin layer chromatography. Therefore, cells were mechanically disrupted using glass beads. Subsequently, samples were applied on TLC Silica Gel 60 F.sub.254 (Merck KGaA, Darmstadt, Germany). The mobile phase was composed of acetone:butanol:acetic acid:water (35:35:7:23).
(112) The results of the TLC analyses are shown in
Example 6
(113) Development of an Improved Plasmid-Free E. coli Lacto-N-Tetraose Production Strain
(114) Escherichia coli BL21(DE3) strain 724 was used to construct a lacto-N-tetraose (LNT) producing strain. Metabolic engineering included the genomic integration of the transposon cassettes <P.sub.tet-wbdO-P.sub.T5-galE-FRT-cat-FRT> (SeqID5), flanked by the inverted terminal repeats specifically recognized by the mariner-like element Himarl transposase, which was inserted from pEcomar-wbdO-galE. The resulting strain 1353 was further metabolically engineered to exhibit an increased intracellular LNT-II pool resulting in the elevated production of LNT. Therefore, the mayor facilitator superfamily transporter yjhB (acc. no. YP_003001824) was deleted from the genome of the E. coli strain, generating strain 1431 (table 2 below).
(115) Batch fermentation of the E. coli BL21(DE3) strains 1353 and 1431 was conducted for 48 hours at 30 C. in mineral salts medium (Samain et al., J. Biotech. 1999, 72:33-47) containing 2% (wt/vol) glucose as sole carbon and energy source. When the cultures reached an OD660 nm of 0.5, 2.5 mM lactose was added. The presence of LNT-II and LNT in the culture supernatant was detected by high performance liquid chromatography (HPLC).
(116) Analysis by HPLC was performed using a refractive index detector (RID-10A) (Shimadzu, Duisburg, Germany) and a ReproSil Carbohydrate, 5 m (250 mm4.6 mm) (Dr. Maisch GmbH, Germany) connected to an HPLC system (Shimadzu, Duisburg, Germany). Elution was performed isocratically with acetonitril:H.sub.2O (68/32 (v/v)) as eluent at 35 C. and a flow rate of 1.4 ml/min. 40 l of the sample were applied to the column. Samples were filtered (0.22 m pore size) and cleared by solid phase extraction on an ion exchange matrix (Strata ABW, Phenomenex, Aschaffenburg, Germany).
(117) The results of the HPLC analyses are shown in
Example 7
(118) Generation of an E. coli Lacto-N-Tetraose Production Strain Overexpressing a Sugar Efflux Transporter
(119) Since an export of lacto-N-tetraose into the medium is only moderate for production strains, a screening of a sugar efflux transporter library was conducted. In accordance to example 3 putative SET proteins were either amplified from E. coli genomic DNA or were codon-optimized and synthetically synthesized by GenScript Cooperation (Piscataway, USA). Following amplification genes were integrated into vector pINT by sequence and ligation-independent cloning. The primer design for the cloning of E. coli genes was in accordance to example 3. Synthetic genes were synthesized with standardized nucleotide overhangs and likewise integrated into the expression vector using the primer 2527, 2444, 2526 and 2443. The primer sequences used for cloning are listed in table 3 below.
(120) E. coli BL21(DE3) 1353 (table 2 below) harbouring plasmids encoding for 66 different transporters were grown at 30 C. in mineral salts medium (Samain et al., J. Biotech. 1999, 72:33-47) supplemented with 3% (w/v) glucose, 5 g l.sup.1 NH.sub.4Cl.sub.2, ampicillin 100 g ml.sup.1 and kanamycin 15 g ml.sup.1. Precultivation appeared in 96-well plates harbouring a total volume of 200 l. After 24 h of incubation at 30 C. by continuous shaking, 50 l per well was transferred into 96-well deep well plates harbouring a total volume of 400 l mineral salts medium additionally supplemented with 200 ng ml.sup.1 anhydrotetracycline and 10 mM lactose. After a sustained incubation for 24 to 48 hours the LNT concentrations in the supernatant were determined by LC-MS. Mass analysis was performed as described in example 3.
