High-level production of diacetyl in a metabolically engineered lactic acid bacterium
10563271 · 2020-02-18
Assignee
Inventors
Cpc classification
C12Y207/01069
CHEMISTRY; METALLURGY
C12Y101/01303
CHEMISTRY; METALLURGY
C12Y401/0204
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention provides a genetically modified lactic acid bacterium capable of producing diacetyl under aerobic conditions. Additionally the invention provides a method for producing diacetyl using the genetically modified lactic acid bacterium under aerobic conditions in the presence of a source of iron-containing porphyrin and a metal ion selected from Fe.sup.3+, Fe.sup.2+ and Cu.sup.2+. The lactic acid bacterium is genetically modified by deletion of those genes in its genome that encode polypeptides having lactate dehydrogenase (E.C 1.1.1.27/E.C.1.1.1.28); -acetolactate decarboxylase (E.C 4.1.1.5); water-forming NADH oxidase (E.C. 1.6.3.4); phosphotransacetylase (E.C.2.3.1.8) activity; and optionally devoid of or deleted for genes encoding polypeptides having diacetyl reductase ((R)-acetoin forming; EC:1.1.1.303); D-acetoin reductase; butanediol dehydrogenase ((R,R)-butane-2,3-diol forming; E.C. 1.1.1.4/1.1.1.-) and alcohol dehydrogenase (E.C. 1.2.1.10) activity. The invention provides for use of the genetically modified lactic acid bacterium for the production of diacetyl and a food product.
Claims
1. A genetically modified lactic acid bacterium for production of diacetyl, wherein the genome of said lactic acid bacterium is deleted for all of the genes or lacks all of the endogenous genes encoding polypeptides having an enzymatic activity of: a. lactate dehydrogenase having E.C.1.1.1.27 or E.C.1.1.1.28; b. -acetolactate decarboxylase having E.C. 4.1.1.5; c. phosphotransacetylase having E.C.2.3.1.8; d. NADH oxidase having E.C. 1.6.3.4; e. alcohol dehydrogenase having E.C. 1.2.1.10; and f. a butanediol dehydrogenase having E.C. 1.1.1.4; and wherein said microorganism is devoid of transgenes encoding polypeptides having an enzymatic activity of: g. a (S)-acetoin forming diacetyl reductase having E.C.1.1.1.304; and h. a L-butanediol dehydrogenase having E.C. 1.1.1.76, wherein the lactic acid bacteria belongs to a genus selected from the group consisting of Lactococcus, Lactobacillus, Pediococcus, Leuconostoc, Streptococcus, Oenococcus, and Bacillus.
2. A genetically modified lactic acid bacterium according to claim 1, wherein the genome of said lactic acid bacterium is additionally deleted for one or more genes or lacks one or more genes encoding polypeptides having an enzymatic activity selected from the group of: i. a (R)-acetoin forming diacetyl reductase having E.C.1.1.1.303; and j. D-acetoin reductase.
3. A genetically modified lactic acid bacterium according to claim 1, wherein the lactic acid bacteria belongs to the genus Lactococcus.
4. A genetically modified lactic acid bacterium according to claim 1, wherein: a. the amino acid sequence of the polypeptide having lactate dehydrogenase activity has at least 80% sequence identity to an amino acid sequence selected from among SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36.
5. A genetically modified lactic acid bacterium according to claim 1, wherein: a. the amino acid sequence of the polypeptide having phosphotransacetylase activity has at least 80% sequence identity to an amino acid sequence selected from among SEQ ID NO: 42, 44, 46, 48, 50, 52, 54, 56, 58 and 60.
6. A genetically modified lactic acid bacterium according to claim 1, wherein the amino acid sequence of the polypeptide having -acetolactate decarboxylase activity has at least 80% sequence identity to an amino acid sequence selected from among SEQ ID NO:80, 82, 84, 86, 88, 90, 92, 94 and 96.
7. A genetically modified lactic acid bacterium according to claim 2, wherein: a. the amino acid sequence of the polypeptide having alcohol dehydrogenase activity has at least 80% sequence identity to an amino acid sequence selected from among SEQ ID NO: 62, 64, 66, 68, 70, 72, 74, 76 and 78; b. the amino acid sequence of the polypeptide having diacetyl reductase having E.C.1.1.1.303 activity has at least 80% sequence identity to SEQ ID NO: 98; c. the amino acid sequence of the polypeptide having D-acetoin reductase activity has at least 80% sequence identity to SEQ ID NO: 100, 102, 104, 106 108, 110 and 112; and d. the amino acid sequence of the polypeptide having butanediol dehydrogenase having E.C.1.1.1.4 activity has at least 80% sequence identity to SEQ ID NO: 114.
8. A genetically modified lactic acid bacterium according to claim 1, wherein the amino acid sequence of the polypeptide having a NADH oxidase activity has at least 80% sequence identity to an amino acid sequence selected from among SEQ ID NO: 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 and 136.
9. A genetically modified lactic acid bacterium according to claim 1, wherein the genome of said lactic acid bacterium comprises genes encoding polypeptides having: k. lactose-specific phosphotransferase system (PTS) activity having EC 2.7.1.69; l. phospho--D-galactosidase activity having EC 3.2.1.85; m. galactose-6-phosphate isomerase activity having EC 5.3.1.26; n. D-tagatose-6-phosphate kinase activity having EC 2.7.1.114; and o. tagatose 1,6-diphosphate aldolase activity having EC 4.1.2.40.
10. A genetically modified lactic acid bacterium according to claim 9, wherein: a. the lactose-specific phosphotransferase system (PTS) activity having EC 2.7.1.69 is provided by a first and a second polypeptide, wherein the amino acid sequence of the first polypeptide has at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 210, and the amino acid sequence of the second polypeptide has at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 212; b. the amino acid sequence of the polypeptide having phospho--D-galactosidase activity having EC 3.2.1.85 has at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 214; c. the amino acid sequence of the polypeptide having galactose-6-phosphate isomerase activity having EC 5.3.1.26 is provided by a first and a second polypeptide, wherein the amino acid sequence of the first polypeptide has at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 202 and the amino acid sequence of the second polypeptide has at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 204; d. the amino acid sequence of the polypeptide having D-tagatose-6-phosphate kinase activity having EC 2.7.1.114 has at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 206; and e. the amino acid sequence of the polypeptide having tagatose 1,6-diphosphate aldolase activity having EC 4.1.2.40 has at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 208.
11. A method for the production of diacetyl, comprising the steps of: a. introducing a genetically modified lactic acid bacterium according to claim 1 into a growth medium to produce a culture, wherein the growth medium comprises a source of protoporphyrin IX or iron-containing porphyrin; b. cultivating the culture of (a) under aerobic growth conditions; c. recovering diacetyl produced by said culture; and optionally d. isolating the recovered diacetyl.
12. A method for the production of diacetyl according to claim 11, wherein the source of iron-containing porphyrin is hemin or hematin, wherein the concentration of hemin or hematin is 0.3- 5 g/ml of the growth medium.
13. A method for the production of diacetyl according to claim 11, wherein the combined total Fe.sup.2+, Fe.sup.3+and Cu.sup.2+concentration of the growth medium in step (a) is less than 20 mM.
14. A method for the production of diacetyl according to claim 11, including an additional step of supplementing the culture produced in step (b) with one or more metal ions selected from the group consisting of Fe.sup.2+, Fe.sup.3+and Cu.sup.2+; and incubating the supplemented culture under aerobic conditions prior to step (c).
15. A method for the production of diacetyl according to claim 11, wherein the growth medium comprises a source of lactose, such as whey, whey permeate or residual whey permeate.
Description
DESCRIPTION OF THE INVENTION
(1) Description Of The Figures
(2)
(3)
(4) (A) Cartoon showing the electron transport chain in L. lactis, from electron donor (NoxAB), electron transfer (menaquinone) to electron acceptor (Cyt bd oxidase), which is activated by hemin.
(5) (B) Cell growth as measured by cell density (OD.sub.600 nm) as a function of time (hr) at a range of hemin concentrations rising from 0.2 g/ml to 5.0 g/ml.
(6) (C) Cellular NAD+ levels and NADH/NAD+ ratio in L. lactis cultivate in at a range of hemin concentrations rising from 0.3 g/ml to 5.0 g/ml.
(7) Experiments were carried out in duplicate and error bars indicate standard deviations.
(8)
(9)
(10) Graphical presentation of diacetyl formation from acetolactate as a function of time (hr) when L. lactis strain CS4616m is cultured in the presence of increasing levels of either Fe.sup.3+ (B); Fe.sup.2+ (C); and Cu.sup.2+ (D). The time point for adding the metal catalysts during the cultivation of L. Lactis strain CS4616m was at 9.8 h, which is indicated by the straight line in (A). Experiments were conducted in duplicate and error bars indicate standard deviations.
(11)
(12)
ABBREVIATIONS AND TERMS
(13) gi number: (genInfo identifier) is a unique integer which identifies a particular sequence, independent of the database source, which is assigned by NCBI to all sequences processed into Entrez, including nucleotide sequences from DDBJ/EMBL/GenBank, protein sequences from SWISS-PROT, PIR and many others.
