METHOD FOR CONSTRUCTING LACTIC ACID-PRODUCING STRAINS, LACTIC ACID-PRODUCING STRAINS, AND USE THEREOF

Abstract

Provided are a method for constructing a lactic acid-producing strain, a lactic acid-producing strain and use thereof. The method for constructing lactic acid-producing strains is characterized by genetically engineering a starting strain to increase lactic acid production, wherein the engineering includes: 1) introducing a lactic acid synthesis pathway; 2) optimizing the lactic acid synthesis pathway; and 3) inhibiting by-product synthesis pathways.

Claims

1. A method for constructing a lactic acid-producing strain, characterized by genetically engineering a starting strain to increase lactic acid production, wherein the engineering comprises: 1) introducing a lactic acid synthesis pathway; 2) optimizing the lactic acid synthesis pathway; and 3) inhibiting by-product synthesis pathways.

2. The method according to claim 1, wherein the starting strain is a lactic acid- and/or pyruvic acid-producing strain; preferably, the starting strain is able to produce lactic acid and/or pyruvic acid at a temperature of 37 C. to 70 C.

3. The method according to claim 2, wherein the starting strain includes microorganisms of genus Bacillus and Geobacillus.

4. The method according to claim 3, wherein the starting strain includes Bacillus lichenformis, Bacillus coagulans, Bacillus smithii, Bacillus pallidus and Geobacillus thermoglucosidasius.

5. The method according to claim 1, wherein introducing the lactic acid synthesis pathway comprises introducing an L-lactate dehydrogenase gene L-ldh or a D-lactate dehydrogenase gene D-ldh into the starting strain.

6. The method according to claim 1, wherein optimizing the lactic acid synthesis pathway comprises introducing, into the starting strain, coding sequences of one or more genes selected from: a lactate dehydrogenase gene ldh, a 6-phosphofructokinase gene pfk and a pyruvate kinase gene pyk, wherein the lactate dehydrogenase gene ldh is an L-lactate dehydrogenase gene L-ldh or a D-lactate dehydrogenase gene D-ldh.

7. The method according to claim 5, wherein the introduction into the starting strain comprises: integration into a genome of the starting strain or expression in a plasmid form in the starting strain; preferably, the introduction comprises introducing single-copy or multi-copy the coding sequences of the genes; preferably, the coding sequences of the genes are introduced as individual single-gene expression fragments or as tandem expression fragments of the coding sequences of the genes.

8. The method according to claim 1, wherein inhibiting the by-product synthesis pathways comprises knocking out or down one or more coding genes in the starting strain selected from: a lactate dehydrogenase gene ldh, a formate acetyltransferase gene pflB, an acetaldehyde dehydrogenase gene acdh, an aldehyde-alcohol dehydrogenase gene aadh and a phosphate acetyltransferase gene pta, wherein the lactate dehydrogenase gene ldh is an L-lactate dehydrogenase gene L-ldh or a D-lactate dehydrogenase gene D-ldh.

9. The method according to claim 1, wherein the lactic acid is L-lactic acid or D-lactic acid.

10. The method according to claim 5, wherein the D-lactate dehydrogenase gene ldh encodes an amino acid sequence as shown in SEQ ID No. 9 or SEQ ID No. 10, or encodes an amino acid sequence exhibiting 30% or higher identity to the amino acid sequence as shown in SEQ ID No. 9 or SEQ ID No. 10.

11. The method according to claim 5, wherein the L-lactate dehydrogenase gene L-ldh is derived from Bacillus coagulans and/or Geobacillus thermoglucosidasius, the D-lactate dehydrogenase gene D-ldh is derived from Bacillus lichenformis, the 6-phosphofructokinase gene pfk is derived from Bacillus coagulans, and the pyruvate kinase gene pyk is derived from Bacillus coagulans.

12. A lactic acid-producing strain, characterized in being constructed in accordance with the method according to claim 1.