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(122) Furthermore, the exporters encoded by the genes spoVB of Bacillus amyloliquefaciens (TP1, acc. no. AFJ60154), yabMof Erwinia pyrilfoliae (TP2, acc. no. CAY73138), bcr of E. coli MG1655 (TP18, acc. no. AAC75243), ydeA of E. coli MG1655 (TP20, acc. no. AAC74601), proP2 of Haemophilus parainfluenzae (TP54, acc. no. EGC72107), setA of Pectobacterium carotovorum (TP55, acc. no. ZP_03829909), fucP of E. coli MG1655 (TP59, acc. no. AIZ90162), mdeA of Staphylococcus aureus Bmb9393 (TP61, acc. no. SABB_01261), lmrA of Lactococcus lactis (TP62, acc. no. L116532), setA of Pseudomonas sp. MT-1 (TP72, acc. no. BAP78849) and setA of Beauveria bassiana D1-5 (TP73, acc. no. KGQ13398) resulted in an increased LNT production when overexpressed in the E. coli production strain 1353.
Example 8
(123) Generation of an E. coli Lacto-N-Triose II Production Strain by Overexpression of Heterologous Sugar Efflux Transporters
(124) The LNT exporter screening described in example 6 interestingly disclosed two proteinsTP11 from Mannheimia succiniciproducens MBEL55E (proP, acc. no. AAU37785) and TP70 from Cedecea neteri M006 (setA, acc. no. WP_039290253)whose overexpression resulted in a significantly increased production of LNT-II and consequently in a decreased LNT production (data not shown). This observation was confirmed in an experimental setup as described in example 3. The overexpression of the sugar efflux transporter YjhB served as a positive control. The overexpression of TP11 as well as TP70 resulted in an approximately 4-fold increase in LNT-II production which was even slightly more than for YjhB:
(125) TABLE-US-00002 TABLE 2 Strains and plasmids Strain Genotype Ref. E. coli BL21(DE3) F-ompT hsdSB(rB-, mB-) gal dcm (DE3) Merck KGaA, Darmstadt, Germany E. coli BL21(DE3) 534 E. coli BL21(DE3) lacZ ara wcaJ This study fuclK nagAB harbouring genomic integrations of: galETKM, lacy E. coli BL21(DE3) 724 E. coli BL21(DE3) lacZ ara wcaJ This study fuclK nagAB harbouring genomic integrations of: galETKM, lacY, lgtA-galT- kanR, glmUM-glmS-dhfr E. coli BL21(DE3) 1326 E. coli BL21(DE3) lacZ ara wcaJ This study fuclK nagAB harbouring genomic integrations of: galETKM, lacY, lgtA-galT- kanR, glmUM-glmS-dhfr, lacy(6HIS)-aadA E. coli BL21(DE3) 707 E. coli BL21(DE3) lacZ ara wcaJ This study fuclK nagAB harbouring genomic integrations of: galETKM, lacY, glmUM- glmS-dhfr E. coli BL21(DE3) 1353 E. coli BL21(DE3) lacZ ara wcaJ This study fuclK nagAB harbouring genomic integrations of: galETKM, lacY, lgtA-galT- kanR, glmUM-glmS-dhfr, wbdO-galE-cat E. coli BL21(DE3) 1431 E. coli BL21(DE3) lacZ ara wcaJ This study fuclK nagAB harbouring genomic integrations of: galETKM, lacY, lgtA-galT- kanR, glmUM-glmS-dhfr, wbdO-galE-cat, yjhB-aacC1 pCDF-galE galE of E. coli K12 integrated into vector EP 14 162 869.3 pCDFDuet pET-lgtA (SeqID7) lgtA of Neisseria meningitidis integrated This study into vector pETDuet pCDF-galT-galE (SeqID8) galT and galE of Escherichia coli K12 This study integrated into vector pCDFDuet pCOLA-glmUM-glmS glmU, glmM and glmS of Escherichia coli This study (SeqID9) K12 integrated into vector pCOLADuet pINT-malE-lex1 Gene fusion of malE with lex-1 of EP 14 162 869.3 Aggregatibacter aphrophilus NJ8700 integrated into vector pINT pINT-malE-PmgalT7 Gene fusion of PmgalT7 of Pasteurella This study (SeqID10) multocida subsp. multocida str. HN06 integrated into vector pINT pINT-malE-MsgalT8 Gene fusion of MsgalT8 of Myxococcus This study (SeqID11) stipitatus DSM14675 integrated into vector pINT pINT-malE-KdgalT10 Gene fusion of KdgalT10 of Kingella This study (SeqID12) denitrificans ATCC 33394 integrated into vector pINT pINT-malE-gatD (SeqID13) Gene fusion of gatD of Pasteurella This study multocida M1404 integrated into vector pINT pINT-malE-BFgalT2 Gene fusion of BfgalT2 of Bacterioidis This study (SeqID14) fragilis NCTC9343 integrated into vector pINT pINT-malE-IsgD (SeqID15) Gene fusion of IsgD of Haemophilus This study influenza integrated into vector pINT pINT-malE-HPgalT Gene fusion of HpgalT of Helicobacter This study (SeqID16) pylori integrated into vector pINT pACYC-waaX (SeqID17) waaX of Pectobacterium atrosepticum This study JG10-08 integrated into vector pACYCDuet pACYC-wbdO (SeqID18) wbdO of Salmonella enterica subsp. This study salamae serovar Greenside integrated into vector pACYCDuet pACYC-furA (SeqID19) furA of Lutiella nitroferrum 2002 integrated This study into vector pACYCDuet pET-PmnagT (SeqID20) PmnagT of Pasteurella multocida subsp. This study multocida str. HN06 integrated into vector pETDuet PINT-yjhB (SeqID21) yjhB of E. coli BL21 DE3 integrated into This study vector pINT pINT-yebQ (SeqID22) yebQ of E. coli BL21 DE3 integrated into This study vector pINT pINT-proP (SeqID23) proP of Mannheimia succiniciproducens This study MBEL55E integrated into vector pINT pINT-Cn-setA (SeqID24) setA of Cedecea neteri M006 integrated This study into vector pINT pINT-spoVB (SeqID25) spoVB of Bacillus amyloliquefaciens This study integrated into vector pINT pINT-yabM (SeqID26) yabM of Erwinia pyrifoliae integrated into This study vector pINT pINT-ydeA (SeqID27) ydeA of E. coli MG1655 integrated into This study vector pINT pINT-proP2 (SeqID28) proP2 of Haemophilus parainfluenzae This study integrated into vector pINT pINT-Pc-setA (SeqID29) setA of Pectobacterium carotovorum This study integrated into vector pINT pINT-fucP (SeqID30) fucP of Escherichia coli BL21 (DE3) This study integrated into vector pINT pINT-mdeA (SeqID31) mdeA of Staphylococcus aureus Bmb9393 This study integrated into vector pINT pINT-lmrA (SeqID32) lmrA of Lactococcus lactis integrated into This study vector pINT pINT-Ps-setA (SeqID33) setA of Pseudomonas sp. MT-1 integrated This study into vector pINT pINT-Bb-setA (SeqID34) setA of Beauveria bassiana D1-5 This study integrated into vector pINT
(126) TABLE-US-00003 TABLE3 OligonucleotidesusedforPCR Primer Sequence5-3 605KIgalfwd TTACTCAGCAATAAACTGATATTCCGTCAGGCTGG(SeqID35) 606KIgalrev TTGTAATCTCGCGCTCTTCACATCAGACTTTCCATATAGAGCGTAATTTC CGTTAACGTCGGTAGTGCTGACCTTGCCGGAGG(SeqID36) 1119ME-for CTGTCTCTTATCACATCTCCTGAAATGGCCAGATGTAATTCCTAATTTTT GTTG(SeqID37) 1120MErev CTGTCTCTTATCACATCTCACATTACATCTGAGCGATTGTTAGG (SeqID38) 1163galE_NdeI-for GATCACATATGAGAGTTCTGGTTACCGGTG(SeqID39) 1164galE_XhoI-rev GATCACTCGAGTCATTAATCGGGATATCCCTGTGGATGGC(SeqID40) 5176lex1pINT-f GTCGATGAAGCCCTGAAAGACGCGCAGACTATGCACTTCATTGAAAAC AAAAACTTCGTC(SeqID41) 5177lex1pINT-r GATGGCCTTTTTGCGTGTCGACGCGGCCGCCTAGATAAACAGGATGAT ATTTTTGCCTIG(SeqID42) 5178pINTlexl-f CAAGGCAAAAATATCATCCTGTTTATCTAGGCGGCCGCGTCGACACGC AAAAAGGCCATC(SeqID43) 5179pINTlexl-r GACGAAGTTTTTGTTTTCAATGAAGTGCATAGTCTGCGCGTCTTTCAGG GCTTCATCGAC(SeqID44) 5192waaXpINTfor GTCGATGAAGCCCTGAAAGACGCGCAGACTATGATTGATAACCTGATTA AGCGTACCCCG(SeqID45) 5193waaXpINTrev ATGGCCTTTTTGCGTGTCGACGCGGCCGCTTAATTCGAGCGGGTAAAG ATCTTCATCAGG(SeqID46) 5194pINTwaaXfor CTGATGAAGATCTTTACCCGCTCGAATTAAGCGGCCGCGTCGACACGC AAAAAGGCCATC(SeqID47) 5195pINTwaaXrev CGGGGTACGCTTAATCAGGTTATCAATCATAGTCTGCGCGTCTTTCAGG GCTTCATCGAC(SeqID48) 5164PmgalT7pINT GTCGATGAAGCCCTGAAAGACGCGCAGACTATGAGCGGTGAACACTAT for GTCATTAGCCTG(SeqID49) 5165PmgalT7pINT