(14) Amino acid sequence identity: The term sequence identity as used herein, indicates a quantitative measure of the degree of homology between two amino acid sequences of substantially equal length. The two sequences to be compared must be aligned to give a best possible fit, by means of the insertion of gaps or alternatively, truncation at the ends of the protein sequences. The sequence identity can be calculated as ((Nref-Ndif)100)/(Nref), wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. Sequence identity can alternatively be calculated by the BLAST program e.g. the BLASTP program (Pearson W.R and D.J. Lipman (1988)) (www.ncbi.nlm.nih.gov/cgi-bin/BLAST). In one embodiment of the invention, alignment is performed with the sequence alignment method ClustalW with default parameters as described by Thompson J., et al 1994.
(15) Preferably, the numbers of substitutions, insertions, additions or deletions of one or more amino acid residues in the polypeptide as compared to its comparator polypeptide is limited, i.e. no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additions, and no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deletions. Preferably the substitutions are conservative amino acid substitutions: limited to exchanges within members of group 1: Glycine, Alanine, Valine, Leucine, Isoleucine; group 2: Serine, Cysteine, Selenocysteine, Threonine, Methionine; group 3: Proline; group 4: Phenylalanine, Tyrosine, Tryptophan; Group 5: Aspartate, Glutamate, Asparagine, Glutamine.
(16) Deleted gene: the deletion of a gene from the genome of a microbial cell leads to a loss of function (knockout) of the gene and hence where the gene encodes a polypeptide the deletion results in a loss of expression of the encoded polypeptide. Where the encoded polypeptide is an enzyme, the gene deletion leads to a loss of detectable enzymatic activity of the respective polypeptide in the microbial cell. A deleted gene in the genome of a microbial cell is characterized by a loss of function due to the deletion of, or substitution of, or addition of, at least one nucleotide leading to a loss of expression of a polypeptide encoded by the gene.
(17) Diacetyl reductase (E.C. 1.1.1.304) has (S)-acetoin forming activity, and is capable of converting diacetyl (DA) to L-acetoin (L-AC)
(18) L-butanediol dehydrogenase (E.C. 1.1.1.76) is capable of converting L-acetoin (L-AC) to S,S-2,3-butanediol (L-BD).
(19) Native gene: endogenous gene in a microbial cell genome, homologous to host micro-organism.
(20) Transgenes encoding polypeptides having diacetyl reductase activity (E.C. 1.1.1.304) and L-butanediol dehydrogenase activity (E.C.1.1.1.76) confer on a cell the ability to convert diacetyl (L-AC) to S,S-2,3-butanediol (L-BD).
(21) Whey and whey permeate and residual whey permeate: whey is a byproduct of cheese manufacture; and comprises whey proteins having a high nutritional value and lactose. Removal of whey proteins, typically by means of ultrafiltration or diafiltration produces a whey protein concentrate and whey permeate that is lactose-rich. The lactose content of the whey permeate is dependent on the treatment conditions and typically it can reach as high as hundreds of grams per liter by reverse osmosis, such as 200 g/L. Removal of fat from whey, or from lactose-rich permeate, typically by centrifugation, yields a fat-free composition (whey or permeate). Residual whey permeate (also called permeate mother liquor) is obtained after the extraction of lactose from whey permeate (typically by lactose crystallisation); and has a lower lactose content of about 150 g/L.
DETAILED DESCRIPTION OF THE INVENTION
(22) I: A Genetically Modified Lactic Acid Bacterium for the Production of Diacetyl
(23) Endogenous Genes Deleted to Enhance Metabolic Flux from Pyruvate to Diacetyl
(24) The lactic acid bacterium of the invention is adapted to produce diacetyl from glucose under aerobic conditions. The lactic acid bacterium of the invention is characterised by an enhanced metabolic flux from pyruvate to diacetyl, due to reduced activity in the enzymes in the pathways leading to the synthesis of lactate, acetate and ethanol. The production of acetate and ethanol by a lactic acid bacterium is reduced when the bacterium is cultivated under aerobic conditions, in a defined growth medium lacking lipoic acid. When the bacterium is cultivated under aerobic conditions, this inactivates the enzyme pyruvate formate lyase that forms formate and acetyl-CoA, which are the precursors of the acetate and ethanol pathways. Since the enzyme, pyruvate dehydrogenase, requires lipoic acid for activity, the use of a lipoic acid-deficient growth medium (supplemented with acetate) inactivates the synthesis of acetyl-CoA by pyruvate dehydrogenase and the down-stream production of acetate and ethanol. When the lactic acid bacterium of the invention is grown under anaerobic conditions in a minimal medium deficient in lipoic acid, the requirement for acetyl-CoA is met by adding acetate to the growth medium.
(25) In one embodiment, the metabolic flux towards lactate, acetate and ethanol in the lactic acid bacterium of the invention is reduced by deletion of one or more genes encoding enzymes of both the lactate, acetate and optionally ethanol pathways.
(26) Ii Deletion of an endogenous lactate synthesis pathway: The lactic acid bacterium of the invention is characterised by knockouts of one or more endogenous native genes encoding polypeptides having lactate dehydrogenase activity causing a block in the lactate synthesis pathway in the bacterium. Deletion of at least one gene (e.g. ldh) encoding a lactate dehydrogenase enzyme (E.C 1.1.1.27 or E.C.1.1.1.28) provides a lactic acid bacterium of the invention that is depleted in lactate production. For example, where the lactic acid bacterium of the invention belongs to a given genus, the deleted endogenous gene is one encoding a polypeptide having lactate dehydrogenase activity in that genus. Preferably the polypeptide having lactate dehydrogenase activity (E.C 1.1.1.27 or E.C.1.1.1.28) has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity to one of the following sequences: SEQ ID NO: 2 in a Lactococcus species (e.g. Lactococcus lactis); SEQ ID NO: 4, 6, or 8 in a Lactobacillus species (e.g. Lactobacillus acidophilus); SEQ ID NO: 10 in a Lactobacillus species (e.g. Lactobacillus delbrueckii); SEQ ID NO. 12, 14 or 16 in a Lactobacillus species (e.g. Lactobacillus casei), SEQ ID NO. 18 or 20 in a Lactobacillus species (e.g. Lactobacillus plantarum); SEQ ID NO: 22 in a Pediococcus species (e.g. Pediococcus pentosaceus), SEQ ID NO: 24 or 26 in a Leuconostoc species (e.g. Leuconostoc mesenteroides), SEQ ID NO: 28 in a Streptococcus species (e.g. Streptococcus thermophilus), SEQ ID NO: 30 or 32 in a Oenococcus species (e.g. Oenococcus oeni), and SEQ ID NO: 34 or 36 in a Bacillus species (e.g. Bacillus coagulans).
(27) In one embodiment, an additional endogenous gene, encoding a polypeptide having lactate dehydrogenase enzymatic activity (E.C 1.1.1.27 or E.C.1.1.1.28), is deleted from the lactic acid bacterium of the invention. For example, where the lactic acid bacterium of the invention belongs to the genus Lactococcus, the deleted gene (ldhX) encodes a polypeptide having at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity to SEQ ID NO: 38.
(28) In one embodiment, an additional endogenous gene, encoding a polypeptide having lactate dehydrogenase enzymatic activity (E.C 1.1.1.27 or E.C.1.1.1.28), is deleted from the lactic acid bacterium of the invention. For example, where the lactic acid bacterium of the invention belongs to the genus Lactococcus, the deleted gene (ldhB) encodes a polypeptide having at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity to SEQ ID NO: 40. Further, where the lactic acid bacterium of the invention belongs to the genus Lactococcus, the three genes (ldh, ldhB and ldhX) encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO: 2, 38 and 40 respectively may be deleted.
(29) Iii Deletion of an endogenous acetate synthesis pathway: In one embodiment, the lactic acid bacterium of the invention is characterised by knockout of the endogenous native gene encoding a phosphotransacetylase (E.C.2.3.1.8), causing a block in the acetate synthesis pathway in the bacterium. Deletion of a gene (e.g. pta) encoding a phosphotransacetylase enzyme provides a lactic acid bacterium of the invention that is blocked in acetate production. For example, where the lactic acid bacterium of the invention belongs to a given genus, the deleted endogenous gene is one encoding a polypeptide having phosphotransacetylase activity (E.C.2.3.1.8) in that genus. Preferably the polypeptide having phosphotransacetylase activity has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity to one of the following sequences: SEQ ID NO: 42 in a Lactococcus species (e.g. Lactococcus lactis); SEQ ID NO: 44, 46, 48, and 50 in a Lactobacillus species (e.g. Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus plantarum), SEQ ID NO: 52 in a Pediococcus species (e.g. Pediococcus pentosaceus), SEQ ID NO: 54 in a Leuconostoc species (e.g. Leuconostoc mesenteroides), SEQ ID NO: 56 in a Streptococcus species (e.g. Streptococcus thermophilus), SEQ ID NO: 58 Oenococcus species (e.g. Oenococcus oeni), and SEQ ID NO: 60 in a Bacillus species (e.g. Bacillus coagulans).
(30) Iiii Deletion of an endogenous ethanol synthesis pathway: In one embodiment, the lactic acid bacterium of the invention is characterised by knockout of the endogenous native gene encoding alcohol dehydrogenase (E.C.1.2.1.10) causing a block in the ethanol synthesis pathway in the bacterium. Deletion of the gene encoding an alcohol dehydrogenase enzyme provides a lactic acid bacterium of the invention that is blocked in ethanol production.