13. The strain according to claim 12, wherein the strain is Geobacillus thermoglucosidasius with deposition Nos. CCTCC M 20221822, CCTCC M 20221823, CCTCC M 20221824 and CCTCC M 20221825.

14. Use of the strain according to claim 12 in lactic acid production.

15. The use of claim 14, wherein the lactic acid is L-lactic acid or D-lactic acid.

16. The use of claim 14, wherein the lactic acid has a titer of at least 80 g L.sup.1, and a yield of at least 80%; preferably, the lactic acid has a chiral purity of at least 99.04%.

17. The use of claim 14, wherein a method of the lactic acid production comprises steps of: 1) providing the lactic acid-producing strain according to claim 12; 2) culturing the strain at 37 C. to 70 C. for 6 hours to 15 hours, thereby providing a seed; and 3) subjecting the seed to fermentation culture at 37 C. to 70 C. in a presence of a carbon source, thereby obtaining the lactic acid.

18. The use of claim 17, wherein the carbon source is selected from one or more of glucose, xylose, sucrose, glycerol, arabinose and mannitol; preferably, the lactic acid is L-lactic acid or D-lactic acid.

19. The use of claim 17, wherein in step 3), an inoculum volume of the seed has an OD.sub.620 nm value of 0.2 to 0.8; preferably, an initial concentration of the carbon source is 40 g/L to 100 g/L; preferably, the fermentation culture is carried out at a pH value of 6.5 to 7.5 for a period of up to 50 hours; preferably, the fermentation culture is performed with agitation at a speed of 50 rpm to 150 rpm.

20. The use of claim 17, wherein the lactic acid has a titer of at least 80 g L.sup.1 and a yield of at least 80%; preferably, the lactic acid has a chiral purity of at least 99.04%.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0038] FIG. 1 shows a schematical diagram of a method for constructing an efficient lactic acid-producing strain and use thereof according to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Below, reference is made to accompanying drawings of the specification to introduce a few preferred embodiments of the present invention so that its techniques will become more apparent and more readily understood. The present invention can be embodied in various different forms of embodiment, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0040] It should be understood that the technical features described above and the technical features detailed below (including, but not limited to, the embodiments and examples) can be mutually combined in an arbitrary appropriate manner, thereby creating new or preferred embodiments, as long as there is no contradiction and the combined technical solutions can be smoothly implemented and solve the problems of the present invention. The arbitrary appropriate manner is such as to be able to achieve the technical solutions of the present invention and solve the problems of the present invention and be able to achieve corresponding technical effects.

[0041] In the present invention, the terms including, further, having, furthermore and the like only mean embodiments or examples with good effects or with certain degrees of particularity. It should be understood that they do not constitute a limitation on the scope of protection of the invention.

[0042] In the present invention, and/or means any one or any combination of the listed items.

[0043] Numerical ranges in the present invention, unless otherwise specifically stated, all include the two endpoints.

[0044] In the present invention, at least, unless otherwise specifically defined, in all cases, includes the stated number.

[0045] The present invention involves temperature control, allows constant temperatures, and also allows the existence of a certain temperature variation range.

[0046] Those skilled in the art should understand that L-lactic acid of the present invention can also be referred to as L(+)-lactic acid or (S)-lactic acid, and these terms are interchangeably used; similarly, D-lactic acid can also be referred to D()-lactic acid or (R)-lactic acid.

[0047] In the present invention, a yield of lactic acid refers to a ratio of the produced lactic acid (the unit is gram) and the consumed carbon source (the unit is gram, for example, glucose).

[0048] In the present invention, chiral purity of lactic acid refers to the percentage of a target product (L-lactic acid or D-lactic acid) in the total of the two species of chiral lactic acid. Specifically, chiral purity of lactic acid refers to the percentage of the area of a peak for a target product (L-lactic acid or D-lactic acid) in the total area of peaks for the two species of chiral lactic acid obtained by a method for detecting chiral purity of lactic acid using a high-performance liquid chromatography system.