GATGGCCTTTTTGCGTGTCGACGCGGCCGCTCATTTAAATTCGATGATC rev ATCTTGTCGTT(SeqID50) 5166pINTPmgalT7 AACGACAAGATGATCATCGAATTTAAATGAGCGGCCGCGTCGACACGC for AAAAAGGCCATC(SeqID51) 5167pINTPmgalT7 CAGGCTAATGACATAGTGTTCACCGCTCATAGTCTGCGCGTCTTTCAGG rev GCTTCATCGAC(SeqID52) 5168MsgalT8pINT GTCGATGAAGCCCTGAAAGACGCGCAGACTATGGATGAAATCAAACTG for TCGGTGGTTATG(SeqID53) 5169MsgalT8pINT GATGGCCTTTTTGCGTGTCGACGCGGCCGCTCATTGGCGACGCCAATC rev GAACGCAACGCG(SeqID54) 5170pINTMsgalT8 CGCGTTGCGTTCGATTGGCGTCGCCAATGAGCGGCCGCGTCGACACG for CAAAAAGGCCATC(SeqID55) 5171pINTMsgalT8 CATAACCACCGACAGTTTGATTTCATCCATAGTCTGCGCGTCTTTCAGG rev GCTTCATCGAC(SeqID56) 5561KdgalT10 GTCGATGAAGCCCTGAAAGACGCGCAGACTATGGAAAACTATGTCGTC pINTfor TCTATCCGCACC(SeqID57) 5562KdgalT10 GATGGCCTTTTTGCGTGTCGACGCGGCCGCTCATTTGAACGGAACAAT pINT-rev CTTTTTGTCATC(SeqID58) 5563pINT- GATGACAAAAAGATTGTTCCGTTCAAATGAGCGGCCGCGTCGACACGC KdgalT10for AAAAAGGCCATC(SeqID59) 5564pINT- GGTGCGGATAGAGACGACATAGTTTTCCATAGTCTGCGCGTCTTTCAG KdgalT10rev GGCTTCATCGAC(SeqID60) 5172gatDpINTfor GTCGATGAAGCCCTGAAAGACGCGCAGACTATGTCCTCAGCTTTCCATT ACGTCATTAGC(SeqID61) 5173gatDpINTrev GATGGCCTTTTTGCGTGTCGACGCGGCCGCTCATTCAAATTCGATAATC ATGGTGATTTT(SeqID62) 5174pINTgatDfor AAAATCACCATGATTATCGAATTTGAATGAGCGGCCGCGTCGACACGCA AAAAGGCCATC(SeqID63) 5175pINTgatDrev GCTAATGACGTAATGGAAAGCTGAGGACATAGTCTGCGCGTCTTTCAG GGCTTCATCGAC(SeqID64) 5160BfglaT2pINT GTCGATGAAGCCCTGAAAGACGCGCAGACTATGAACGTGAATAAGCCG for ACCACCGAAAAG(SeqID65) 5161BfgalT2pINT GATGGCCTTTTTGCGTGTCGACGCGGCCGCTCAGTATTCTTCAATTTTG rev TCCAGTTGATA(SeqID66) 5162pINTBfgalT2 TATCAACTGGACAAAATTGAAGAATACTGAGCGGCCGCGTCGACACGC for AAAAAGGCCATC(SeqID67) 5163pINTBfgalT2 CTTTTCGGTGGTCGGCTTATTCACGTTCATAGTCTGCGCGTCTTTCAGG rev GCTTCATCGAC(SeqID68) 5746 GTGATCAACGCCGCCAGCGGTCGTCAGACTGTCGATGAAGCCCTGAAA GACGCGCAGACT(SeqID69) 5747 GCGGCCGCGTCGACACGCAAAAAGGCCATCCATCCGTCAGGATGGCC TTCTGCTTAATTT(SeqID70) 5748 AAATTAAGCAGAAGGCCATCCTGACGGATGGATGGCCTTTTTGCGTGT CGACGCGGCCGC(SeqID71) 5749 AGTCTGCGCGTCTTTCAGGGCTTCATCGACAGTCTGACGACCGCTGGC GGCGTTGATCAC(SeqID72) 1886SLICwbdO GTTTAACTTTAATAAGGAGATATACCATGCTGACGGAAGTGCGCCCGGT pACYCfor CTCTACGACGAAACCGC(SeqID73) 1887SLICwbdO CGACCTGCAGGCGCGCCGAGCTCGAATTCATTTGATGTATTTGCAATA pACYCrev GAACACAGAAAAGACCGT(SeqID74) 1888SLICpACYC GTGTTCTATTGCAAATACATCAAATGAATTCGAGCTCGGCGCGCCTGCA wbdorev GGTCGACAAGCTTGCGG(SeqID75) 1889SLICpACYC GAGACCGGGCGCACTTCCGTCAGCATGGTATATCTCCTTATTAAAGTTA Wbd0For AACAAAATTATTTCTACAGG(SeqID76) 