(31) For example, where the lactic acid bacterium of the invention belongs to a given genus, the deleted endogenous gene (e.g. adhE) is one encoding a polypeptide having alcohol dehydrogenase activity (E.C.1.2.1.10) in that genus. Preferably the polypeptide having alcohol dehydrogenase activity has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity to one of the following sequences: SEQ ID NO: 62 in a Lactococcus species (e.g. Lactococcus lactis); SEQ ID NO: 64 in a Lactobacillus species (e.g. Lactobacillus acidophilus); SEQ ID NO: 66 or 68 in a Lactobacillus species (e.g. Lactobacillus casei); SEQ ID NO: 70 in a Lactobacillus species (e.g., Lactobacillus plantarum), SEQ ID NO: 72 in a Leuconostoc species (e.g. Leuconostoc mesenteroides), SEQ ID NO: 74 in a Streptococcus species (e.g. Streptococcus thermophilus), SEQ ID NO: 76 in a Oenococcus species (e.g. Oenococcus oeni), and SEQ ID NO: 78 in a Bacillus species (e.g. Bacillus coagulans).
(32) Endogenous Genes Deleted to Enhance Diacetyl Accumulation
(33) Iiv Deletion of an Endogenous 2,3-butanediol Synthesis Pathway
(34) The lactic acid bacterium of the invention may be characterized by additional knockouts of the endogenous native genes encoding enzymes having -acetolactate decarboxylase (E.C 4.1.1.5), a diacetyl reductase (EC:1.1.1.303); D-acetoin reductase, and a 2,3-butanediol dehydrogenase ((R,R)-butane-2,3-diol forming; E.C 1.1.1.4/1.1.1.-) activity, thereby causing a block in the 2,3-butanediol synthesis pathway in the bacterium for conversion of -acetolactate, via D-acetoin or diacetyl, to 2,3-butanediol. When the lactic acid bacterium of the invention is devoid of these endogenous native genes encoding the native 2,3-butanediol pathway; and is also devoid of transgenes encoding a diacetyl reductase (E.C.1.1.1.304) and a L-butanediol dehydrogenase (E.C. 1.1.1.76), the cells are unable to produce meso 2,3-butanediol or its chiral forms.
(35) In the case where the lactic acid bacterium of the invention belongs to a given genus, that lacks one or more endogenous native gene encoding one or more polypeptide having -acetolactate decarboxylase activity (E.C 4.1.1.5), diacetyl reductase (EC:1.1.1.303); D-acetoin reductase, 2,3-butanediol dehydrogenase (E.C 1.1.1.4/1.1.1.-) activity or any combination thereof; the step of deletion of the respective gene in order to produce the bacterium of the invention is not required. Accordingly the lactic acid bacterium of the invention lacking endogenous native genes that express enzymes having -acetolactate decarboxylase (E.C 4.1.1.5), diacetyl reductase (EC.1.1.1.303); D-acetoin reductase, and a 2,3-butanediol dehydrogenase ((R,R)-butane-2,3-diol forming; E.C 1.1.1.4/1.1.1.-) activity, may be due either to the absence of genes encoding and expressing said enzymes in the lactic acid bacterium of the invention, or due to deletion of the respective gene from the genome of the bacterium.
(36) Deletion of an endogenous native gene (e.g. aldB) encoding an -acetolactate decarboxylase enzyme (E.C 4.1.1.5) provides a lactic acid bacterium of the invention that is blocked in D-acetoin production. For example, where the lactic acid bacterium of the invention belongs to a given genus, the deleted endogenous gene is one encoding a polypeptide having -acetolactate decarboxylase activity (E.C 4.1.1.5) in that genus. Preferably the polypeptide having -acetolactate decarboxylase activity has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity to one of the following sequences: SEQ ID NO: 80 in a Lactococcus species (e.g. Lactococcus lactis); SEQ ID NO: 82, or 84 in a Lactobacillus species (e.g. Lactobacillus casei, Lactobacillus plantarum), SEQ ID NO: 86 in a Pediococcus species (e.g. Pediococcus pentosaceus), SEQ ID NO: 88 or 90 Leuconostoc species (e.g. Leuconostoc mesenteroides), SEQ ID NO: 92 in a Streptococcus species (e.g. Streptococcus thermophilus), SEQ ID NO: 94 in a Oenococcus species (e.g. Oenococcus oeni), and SEQ ID NO: 96 in a Bacillus species (e.g. Bacillus coagulans).
(37) Deletion of an endogenous native gene (e.g. dar) encoding diacetyl reductase (EC:1.1.1.303) provides a lactic acid bacterium of the invention that is blocked in D-acetoin production. For example, where the lactic acid bacterium of the invention belongs to the genus Lactococcus (e.g. Lactococcus lactis), the deleted gene (dar) encodes a polypeptide having at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity to SEQ ID NO: 98.
(38) Deletion of an endogenous native gene (e.g. ar) encoding a D-acetoin reductase enzyme provides a lactic acid bacterium of the invention that is blocked in D-acetoin production. For example, where the lactic acid bacterium of the invention belongs to a given genus, the deleted endogenous gene is one encoding a polypeptide having D-acetoin reductase activity in that genus. Preferably the polypeptide having D-acetoin reductase activity has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity to one of the following sequences: SEQ ID NO: 100 in a Lactococcus species (e.g. Lactococcus lactis); SEQ ID NO: 102 in a Pediococcus species (e.g. Pediococcus pentosaceus), SEQ ID NO: 104 or 106 in a Leuconostoc species (e.g. Leuconostoc mesenteroides), SEQ ID NO: 108 or 110 Oenococcus species (e.g. Oenococcus oeni), SEQ ID NO: 112 in a Bacillus species (e.g. Bacillus coagulans), SEQ ID NO: 198 or 200 in a Lactobacillus species (e.g. Lactobacillus buchneri).
(39) Deletion of a gene (e.g. butAB) encoding 2, 3-butanediol dehydrogenase activity (E.C 1.1.1.4/1.1.1.-) provides a lactic acid bacterium of the invention that is blocked in meso-2,3-butanediol production. For example, where the lactic acid bacterium of the invention belongs to the genus Lactococcus (e.g. Lactococcus lactis), the deleted gene (butAB) encodes a polypeptide having at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity to SEQ ID NO: 114.
(40) Endogenous Genes Deleted to Maintain a Balance of Redox Equivalents
(41) Iv Deletion of an Endogenous NADH Oxidation Activity
(42) The lactic acid bacterium of the invention is characterized by knockout of the endogenous native gene(s) encoding a water-forming NADH oxidase causing a block in NADH oxidation, and maintenance of reduced NADH levels. Deletion of a gene (e.g. noxE) provides a lactic acid bacterium of the invention that is partially blocked in NADH oxidation.
(43) For example, where the lactic acid bacterium of the invention belongs to a given genus, the deleted endogenous gene is one encoding a polypeptide having water-forming NADH oxidase activity (E.C. 1.6.3.4) in that genus. Preferably the polypeptide having NADH oxidase activity has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity to one of the following sequences: SEQ ID NO: 116 in a Lactococcus species (e.g. Lactococcus lactis); SEQ ID NO: 118 in Lactobacillus casei), SEQ ID NO: 120, 122, 124, 126 and 128 in Lactobacillus plantarum, SEQ ID NO: 130 in a Streptococcus species (e.g. Streptococcus thermophilus), SEQ ID NO: 132, 134 and 136 in a Bacillus species (e.g. Bacillus coagulans).
(44) In the case that the lactic acid bacterium of the invention belongs to a given genus, that lacks an endogenous native gene encoding one or more polypeptide having water-forming NADH oxidase activity (E.C. 1.6.3.4) activity; the step of deletion of the respective gene in order to produce the bacterium of the invention is not required. Accordingly the lactic acid bacterium of the invention lacks endogenous native genes that express an enzyme having water-forming NADH oxidase activity (E.C. 1.6.3.4) activity, either due to the absence of gene encoding said enzyme in the lactic acid bacterium of the invention, or due to deletion of the respective gene from the genome of the bacterium.
(45) The genetically modified lactic acid bacterium of the invention is characterised by the ability to produce diacetyl from a carbon source (e.g. glucose) in surprisingly high amounts (Example 3: product level of 95 mM diacetyl) in very high yields (Example 3: diacetyl yield: 0.87 mol mol1 glucose) and very high productivity (Example 3: diacetyl produced: 0.58 g/L.Math.h).
(46) I.vi Genes Encoding Enzymes of the Lactose Catabolism Pathway:
(47) The lactic acid bacterium of the invention may further comprise the following native genes or transgenes required for lactose assimilation and catabolism:
(48) 1) a first and second gene encoding a first and a second polypeptide component together conferring lactose-specific phosphotransferase system (PTS) activity (EC 2.7.1.69), whereby phosphorylated lactose is assimilated by the cells. The amino acid sequence of the first polypeptide component has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% sequence identity to the amino acid sequence of the phosphotransferase system EIICB component (SEQ ID NO: 210) encoded by the Lactococcus lactis lacE gene; and the amino acid sequence of the second polypeptide component has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% sequence identity to the amino acid sequence of the phosphotransferase system EIIA component (SEQ ID NO: 212) encoded by the Lactococcus lactis lacF gene; and
(49) 2) a gene encoding a polypeptide having phospho--D-galactosidase activity (EC 3.2.1.85) that hydrolyzes lactose-6-phosphate to glucose and galactose-6-phosphate (gal-6-P), whereby the glucose moiety can then enter the glycolytic pathway. The amino acid sequence of the polypeptide has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% sequence identity to the amino acid sequence of the phospho--D-galactosidase (SEQ ID NO: 214) encoded by the Lactococcus lactis lacG gene.