[0049] In the present invention, high-temperature fermentation is used in contrast to medium temperature (means lower than 37 C.). High-temperature fermentation production herein, unless otherwise specifically defined, refers to fermentation production under a 37 C. to 70 C. high temperature condition. Temperatures for high-temperature fermentation include, but are not limited to, 50 C., 55 C., 56 C., 57 C., 58 C., 59 C., 60 C., 61 C., 62 C., 63 C., 64 C., 65 C. and 70 C. Temperature ranges for high-temperature fermentation include, but are not limited to, 40 C. to 70 C., 50 C. to 65 C., 55 C. to 65 C. and 60 C. to 65 C.

[0050] As used herein, the term starting strain refers to a strain that has not undergone any genetic modification described in the present invention. It may be a wild-type strain, and may also be a recombinant strain that has previously experienced one or some certain known modifications.

[0051] The term exogenous, as used herein to refer to genes, coding sequences, proteins, enzymes and the like, refers to substances that, when in a natural state, do not belong to a starting strain. For example, an exogenous gene refers to a gene introduced from an external source into the starting strain. The exogenous gene may be a gene already in the strain's genome, and may also be a gene not in its genome. For example, according to a requirement, from an external source, into the genome of the starting strain, a gene that is already in it is introduced to overexpress the gene.

[0052] As used herein, the term inhibit refers to causing a function of an object being inhibited, compared to before the inhabitation is carried out, to be completely lost or weakened.

[0053] As used herein, the term knockout or knockdown refers to, by means of genetic manipulations, causing a function of a selected gene to be completely lost or weakened. The genetic manipulations include those commonly used in the art, such as insertion, substitution or deletion of one or more nucleic acid fragments from the selected gene.

[0054] The nucleotide sequences involved in the present invention are as follows:

TABLE-US-00001 (D-ldhcodingsequence-Bacilluslicheniformis): SEQIDNo.1 ATGAAAGTAATTTTTTTTTCTATGCACCCGTATGAAGAGGAATTTCTGGGTC CGATTCTGCCGTCTGACTGGGACGTAGAAATGACCCCGGACTTTCTGGACG AAACCACCGTGGAAAAGGCTAAAGGTGCCCAGGTAGTAAGCCTGTTTGTT TCTGACAAAGCTGATGGTCCGGTACTGGAAGCGCTGCATTCTTACGGTGTG GGCCTGCTGGCCCTGCGTTCTGCTGGCTATGATCACATCGATATTGAGACCG CAAAACGCCTGGGTATCAAAGTAGTTAACGTGCCAGCCTATTCTCCGCACG CTATCGCTGACCATACTCTGGCTATCATGCTGGCTCTGATTCGTCGTCTGCA CCGTGCCCATGATAAAGTGCGCCTGGGTGATTTTGATCTGGATGGTCTGATG GGCTTTGATCTGAACGGCAAAGTTGCTGGTGTAATTGGTCTGGGTAAAATC GGTCGCCTGGTAGCTACCCGCCTGAAAGCGTTTGGTTGCAAAGTTCTGGGC TATGATCCATACATTCAGCCGGAAATCGTAGAAAACGTTGATCTGGATACCC TGATCACTCAGGCTGATATCATTTCTATTCATTGTCCGCTGACCCGTGAAAA CTTTCATATGTTTAACGAAGAGACTTTTAAGCGTATGAAACCGGGTGCTATT CTGGTTAACACCGCGCGTGGTGGTCTGATCGATACCAAGGCCCTGCTGGAG GCCCTGAAGTCTGGTAAACTGGGCGGCGCAGCCCTGGATGTGTATGAATAT GAACGTGGCCTGTTTTTTAAAAACCACCAAAAAGAAGGTATCAAAGACCC GTATCTGGCCCAGCTGCTGGGTCTGGCCAACGTAGTGCTGACCGGTCATCA GGCCTTTCTGACCCGTGAGGCTGTAAAAAACATCGAAGAAACTACCGTAG AAAACATTCTGGAATGGCAAAAGAACCCGCAGGCAAAGCTGAAAAACGA AATCTAA (D-ldh.sup.P101Qcodingsequence-Bacilluslicheniformis): SEQIDNo.2 ATGAAAGTAATTTTTTTTTCTATGCACCCGTATGAAGAGGAATTTCTGGGTC CGATTCTGCCGTCTGACTGGGACGTAGAAATGACCCCGGACTTTCTGGACG AAACCACCGTGGAAAAGGCTAAAGGTGCCCAGGTAGTAAGCCTGTTTGTT TCTGACAAAGCTGATGGTCCGGTACTGGAAGCGCTGCATTCTTACGGTGTG GGCCTGCTGGCCCTGCGTTCTGCTGGCTATGATCACATCGATATTGAGACCG CAAAACGCCTGGGTATCAAAGTAGTTAACGTGCCAGCCTATTCTCAGCACG CTATCGCTGACCATACTCTGGCTATCATGCTGGCTCTGATTCGTCGTCTGCA CCGTGCCCATGATAAAGTGCGCCTGGGTGATTTTGATCTGGATGGTCTGATG GGCTTTGATCTGAACGGCAAAGTTGCTGGTGTAATTGGTCTGGGTAAAATC GGTCGCCTGGTAGCTACCCGCCTGAAAGCGTTTGGTTGCAAAGTTCTGGGC TATGATCCATACATTCAGCCGGAAATCGTAGAAAACGTTGATCTGGATACCC TGATCACTCAGGCTGATATCATTTCTATTCATTGTCCGCTGACCCGTGAAAA CTTTCATATGTTTAACGAAGAGACTTTTAAGCGTATGAAACCGGGTGCTATT CTGGTTAACACCGCGCGTGGTGGTCTGATCGATACCAAGGCCCTGCTGGAG GCCCTGAAGTCTGGTAAACTGGGCGGCGCAGCCCTGGATGTGTATGAATAT GAACGTGGCCTGTTTTTTAAAAACCACCAAAAAGAAGGTATCAAAGACCC GTATCTGGCCCAGCTGCTGGGTCTGGCCAACGTAGTGCTGACCGGTCATCA GGCCTTTCTGACCCGTGAGGCTGTAAAAAACATCGAAGAAACTACCGTAG AAAACATTCTGGAATGGCAAAAGAACCCGCAGGCAAAGCTGAAAAACGA AATCTAA (codon-optimizedsequenceofSEQIDNo.2for Geobacillusthermoglucosidasius): SEQIDNo.3 ATGAAAGTAATTTTTTTTAGCATGCATCCGTATGAAGAAGAATTTTTAGGCC