1890SLICpACYC GTATGGTGACCCTGTGGCGCAAATGAGAATTCGAGCTCGGCGCGCCTG furArev CAGGTCGACAAGCT(SeqID77) 1891SLICpACYC GCGCTGCCCTGTTTGATTTTATCCATGGTATATCTCCTTATTAAAGTTAA furAfor ACAAAATTATTTCT(SeqID78) 1892SLICfurA CCTGCAGGCGCGCCGAGCTCGAATTCTCATTTGCGCCACAGGGTCACC pACYCrev ATACGTGCCGGCAGG(SeqID79) 1893SLICfurA GITTAACTTTAATAAGGAGATATACCATGGATAAAATCAAACAGGGCAG pACYCfor CGCCTCTCTGGTTGTCG(SeqID80) 3055SLICPmnagT CAGACTCGAGGGTACCGACGTCCTAATAAGTAGATGAATATTTATCAGG pETrev ACGAAGAT(SeqID81) 3056SLICpET AACTAAAGGTTTATTTTCCATATGTATATCTCCTTCTTATACTTAACTAAT PmnagTfor ATAC(SeqID82) 3057SLICpET TAAATATTCATCTACTTATTAGGACGTCGGTACCCTCGAGTCTGGTAAA PmnagTrev GAAACCGCTGCTGCG(SeqID83) 3058SLICPmnagT GTATAAGAAGGAGATATACATATGGAAAATAAACCTTTAGTTTCAGTTTT pETfor GATTTGTGC(SeqID84) 2567_SLIC_yjhB-for TAACTTTAAGAAGGAGATATACAAGAGCTCGAGTCGAAGGAGATAGAAC CATGGCAACAGCATGGTATAAACAAG(SeqID85) 2568_SLIC_yjhB- GCGTGTCGACGCGTTTAGAGGCCCCAAGGGGTTATGCTAGTATCGATT rev TATCATTTAGCCACGGATAGTTTATAAATTTTAC(SeqID86) 2526_SLIC_pINT_ GGTTCTATCTCCTTCGACTCGAGCTCTTGTATATCTCCTTCTTAAAGTTA TP-rev AACAAAATTATTTCTAGATTTTTGTCGAAC(SeqID87) 2443_SLIC_pINT_ TAAATCGATACTAGCATAACCCCTTGGGGCCTCTAAACGCGTCGACAC TP-forw GCAAAAAGGCCATCC(SeqID88) 2527_SLIC_TP_pINT- GTTCGACAAAAATCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATAT forw ACAAGAGCTCGAGTCGAAGGAGATAGAACC(SeqID89) 2444_SLIC_TP_pINT- GGATGGCCTTTTTGCGTGTCGACGCGTTTAGAGGCCCCAAGGGGTTAT rev GCTAGTATCGATTTA(SeqID90) 688IgtAAatIIrev ATATGACGTCTCATTAGCGGTTTTTCAGGAGACG(SeqID91) 689IgtANdeIfor ATATCATATGCCGTCCGAAGCATTCCGTCGTCACC(SeqID92) 991galT-pCDFfor TAACTTTAATAAGGAGATATACCATGACGCAATTTAATCCCGTTGATCAT CCACATCGCCGC(SeqID93) 992pCDF-galTfor ATTTTCGCGAATCCGGAGTGTAAAAGCTTGCGGCCGCATAATGCTTAAG TCGAACAGAAAGTAATCG(SeqID94) 993galT-pCDFrev AAGCATTATGCGGCCGCAAGCTTTTACACTCCGGATTCGCGAAAATGG ATATCGCTGACTGCGCGCAAACGC(SeqID95) 994pCDF-galTrev TCAACGGGATTAAATTGCGTCATGGTATATCTCCTTATTAAAGTTAAACA AAATTATTTCTACAGGGG(SeqID96) 848gImMpCOLA ATGGTGATGGCTGCTGCCCATTTAAACCGCTTTGACTGCGTCGGCAATA SLICrev CGGTGCGC(SeqID97) 849glmUpCOLA GTTTAACTTTAATAAGGAGATATACCATGCTGAACAACGCGATGTCTGTT SLICfor GTTATCCTGG(SeqID98) 850pCOLAglmM CGCAGTCAAAGCGGTTTAAATGGGCAGCAGCCATCACCATCATCACCA SLICrev CAGCC(SeqID99) 851pCOLAglmU TCGCGTTGTTCAGCATGGTATATCTCCTTATTAAAGTTAAACAAAATTAT SLICfor TTCTACAGG(SeqID100) 852glmScopCOLA ATATATCATATGTGCGGTATCGTTGGTGCTATCGC(SeqID101) forNdel 853glmScopCOLA ATATATGACGTCTTATTCCACGGTCACGGATTTCGC(SeqID102) revAatII