(50) Additionally, the following genes encoding enzymes in the tagatose-6-P pathway, whereby gal-6-P is degraded and enters the glycolytic pathway as glyceraldehyde-3-phosphate, are required:
(51) 3) a first and second gene encoding a first and a second polypeptide subunit together conferring galactose-6-phosphate isomerase activity (EC 5.3.1.26). The amino acid sequence of the first polypeptide subunit has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% sequence identity to the amino acid sequence of the first subunit of the galactose-6-phosphate isomerase (SEQ ID NO: 202) encoded by the Lactococcus lactis lacA gene; and the amino acid sequence of the second polypeptide subunit has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% sequence identity to the amino acid sequence of the second subunit of the galactose-6-phosphate isomerase (SEQ ID NO: 204) encoded by the Lactococcus lactis lacB gene; and
(52) 4) a gene encoding a polypeptide having D-tagatose-6-phosphate kinase activity (EC 2.7.1.114). The amino acid sequence of the polypeptide has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% sequence identity to the amino acid sequence of the D-tagatose-6-phosphate kinase (SEQ ID NO: 206) encoded by the Lactococcus lactis lacC gene; and
(53) 5) a gene encoding a polypeptide having tagatose 1,6-diphosphate aldolase activity (EC 4.1.2.40). The amino acid sequence of the polypeptide has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% sequence identity to the amino acid sequence of the tagatose 1,6-diphosphate aldolase (SEQ ID NO: 208) encoded by the Lactococcus lactis lacD gene; and
(54) 6) optionally a gene encoding a polypeptide having lactose transport regulator activity. The amino acid sequence of the polypeptide has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% sequence identity to the amino acid sequence of the lactose transport regulator (SEQ ID NO: 216) encoded by the Lactococcus lactis lacR gene.
(55) A genetically modified lactic acid bacterium according to the invention, comprising genes encoding and capable of expressing a functional lactose pathway, are able to use lactose as sole carbon source for the production of diacetyl. When whey permeate or residual whey permeate is used as a source of lactose and is a component of the growth medium; then the medium should be supplemented with a source of complex amino nitrogen (comprising soluble proteins and/or peptides; peptides and/or amino acids). Yeast extract is a suitable source of complex amino nitrogen; as well as CSL provided that it is provided in a hydrolysed form as described herein.
(56) II A Genetically Modified Lactic Acid Bacterium Comprising a Pathway for Diacetyl Production
(57) The genetically modified lactic acid bacterium according to the invention, comprising a pathway for synthesis of diacetyl, is a member of a genus of lactic acid bacteria selected from the group consisting of Lactococcus, Lactobacillus, Pediococcus, Leuconostoc, Streptococcus, Oenococcus, and Bacillus. The lactic acid bacterium of the invention may for example be a species of lactic acid bacteria selected from the group consisting of Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus plantarum, Pediococcus pentosaceus, Leuconostoc mesenteroides, Streptococcus thermophilus, Oenococcus oeni and Bacillus coagulans. Preferably the lactic acid bacterium of the invention is a species of Lactococcus.
(58) III Methods for Producing Diacetyl Using the Genetically Modified Micro-Organism of the Invention
(59) Diacetyl can be produced using a lactic acid bacterium of the invention by introducing the bacterium into a culture medium comprising a carbon source for diacetyl biosynthesis; providing the culture with a source of protoporphyrin IX or iron-containing porphyrin (e.g. hemin and hematin) and culturing the bacterium under aerobic conditions (for example: to allow for bacterial cell growth and multiplication to reach a desired biomass); and then adding one or more metal ion selected from the group of Fe.sup.2+, Fe.sup.3+ and Cu.sup.2+ to the culture and incubating the culture further under aerobic culture conditions; and finally recovering the diacetyl produced by the culture, as illustrated in the Examples.
(60) During the cultivation step, cells of the lactic acid bacterium of the invention grow and multiply, where a supply of an iron-containing porphyrin, in limited amounts, is essential for growth of the cells under aerobic conditions. In a preferred embodiment, the protoporphyrin IX or iron-containing porphyrin is provided to the culture, by addition to the culture medium either prior to and subsequent to the introduction of the lactic acid bacterium into the culture medium. The protoporphyrin IX or iron-containing porphyrin may be added continuously or as a batch addition to the culture during growth of the culture. For example hemin is preferably added in amounts to provide a final concentration in the liquid culture medium of 0.1-5 g/ml. Surprisingly, even though the cells are grown under aerobic conditions during this cultivation step, the major fermentation product produced by the cells is -acetolactate, while the accumulation of diacetyl is limited. Further it has surprisingly been observed, that the growth and multiplication of the cells during the cultivation step is not inhibited by the accumulation of -acetolactate in the cells and culture medium. The method of producing diacetyl using cells of the lactic acid bacterium of the invention, takes advantage of the -acetolactate tolerance of the growing cells; since the levels of -acetolactate (the direct precursor for diacetyl) can accumulate during this cultivation step without compromising growth or eventual diacetyl production. Since diacetyl itself, is shown to inhibit cell growth (see Example 3.2), which in turn would limit -acetolactate production, the method of the invention employs growth conditions during the cultivation step that support cell growth and -acetolactate production, but limit the chemical oxidation of -acetolactate to diacetyl. For the reasons set forth below, the concentration of the following metal ions in the growth medium, provided during the cultivation step, is preferably 10 mM Fe.sup.2+ (e.g. 1 mM Fe.sup.2+; 0.1 mM Fe.sup.2+); 10 mM Fe.sup.3+ (e.g. 5 mM Fe.sup.3+; 1 mM Fe.sup.3+; 0.1 mM Fe.sup.3+; 0.01 mM Fe.sup.3+;) and 1 mM Cu.sup.2+ (e.g. 0.1 mM Cu.sup.2+; 0.01 mM Cu.sup.2+, 0.001 mM Cu.sup.2+). A typical growth medium used for cultivation of lactic acid bacterium of the invention comprises one or more of the following metal ions in a concentration range of 0.005-10 mM Fe.sup.2+, 0.005-10 mM Fe.sup.3+ and 0-1 mM Cu.sup.2+; corresponding to a total combined concentration of Fe.sup.2+, Fe.sup.3+ and Cu.sup.2+ in the range of 0.005-20 mM.
(61) The lactic acid bacterium of the invention will grow and produce -acetolactate (and diacetyl in limited amounts), when supplied with a suitable carbon source including glucose, maltose, galactose, fructose, sucrose, arabinose, xylose, raffinose, mannose, and lactose.
(62) When the lactic acid bacterium of the invention is a strain of Lactococcus lactic, the preferred temperature for cultivation is 30 C.; while the selection of a suitable temperature for growth of lactic acid bacteria of the invention belonging to other Genus lies within the competence of the skilled man.
(63) Following the cultivation step, (where the cells of the lactic acid bacterium of the invention have been cultivated to reach a desired biomass), the cells are then further incubated. In this incubation step, the -acetolactate produced by the cells and released into the growth medium, is slowly converted to diacetyl by chemical oxidation. The method of diacetyl production may thus include an incubation step prior to recovery of the diacetyl. Surprisingly, it has been observed that the rate of chemical oxidation of the -acetolactate (produced by the cells) to diacetyl can be enhanced during this incubation step, by the addition of metal ions, in the form of Fe.sup.2+, Fe.sup.3+ and Cu.sup.2+. Accordingly, in one embodiment one or more of the combined final concentration of these one or more metal ions is 5 mM. Preferably the metal ions is Fe.sup.3+ and/or Cu.sup.2+ in a combined final concentration of at least 6 mM, 8 mM, 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, 22 mM, 24 mM, 26 mM, 28 mM and 30 mM; more preferably between 15 mM and 30 mM.
(64) Diacetyl produced by the lactic acid bacterium of the invention, can be recovered from the growth medium; and where the diacetyl is an intracellular product, it can be recovered from cells of the micro-organism of the invention by permeabilization of cell membranes combined with extraction of the diacetyl, employing standard methods for extraction, as illustrated in the examples.
(65) Using the lactic acid bacterium of the invention, a yield of 0.87 mol mol.sup.1 of glucose, and a titer of 78 mM diacetyl was achieved. This productivity greatly exceeded that reported for other diacetyl producing microorganisms.
(66) IV Methods for Producing a Micro-organism of the Invention
(67) Deletion of endogenous genes in a host lactic acid bacterium to obtain a genetically modified lactic acid bacterium according to the invention can be achieved by a variety of methods, for example by transformation of the host cell with linear DNA fragments containing a locus for resistance to an antibiotic, or any other gene allowing for rapid phenotypic selection, flanked by sequences homologous to closely spaced regions on the cell chromosome on either side of the gene to be deleted, in combination with the immediate subsequent deletion or inactivation of the recA gene. By selecting for a double-crossover event between the homologous sequences, shown by the antibiotic resistance or other detectable phenotype, a chromosome disruption can be selected for which has effectively deleted an entire gene. Inactivation or deletion of the recA gene prevents recombination or incorporation of extrachromosomal elements from occurring, thereby resulting in a bacterial strain which is useful for screening for functional activity or production of genetically engineered proteins in the absence of specific contaminants.
(68) An alternative method for the deletion of genes in order to produce the lactic acid bacterium of the invention employs the non-replicating vector pCS1966. This method involves PCR-amplifying 800 base pairs (bp) dsDNA molecules, corresponding to DNA regions located upstream and downstream of the gene to be deleted, and inserting the amplified molecules into pCS1966 using restriction enzymes and T4 ligase. The recombinant plasmid is then introduced into L. lactis, where integration gave rise to erythromycin resistance. Subsequently, counter selection is performed in the presence of 5-fluoroorotate (5FO) where excision and loss of the plasmid gives rise to 5FO resistance. This method for the deletion of ldhX, ldhB, ldh, pta, adhE, butBA, aldB and noxE from L.lactis is described in further detail Example 1.
(69) The deletion of endogenous genes in a host lactic acid bacterium to obtain a genetically modified lactic acid bacterium according to the invention can also be achieved by the more traditional approach involving mutagenesis and screening/selection. For instance, LDH (lactate dehydrogenase) mutants can be screened out using solid medium containing 2,3,5-triphenyl tetrazolium following mutagenesis using for instance N-methyl-N-nitro-N-nitrosoguanidine (NTG) or UV radiation. Alternatively, after mutagenesis, low-lactic acid producing strains can be selected using a combination of bromide and bromate as described by Han et al., 2013. ALDB (-acetolactate decarboxylase) mutants can be obtained easily after mutagenesis, for instance using NTG, or grown in the medium containing an unbalanced concentration of leucine versus valine and isoleucine in the medium (Goupil et al., 1996). ADHE (ethanol dehydrogenase) mutants can be screened and selected for in the presence of various concentrations of acetaldehyde. A phosphotransacetylase (E.C.2.3.1.8) mutant, induced by mutagenesis (e.g. using NTG), can subsequently be selected by penicillin enrichment in defined medium lacking lipoic acid. Under these conditions acetyl-CoA, needed for fatty acid biosynthesis, can only be obtained from acetate. Those cells that are able to assimilate acetate will proliferate and will subsequently be killed by the presence of penicillin. A mutant, having an inactivated phosphotransacetylase, can then be isolated by screening the survivors. An NADH oxidase (E.C. 1.6.3.4) mutant, induced by mutagenesis (e.g. using NTG), can be selected for based on oxygen level sensitivity. Diacetyl reductase (E.C.1.1.1.303), D-acetoin reductase and butanediol dehydrogenase (E.C. 1.1.1.4) mutants can be selected for after UV or chemical mutagenesis. For example, a filter assay for detecting BDH activity is described by Zhang et al. (2013) where single colonies are picked onto sterile membrane filters and then the filters are stained deep blue by treatment with 2,6-dichlorophenolindophenol. The filters are then floated on a solution of 2,3-butanediol, where only the BDH positive strains turn white, allowing the selection of BDH deficient mutants.
(70) By combining the different approaches mentioned, a non-GMO diacetyl producer is readily obtainable. Accordingly, a genetically modified lactic acid bacterium of the invention is characterised by the deletion or knockout of endogenous genes in the lactic acid bacterium, where the functional deletion or functional knockout can also be obtained by means of induced mutagenesis and screening/selection.
(71) V A Method of Detecting Diacetyl Production
(72) Methods for detecting and quantifying diacetyl produced by a micro-organism of the invention include high performance liquid chromatography (HPLC) combined with Refractive Index detection to identify and quantify diacetyl (as describe by Benson et al., 1996), relative to a standard, as described and illustrated in the examples.
(73) VI Use of the Genetically Modified Micro-organism of the Invention for Production of a Food Product
(74) Fermented food products rely on diacetyl produced during fermentation for their typical butter flavor and aroma; and the absence of this flavor reduces consumer appeal. Lactococci, typically used in the manufacture of milk products, can reduce diacetyl to acetoin and 2,3-butandiol, even at low temperatures, and thereby reduce product quality. Use of the genetically modified micro-organism of the invention overcomes this problem, since the micro-organism produces the desired diacetyl during fermentation in the production of the milk product; while the product can subsequently be safely stored without risk of a reduction of diacetyl content. Accordingly, the present invention encompasses both the production of a fermented food product using the genetically modified micro-organism of the invention; and a fermented milk product comprising the genetically modified micro-organism of the invention. Examples of fermented food products include: cottage cheese, yogurt, sour cream, and kefir.
EXAMPLES
Example 1
Genetic Modification of a Lactococcus lactis Strain for Production of Diacetyl
(75) The genetic modifications required to produce a Lactococcus lactis strain that is capable of producing diacetyl from glucose and to efficiently direct the flux towards this compound include the inactivation of all alternative product pathways, as described below.
(76) 1.1 Host Strains and Plasmids:
(77) The plasmid-free strain Lactococcus lactis subsp. cremoris MG1363 (Gasson, 1983) or derivatives thereof were used as the parent strain for the genetic construction of a strain capable of producing diacetyl. E. coli strain ABLE-C (E. coli C lac(LacZ-)[Kanr McrA- McrCB- McrF- Mrr- HsdR (rk- mk-)][F proAB lacIqZM15 Tn10(Tetr)]) (Stratagene) was used for cloning purposes. The plasmid pCS1966 (Solem et al., 2008), was used for the purpose of deleting various genes in L. lactis.
(78) 1.2 DNA Techniques:
(79) All manipulations were performed according to Sambrook et al., 1989. PCR primers used can be seen in TABLE 1. PfuX7 polymerase (Nrholm, 2010) was used for PCR applications. Chromosomal DNA from L. lactis was isolated using the method described for E. coli with the modification that cells were treated with 20 g of lysozyme per ml for 2 hours before lysis. Cells of E. coli were transformed using electroporation. Cells of L. lactis were made electro-competent by growth in GM17 medium (Terzaghi et al., 1975) containing 1% glycine and transformed by electroporation as previously described by Holo and Nes (1989). The plasmid vector pCS1966 (Solem et al., 2008) was used for deleting genes in L. lactis. Plasmids employed for deleting chromosomal genes were prepared by PCR amplifying approximately 800 base pairs (bp) regions upstream and downstream of the L. lactis chromosomal region to be deleted using the PCR primers and chromosomal DNA isolated from L. lactis. The primers used for amplifying the upstream and downstream regions are indicated in TABLE 1 as geneX ups. and geneX dwn. The amplified fragments and the plasmid, pCS1966, were then digested with the respective restriction enzymes indicated in the primer table, prior to inserting the fragment into the plasmid. The resulting plasmids were transformed into the parent strain individually and gene deletion was performed as described by Solem C, et al. (2008). Specifically, the plasmids were transformed into the strains via electroporation, and the strains comprising the plasmids integrated into the chromosome were selected for on GM17 agar plates (Difco Cat. No. 218571: M17 broth from Becton, Dickinson and Company) supplemented with glucose and erythromycin. Afterwards, the transformants were purified and plated on SA glucose plates supplemented with 5-fluoroorotate, thereby selecting for strains in which the plasmid had been lost by homologous recombination. The successful deletions were verified by PCR (Solem et al., 2008).
(80) TABLE-US-00001 TABLE1 Primers Primer name Primeruse Primersequence(5.fwdarw.3) 43(T3) Verifyinsert AATTAACCCTCACTAAAGGG[SEQIDNO:137] inpCS1966 603 Verifyinsert ATCAACCTTTGATACAAGGTTG[SEQIDNO:138] inpCS1966 710 SP-ldh,XbaI CTAGTCTAGANNNNNAGTTTATTCTTGACANNNNNNNNNNNN NNTGRTATAATNNNNAAGTAATAAAATATTCGGAGGAATTTT GAAATGGCTGATAAACAACGTAAG[SEQIDNO:139] 768 ldhBups., AATTCCTGCAGCATATTAAATAATGAACAAGTCATTC[SEQID PstI NO:140] 769 ldhBups., TAGTGGATCCTGGTAAATCCAAACACAACAAC[SEQIDNO: BamHI 141] 770 ldhBdwn., AATTCCTGCAGTAATTTCCAGCTCTTACAATAAC[SEQIDNO: PstI 142] 771 ldhBdwn., GACCTCGAGTCAGAAACTTTCTTTACCAGAG[SEQIDNO:143] XhoI 772 pCS1966, GCGGGGATCCACTAGTTCTAG[SEQIDNO:144] BamHI 773 pCS1966, ATACCGTCGACCTCGAG[SEQIDNO:145] XhoI 774 ldhXups., TAGTGGATCCCTGTTTCAGGTCTTGGATAG[SEQIDNO:146] BamHI 775 ldhXups., CCGATGAATTCTCATTAGCACGTTTAACAAGAG[SEQIDNO: EcoRI 147] 776 ldhXdwn., CCGATGAATTCATCAGCGTAGTCTGCTGC[SEQIDNO:148] EcoRI 777 ldhXdwn., CGGGGTACCATTTAATCCTAAAGTCGTTATTAC[SEQIDNO: KpnI 149] 785 ldhups., CCGATGAATTCTTAAGTCAAGACAACGAGGTC[SEQIDNO: EcoRI 150] 786 ldhdwn, CCGATGAATTCGACCTTGTTGAAAAAAATCTTC[SEQIDNO: EcoRI 151] 787 ldhups., TAGTGGATCCGTACAATGGCTACTGTTAAC[SEQIDNO:152] BamHI 788 ldhdwn., GACCTCGAGGATGAACAGACTTTTTTATTATAG[SEQIDNO: XhoI 153] 789 Verifyldh AAAACCAGGTGAAACTCGTC[SEQIDNO:154] deletion 791 adhBrev, TCGGACTGCAGTTAAAATGCTGATAAAAACAATTCTTC[SEQ PstI IDNO:155] 827 pCS1966, ATACCGTCGACCTCGAG[SEQIDNO:156] BamHI 828 pCS1966, CGATAAGCTTGATATCGAATTC[SEQIDNO:157] XhoI 830 adhBfwd, CCGATGAATTCTATAAGGAGAATTAGAATGGCAAGTAGTACA EcoRI TTTTATATTC[SEQIDNO:158] 878 ptaups., ATCCCTCGGTTACAAGTTTCU[SEQIDNO:159] USER 879 ptadwn., AGAAACTTGTAACCGAGGGAUAATAATAGATTGAAATTCTGT USER CAG[SEQIDNO:160] 880 ptaups., ATTCGATATCAAGCTTATCGAUCAAAAATTGTGGTAGAATATA USER TAG[SEQIDNO:161] 881 ptadwn., AGGTCGACGGTATCGATAAUCCTAGTTCAATTGATGTGAC USER [SEQIDNO:162] 882 pCS1966, ATCGATAAGCTTGATATCGAAU[SEQIDNO:163] USER 883 pCS1966, ATTATCGATACCGTCGACCU[SEQIDNO:164] USER 887 noxEups, ATTCGATATCAAGCTTATCGAUATTTAAAAATGATTGCAACAT USER ATAAC[SEQIDNO:165] 888 noxEups, ATAGGTCTCCTTTAAATGTAAAAU[SEQIDNO:166] USER 889 noxEdwn, ATTTTACATTTAAAGGAGACCTAUTAGAAATCTATCTGCTTGA USER TAG[SEQIDNO:167] 890 noxEdwn, AGGTCGACGGTATCGATAACGUCTTCACCGTCCATTTTGAC USER [SEQIDNO:168] 891 pTD6,USER ACAGATTAAAGGTTGACCAGTAU[SEQIDNO:169] 892 pTD6,USER ACCAATTCTGTGTTGCGCAU[SEQIDNO:170] 893 SP-dar-bdh, ATGCGCAACACAGAATTGGUGGCCNNNNNAGTTTATTCTTGA fwd. CANNNNNNNNNNNNNNTGRTATAATNNNNAAGTAATAAAAT ATTCGGAGGAAT[SEQIDNO:171] 894 adhBrev., ATACTGGTCAACCTTTAATCTGUTTAAAATGCTGATAAAAACA USER ATTCTT[SEQIDNO:172] 920 pCS1966, ATAAGCTUGATATCGAATTCCT[SEQIDNO:173] USER 921 pCS1966, ATTCCCTTUAGTGAGGGTTAAT[SEQIDNO:174] USER 926 ldhrev, TCGACCTCGAGTTTTTTATTTTTAGTTTTTAACTGCAG[SEQID XhoI NO:175] 927 adhEups., ATGTGTACGUTCTCCTTTGTG[SEQIDNO:176] USER 928 adhEdwn., ACGTACACAUATTATAGTATTTGGAACCGAAC[SEQIDNO: USER 177] 929 adhEups., AAGCTTAUGGTCGTCTTGTTACTTGTG[SEQIDNO:178] USER 930 adhEdwn., AAAGGGAAUTCTGCCGGAGCTATATATG[SEQIDNO:179] USER 975 dar-bdhrev. TTAATTATACAACATTCCTCCATC[SEQIDNO:180] 976 butBAups., AATTCCTGCAGATCTATACCTACTTGACCAGC[SEQIDNO:181] PstI 977 butBAups., TAGTGGATCCGAGTATTCGCAAACCTTCAG[SEQIDNO:182] BamHI 978 butBAdwn., AATTCCTGCAGAATAAATGAATGAGGTAAGGTCTA[SEQID PstI NO:183] 979 butBAdwn., GACCTCGAGTTTAAGAGATAAAAGGTTAATTGTG[SEQIDNO: XhoI 184] 991 gusA GAATCGGTACCAATAAAATATTCGGAGGAATTTTGAAATGTTA MG1655 CGTCCTGTAGAAAC[SEQIDNO:185] 992 gusA GGACCGTACGTTAAAAAATAAAAAAGAACCCACTCGGGTTCT MG1655 TTTTTTTATTGTTTGCCTCCCTGCTG[SEQIDNO:186] 1057 aldBups., TAGTGGATCCCTTAATTGCTGGAATCACTG[SEQIDNO:187] BamHI 1058 aldBups., AATTCCTGCAGATGATATTTCTCTTTTCTATCTCA[SEQIDNO: PstI 188] 1059 aldBdwn., AATTCCTGCAGAATTGCTTAAATTTCTTTAGCTAC[SEQIDNO: PstI 189] 1060 aldBdwn., TCGACCTCGAGTTAGACGCTCGGGATAAAG[SEQIDNO:190] XhoI 1112 pCI372 GCAACAACGTGCGCAAAC[SEQIDNO:191] 1113 pCI372 CTGCAGGTCGACTCTAG[SEQIDNO:192] 1117 aldBfwd. AATATTTTAGGACCCAATGATG[SEQIDNO:193] 1119 aldBrev CGAGCTGGAAAGCTTTTATC[SEQIDNO:194] 1130 SP-ldhAE. CTAGTCTAGAGCNNAGTTTATTCTTGACANNNNNNNNNN coli,XbaI NNNNTGRTATAATNNNNAAGTAATAAAATATTCGGAGGAATT TTGAAATGAAACTCGCCGTTTATAG[SEQIDNO:195] 1131 ldhArev, TCGACCTCGAGAAGAATAGAGGATGAAAGGTC[SEQIDNO: XhoI 196]
(81) 1.3 Deleting Genes from the Lactococcus lactis subsp. cremoris
(82) The following genes were deleted from the Lactococcus lactis subsp. cremoris parent strain ldhX, ldhB, ldh, pta, adhE, butBA, aldB and noxE. The genes were deleted using gene deletion plasmids derived from pCS1966 designated as: pCS4026 (ldhX), pCS4020 (ldhB), pCS4104 (ldh), pCS4230 (pta), pCS4273 (adhE), pCS4491 (butBA), pCS4495 (aldB) and pCS4256 (noxE), constructed as described above (Example 1.2).
(83) Deletion of the genes from the Lactococcus lactis subsp. cremoris parent strain was verified by PCR amplification of the respective gene using primers 774/777 (ldhX), 769/771 (ldhB), 788/789 (ldh), 880/881(pta), 929/930 (adhE), 977/979 (butBA), 1117/1119 (aldB), 887/890 (noxE).
(84) Strains and Plasmids
(85) TABLE-US-00002 Designation Genotype or description Reference L. lactis strains CS4363 MG1363 .sup.3ldh pta adhE Solem et al., 2013 CS4311 MG1363 .sup.3ldh pta adhE pCS4268 This work CS4502 *MG1363 .sup.3ldh pta adhE butBA This work pCS4268 CS4525 *MG1363 .sup.3ldh pta adhE butBA aldB This work pCS4268 CS4554 MG1363 ldhX ldhB pta adhE butBA This work aldB noxE pCS4268 CS4562 MG1363 .sup.3ldh adhE butBA aldB noxE This work pCS4564 CS4615 MG1363 .sup.3ldh pta adhE butBA aldB This work noxE pCS4564 CS4634 MG1363 pCS4634 (pCI372::SP-budC-bdh) This work CS4701 MG1363 .sup.3ldh pta adhE butBA aldB This work noxE pCS4634 CS4616m MG1363 .sup.3ldh pta adhE butBA aldB This work noxE CS4616m2 MG1363 .sup.3ldh pta adhE butBA aldB This work noxE pLP712 Plasmids pG.sup.+host8 E. coli/L. lactis shuttle vector, Tet.sup.R, Maguin et thermosensitive replicon al., 1996 pCS4268 pG.sup.+host8::SP-ldh (L. lactis) This work pCS4564 pG.sup.+host8::SP-ldhA (E. coli) This work pCI372 E. coli/L. lactis shuttle vector, Cam.sup.R Hayes et al., 1990 pCS4518 pCI372::gusA This work pCS4634 pCS4518::SP-budC-bdh This work pLP712 The lactose plasmid, isolated from NCDO712 Wegmann et al., 2012 *Indicates that the chromosomal ldh may have reverted to wild-type by recombination with pCS4268; 3ldh = ldh ldhX ldhB; SP signifies Synthetic Promoter.
(86) The strain containing the three lactate dehydrogenase deletions (ldh, ldhB, ldhX) was named CS4099 or MG13633ldh. CS4363 was derived from CS4099, by additionally the deleting a phosphotransacetylase gene, pta and adhE. Strains deleted for the three ldh genes had poor growth properties (CS4363); so to facilitate growth of CS4363 and its subsequent genetic modification, the CS4363 strain was transformed with a plasmid (pCS4268) with a thermosensitive replicon carrying L. lactis ldh expressed from a synthetic promoter (SP), to give strain CS4311. The plasmid was prepared as follows: an SP-ldh fragment was amplified from L. lactis using primers 710/926, was digested with XbaI/XhoI and inserted into pG.sup.+host8 plasmid (Maguin et al., 1996) digested with the same enzymes, and the ligated plasmid was then introduced into the CS4363 strain to give rise to CS4311. Strain CS4502 was derived from strain CS4311 by deletion of butBA, and CS4525 was derived from strain CS4502 by deletion of deleted aldB. CS4554 was derived from strain CS4525 by deletion of noxE, but in this strain ldh was found to have reverted to wild-type (ldh) due to a recombination event between the deleted ldh locus and the intact ldh gene on the pG.sup.+host8 plasmid (pCS4564). CS4562 was derived from strain CS4554, lacking the pG+host8 plasmid, but substituted by another pG+host8 plasmid carrying an E. coli ldhA (pCS4564). The plasmid pCS4564 was constructed in the following manner: SP-ldhA was amplified from E. coli using 1130/1131, digested with XhoI/XbaI and inserted into pG+host8 digested with the same enzymes. The chromosomal ldh was then deleted from CS4562 thus giving rise to CS4615 (MG1363 ldh ldhX ldhB pta adhE butBA aldB noxE pCS4564); whereafter pCS4564 was lost by incubation at 36 C. in the presence of 5 g/ml hemin to yield CS4616m.
(87) 1.4 Cultivation of the Genetically Engineered Strain Lactic Acid Bacterium
(88) L. lactis were grown aerobically at 30 C. in rich M17 broth (Terzaghi et al., 1975) supplemented with 2% (w/v) glucose and different concentrations of hemin in shaken 250 ml conical flasks. When required, antibiotics were added in the following concentrations: erythromycin: 200 g/ml for E. coli and 5 g/ml for L. lactis, tetracycline: 8 g/ml for E. coli and 5 g/ml for L. lactis, chloramphenicol: 20 g/ml for E. coli and 5 g/ml for L. lactis.
(89) 1.5 Measurement of NADH and NAD.sup.+ in the Genetically Engineered Strain Lactic Acid Bacterium
(90) L. lactis cell culture harvested in the mid-exponential growth phase (OD.sub.600 nm=0.6) was frozen in liquid nitrogen and stored at 20 C. Extraction and quantification of NADH and NAD.sup.+ were performed using the kit NAD.sup.+/NADH-Glo Assay (Promega) according to the instructions. Intracellular NADH and NAD.sup.+ concentrations were estimated by assuming that 1 g (dry weight) of cells corresponded to 1.67 ml of intracellular volume and the cell density of 1 OD.sub.600 nm corresponds to 0.35 g/I dry cell mass.
(91) 1.6 Measurement of Fermentation Products of the Genetically Engineered Strain Lactic Acid Bacterium
(92) Glucose, lactate, and ethanol were detected and quantitated using the Ultimate 3000 high-pressure liquid chromatography system (Dionex, Sunnyvale, USA) equipped with a Aminex HPX-87H column (Bio-Rad, Hercules, USA) and a Shodex RI-101 detector (Showa Denko K.K., Tokyo, Japan). Pyruvate was detected and quantitated using the DAD-3000 diode array detector (Dionex, Sunnyvale, USA). The column oven temperature was set at 60 C. with a mobile phase of 5 mM H.sub.2SO.sub.4, and the flow rate was 0.5 ml/min. Acetoin and -acetolactate were measured colorimetrically based on Westerfeld et al. (1945). Diacetyl concentration was determined as described by Benson et al (1996).
(93) 1.7 Properties of the Genetically Engineered Strain Lactic Acid Bacterium
(94) A strain of Lactococcus lactis subsp. cremoris, from which the lactate dehydrogenases (ldh, ldhB, ldhX), phosphotransacetylase (pta), and alcohol dehydrogenase (adhE) have been inactivated by deletion of their genes, results in an efficient redirection of metabolic flux towards diacetyl. However, the main fermentation product of this strain was R-acetoin, and additionally it was only able to grow aerobically. When the chromosomally encoded LDH and PTA genes in the strain were also inactivated, this led to a large decline in specific growth rate and reduced the transformation efficiency needed for the succeeding molecular manipulations. Transformation with of a plasmid having a thermosensitive replicon expressing LDH (pCS4268 or pCS4564) into the strain allowed for its growth under anaerobic conditions and for the further engineering of a strain for homo-diacetyl production by deletion of the native aldB gene and native butBA operon (
(95) In strains deleted for native aldB gene, the formation of diacetyl from acetolactate is dependent on non-enzymatic oxidative decarboxylation, which requires aerobic growth conditions. However, it was not possible to produce L. lactis strains for diacetyl production having the genotype (MG1363 3ldh pta adhE butBA aldB). Under aerobic conditions a large portion of NADH is consumed in NAD.sup.+ regeneration and O.sub.2 elimination due to NADH oxidase activity in L. lactis. The main source of NADH oxidase activity in L. lactis is attributed to NoxE (>95%), which is a water-forming NADH oxidase. Likely, NoxE activity is insufficient to provide the cofactor balance and oxidative stress resistance required in a L. lactis strain for diacetyl production. Accordingly, in order to produce a L. lactis strain for diacetyl production, the NoxE gene was deleted; and instead methods for activating respiration were explored. The L. lactis respiratory chain in the cell membrane includes electron donor (NoxAB), electron transfer (menaquinone) and electron acceptor (Cyt bd). Hemin, which is not synthesized in L. lactis, is an essential cofactor for Cyt bd activity, and could be used to activate respiration in the L. lactis strains engineered for diacetyl production. An external supply of hemin would contribute to NAD+/NADH balance, proton translocation, high biomass yield and low oxidative stress. Thus the viable L. lactis strain CS4616m (MG1363 3ldh pta adhE butBA aldB noxE) was metabolic engineered for diacetyl production under aerobic conditions, where hemin-activated respiration is coupled to glycolysis within the cell (
(96) 1.8 Introducing Genes Encoding the Lactose Catabolism Pathway Into Lactococcus lactis subsp. cremoris Strain CS4616m
(97) The wild type strain L. lactis MG1363, and its derivatives described herein, are plasmid-free strains that cannot utilize lactose as a carbon source. The Lactococcus plasmid, pLP712 (55.395 kbp), comprises genes encoding the entire lactose catabolism pathway (Wegmann et al., 2012). The lactose-metabolism plasmid pLP712 (55,395 bp) was extracted from the dairy isolate NCDO712 based on the method of Andersen (1983); and then transformed into L. lactis strain CS4616m to give strain CS4616m2.
(98) The lactose catabolism pathway genes located on the pLP712 plasmid (
(99) 1. the lacAB genes encoding two subunit polypeptides that together have galactose-6-phosphate isomerase activity (EC 5.3.1.26); wherein the first subunit polypeptide has an amino acid sequence of SEQ ID NO: 202 encoded by the L. lactis lacA gene; and the second subunit polypeptide has an amino acid sequence of SEQ ID NO: 204 encoded by the L. lactis lacB gene;
(100) 2. the lacC gene encoding a polypeptide having D-tagatose-6-phosphate kinase activity (EC 2.7.1.114); wherein the polypeptide has an amino acid sequence of SEQ ID NO: 206);
(101) 3. the lacD gene encoding a polypeptide having tagatose 1,6-diphosphate aldolase activity (EC 4.1.2.40); wherein the polypeptide has an amino acid of SEQ ID NO: 208;
(102) 4. the lacEF genes encoding a two polypeptide components together having lactose-specific phosphotransferase system (PTS) activity (EC 2.7.1.69); wherein the first polypeptide component is a phosphotransferase system EIICB component having an amino acid sequence of SEQ ID NO: 210, encoded by the L. lactis lacE gene; and the second polypeptide component is a phosphotransferase system EIIA component having an amino acid sequence of SEQ ID NO: 212, encoded by the L. lactis lacF gene; and
(103) 5. the lacG gene encoding a polypeptide having phospho--D-galactosidase activity (EC 3.2.1.85); wherein the polypeptide has the amino acid sequence of the phospho--D-galactosidase of SEQ ID NO: 214; and
(104) 6. the lacR gene encoding a lactose transport regulator of SEQ ID NO: 216
Example 2
Respiration-dependent Growth of a Genetically Engineered Lactococcus lactis Strain Adapted for Production of Diacetyl
(105) The genetically engineered L. lactis strain CS4616m was unable to grow anaerobically, implying that it had lost the capacity for fermentation. However, growth of the L. lactis strain was restored under aerobic conditions when hemin was present (
(106) The measured intracellular NAD.sup.+ concentration of the L. lactis strain increased from 0.25 mM when grown in the presence of 0.3 g/ml hemin to 0.47 mM in the presence of 1 g/ml hemin, while the NADH/NAD+ ratio sharply decreased from 0.07 to 0.03 (
Example 3
Growth and Diacetyl Production of a Genetically Engineered Lactococcus lactis Strain
(107) 3.1 Diacetyl Production from Glucose in the Presence of Hemin
(108) When L. lactis strain CS4616m was grown in the presence of 1 g/ml hemin the production of diacetyl increased in response to the concentration of glucose in the growth medium (
(109) TABLE-US-00003 TABLE 2 Fermentation products of L. lactis strain CS4616m grown with 1 g/ml hemin. Yield Yield Initial Glu Pyruvate Lactate Acetolactate Acetoin Diacetyl (mol mol.sup.1).sup.1 (mol mol.sup.1).sup.2 25 0.6 ND 17 ND 5.3 0.89 0.21 50 1.5 ND 31 3 10 0.88 0.2 75 3.7 ND 42 5.3 16 0.85 0.21 108 5.7 ND 65 7.1 25 0.89 0.23 Fermentation product (mM) profiles detected after 15 h fermentation. .sup.1yield for the total acetolactate, diacetyl and acetoin. .sup.2yield for diacetyl.
(110) Carbon flux in L. lactis strain CS4616m is effectively redirected to the acetolactate-formation pathway when the cells are grown on glucose supplemented with hemin. The measured diacetyl yield was around 0.2 mol mol.sup.1, while acetolactate was observed to accumulate (Table 2). Small amounts of acetoin were detected, which may be attributed to the activity of an ALDC (acetolactate decarboxylase C), or to chemical non-oxidative decarboxylation.
(111) When grown with 108 mM glucose in the presence of 1 g/ml hemin, the L. lactis strain CS4616m accumulated acetolactate (
(112) 3.2 Metal Ions Enhance Diacetyl Production from Glucose in the Presence of Hemin
(113) The addition of the metal ions Fe.sup.3+, Fe.sup.2+ and Cu.sup.2+ were found to significantly increase diacetyl production by cells of the L. lactis strain CS4616m. When the cells entered the stationary phase, different concentrations of these metal catalysts were added to the cultivation medium. As shown in
(114) The observed high yields of diacetyl by cells of the L. lactis strain CS4616m are achieved by the addition of metal ions to the cells after they have reached the desired cell biomass. Addition of metal ions during the growth phase of a L. lactis strain CS4616m cell culture was observed to inhibit its growth. Thus Cu.sup.2+ totally abolished cell growth; while Fe.sup.3+ and Cu.sup.2+ (10 or 20 mM) when present in the growth medium from the beginning of the growth phase inhibited growth. The final biomass of a culture grown in the presence of 10 mM Fe.sup.3+ was only 3.3 (OD.sub.600 nm) (
Example 4
Development of a Lactose Medium for Production of Diacetyl from Lactose by Genetically Modified Lactococcus lactis Strain of the Invention
(115) Waste stream residual whey permeate (RWP) is the permeate mother liquor obtained after extracting lactose from whey permeate. The composition of the RWP, which was supplied by Arla Foods Ingredients Group P/S was determined and shown in Table 4. The sugar components of a filtered sample of RWP were determined as described in example 1.8, and the amino acid composition was determined by the steps of hydrolysis of the filtered sample with 6 M HC1, derivatization of the amino acids using o-phthaldialdehyde, and analysis of the derivatives using ion exchange chromatography as described by Barkholt et al., (1989).
(116) TABLE-US-00004 TABLE 4 The composition of residual whey permeate.sup.a Composition Concentration Lactose 150 gL Galactose 3 gL Aspartate 0.252 mM (mmol/L) Threonine 0.076 mM Serine 0.088 mM Glutamate 1.464 mM Proline 0.384 mM Glycine 0.904 mM Alanine 0.24 mM Cysteine 0.096 mM Valine 0.072 mM Methionine 0.124 mM isoleucine 0.04 mM Leucine 0.092 mM Histidine 0.208 mM Lysine 0.304 mM Arginine 0.096 mM .sup.aResidual whey permeate is a concentrate of the residue remaining after lactose extraction from whey permeate.
(117) In view of the relatively low amino acid content of RWP, a nitrogen source should be added to the RWP, to support growth and diacetyl production.
(118) Corn steep liquor (CSL) is a cheap source of complex nitrogen. CSL was purchased from Sigma-Aldrich (St. Louis, Mo.) with a 40-60% solids content. The RWP medium was supplemented with either non-treated CSL, or acid treated CSL in order to enhance the availability of the amino nitrogen content of the CSL. Samples of CSL were subjected to various degrees of acid hydrolysis. The acid hydrolysis was performed with very small amounts of sulfuric acid (0.05-0.5% concentrated H.sub.2SO.sub.4 added to CSL having a 20-30% w/v solids content). The following hydrolysis conditions were applied to produce corn steep liquor hydrolysates (CSLH). H1 condition: original CSL was diluted 2 times with water and then 50 l concentrated sulfuric acid was mixed with 100 ml diluted CSL. The mixture was kept at 121 C. for 15 mins and subsequently the pH was adjusted to 6.8-7.1 with the addition of 10 M NaOH solution.
(119) Analysis of the free amino acid composition of CSL revealed that hydrolysis of corn steep liquor increases the free amino acid content of CSL by circa 2 fold in comparison with untreated corn steep liquor.
(120) TABLE-US-00005 TABLE 5 Free amino acid composition of CSL before and after hydrolysis Amino acids CSL Hydrolyzed CSL (H1) Unit (mM) 25% (wv) 25% (wv) Aspartate 1.8 3.6 Glutamate 0.9 2 Asparagine 1.4 3 Glutamine 0.8 2.1 Histidine 6.1 11 Arginine 4.5 10.5 Alanine 2.1 5 Tyrosine 0.8 2.1 Cysteine 3.7 5.7 Valine 2.1 4.8 Isoleucine 1.8 3.6 Leucine 0.8 2.5 Methionine 0.1 1.2
Example 5
A Method for Producing Diacetyl from Lactose Using the Genetically Engineered Lactococcus lactis Strain of the Invention
(121) Fermentation was performed using CS4616m2 (MG1363 .sup.3ldh pta adhE butBA aldB noxE pLP712) in diluted residual whey permeate (RWP) medium with 1 g/ml hemin supplemented with different nitrogen sources. As shown in table 3, the combination of diluted RWP and 2% (w/v) yeast extract stimulated the highest diacetyl production (57 mM or 4.9 g/L). The hydrolyzed corn steep liquor is also a good nitrogen source (Hydrolysis condition H1) for supporting cell growth and diacetyl production.
(122) TABLE-US-00006 TABLE 3 Diacetyl production from residual whey permeate medium Diacetyl Strain Medium OD.sub.600 (mM) CS4616m2 Diluted RWP (including 20 gL 3.7 39 lactose) and 1% (wv) yeast extract and 1 g/ml hemin and 30 mM Fe.sup.3+ a CS4616m2 Diluted RWP (including 20 gL 5.7 57 lactose) and 2% (wv) yeast extract and 1 g/ml hemin and 30 mM Fe.sup.3+ a CS4616m2 Diluted RWP (including 20 gL 3.5 41 lactose) and 5% (wv) hydrolyzed corn steep liquor.sup.b and 1 g/ml hemin and 30 mM Fe.sup.3+ .sup.a .sup.a 30 mM Fe.sup.3+ was added into the medium after 20 h, at which time the culture had entered the stationary phase. .sup.bHydrolysis condition: the original CSL (about 50% (wv) solid content) was diluted 2 times with water and then 50 l concentrated sulfuric acid was mixed with 100 ml diluted CSL. The mixture was kept at 121 C. for 15 mins and subsequently pH was adjusted to 6.8-7.1 with the addition of 10M NaOH solution.
REFERENCES
(123) Benson, K. H., Godon, J. J., Renault, P., Griffin, H. G. & Gasson, M. J. Effect of ilvBN-encoded -acetolactate synthase expression on diacetyl production in Lactococcus lactis. Appl. Microbiol. Biotechnol. 45, 107-111 (1996).
(124) Holo, H. & Nes, I. High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl. Environ. Microbiol. 55, 3119-3123 (1989).
(125) Nrholm, M. H. H. A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnol. 10, 21 (2010).
(126) Sambrook, J. & Russell, D. Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, 2001).
(127) Solem, C., Defoor, E., Jensen, P. R. & Martinussen, J. Plasmid pCS1966, a new selection/counterselection tool for lactic acid bacterium strain construction based on the oroP gene, encoding an orotate transporter from Lactococcus lactis. Appl. Environ. Microbiol. 74, 4772-4775 (2008).
(128) Terzaghi, B. E. & Sandine, W. E. Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 29, 807-13 (1975).
(129) Westerfeld, W. W. A colorimetric determination of paraldehyde. J. Lab. Clin. Med. 30, 1076 (1945).
(130) Han, S.-H., Lee, J.-E., Park, K., Park, Y.-C. Production of 2,3-butanediol by a low-acid producing Klebsiella oxytoca NBRF4. New Biotechnology 30,166-172 (2013).
(131) Goupil, N., Corthier, G., Ehrlich, S. D., Renault, P. Imbalance of leucine flux in Lactococcus lactis and its use for the isolation of diacetyl-overproducing strains. Appl. Environ. Microbiol. 62, 2636-2640 (1996).
(132) Zhang X, et al. Mutation breeding of acetoin high producing Bacillus subtilis blocked in 2,3-butanediol dehydrogenase. World. J. Mirobiolol. Biotechnol. 29, 1783-9 (2013).