CGATTTTACCGTCGGATTGGGATGTAGAAATGACGCCGGATTTTTTAGATGA AACGACGGTGGAAAAAGCGAAAGGAGCGCAAGTAGTAAGCTTGTTTGTTT CGGATAAAGCGGATGGCCCGGTACTTGAAGCGCTTCATTCGTATGGAGTGG GCCTTTTGGCGCTTCGCAGCGCGGGCTATGATCATATTGATATTGAAACAGC GAAACGCCTGGGCATTAAAGTAGTTAATGTGCCGGCGTATTCGCAACATGC GATTGCGGATCATACATTAGCGATTATGCTTGCGCTTATTCGCCGCCTTCATC GCGCGCATGATAAAGTGCGCCTGGGAGATTTTGATCTTGATGGCCTTATGGG CTTTGATTTAAATGGCAAAGTTGCGGGCGTAATTGGCCTTGGCAAAATTGG CCGCCTGGTAGCGACACGCTTAAAAGCGTTTGGCTGCAAAGTTTTAGGCTA TGATCCGTATATTCAACCGGAAATTGTAGAAAATGTTGATTTGGATACACTTA TCACACAAGCGGATATCATTTCGATTCATTGTCCGCTTACGCGCGAAAATTT TCATATGTTTAATGAAGAAACATTTAAACGCATGAAACCGGGCGCGATTTTG GTTAACACGGCGCGCGGAGGCCTTATAGATACAAAAGCGTTGCTTGAAGCG TTAAAATCGGGAAAACTTGGCGGCGCGGCGCTTGATGTGTATGAATATGAA CGCGGCCTTTTTTTTAAAAACCATCAAAAAGAAGGCATTAAAGATCCGTAT CTTGCGCAACTTTTGGGCTTGGCGAATGTAGTGTTAACAGGCCATCAAGCG TTTCTTACGCGCGAAGCGGTAAAAAACATTGAAGAAACAACAGTAGAAAA TATTTTAGAATGGCAAAAAAATCCGCAAGCGAAACTTAAAAATGAAATCTG A (D-ldhP.sup.101Ncodingsequence-Bacilluslicheniformis): SEQIDNo.4 ATGAAAGTAATTTTTTTTTCTATGCACCCGTATGAAGAGGAATTTCTGGGTC CGATTCTGCCGTCTGACTGGGACGTAGAAATGACCCCGGACTTTCTGGACG AAACCACCGTGGAAAAGGCTAAAGGTGCCCAGGTAGTAAGCCTGTTTGTT TCTGACAAAGCTGATGGTCCGGTACTGGAAGCGCTGCATTCTTACGGTGTG GGCCTGCTGGCCCTGCGTTCTGCTGGCTATGATCACATCGATATTGAGACCG CAAAACGCCTGGGTATCAAAGTAGTTAACGTGCCAGCCTATTCTAACCACG CTATCGCTGACCATACTCTGGCTATCATGCTGGCTCTGATTCGTCGTCTGCA CCGTGCCCATGATAAAGTGCGCCTGGGTGATTTTGATCTGGATGGTCTGATG GGCTTTGATCTGAACGGCAAAGTTGCTGGTGTAATTGGTCTGGGTAAAATC GGTCGCCTGGTAGCTACCCGCCTGAAAGCGTTTGGTTGCAAAGTTCTGGGC TATGATCCATACATTCAGCCGGAAATCGTAGAAAACGTTGATCTGGATACCC TGATCACTCAGGCTGATATCATTTCTATTCATTGTCCGCTGACCCGTGAAAA CTTTCATATGTTTAACGAAGAGACTTTTAAGCGTATGAAACCGGGTGCTATT CTGGTTAACACCGCGCGTGGTGGTCTGATCGATACCAAGGCCCTGCTGGAG GCCCTGAAGTCTGGTAAACTGGGCGGCGCAGCCCTGGATGTGTATGAATAT GAACGTGGCCTGTTTTTTAAAAACCACCAAAAAGAAGGTATCAAAGACCC GTATCTGGCCCAGCTGCTGGGTCTGGCCAACGTAGTGCTGACCGGTCATCA GGCCTTTCTGACCCGTGAGGCTGTAAAAAACATCGAAGAAACTACCGTAG AAAACATTCTGGAATGGCAAAAGAACCCGCAGGCAAAGCTGAAAAACGA AATCTAA (L-ldhcodingsequence-BacilluscoagulansH-2strain): SEQIDNo.5 ATGAAAAAAGTAAACCGTGTTGCAGTGATCGGAACTGGCGCAGTAGGCAC AAGTTATTGCTATGCAATGATTAACCAGGGTGTTGCAGAAGAGCTTGTTTTA ATCGATATTAACGAAGCAAAAGCAGAAGGGGAAGCCATGGACCTGAACCA CGGCCTGCCATTTGCGCCTACGCCGACCCGCGTTTGGAAAGGCGATTATTC CGATTGCGGCACTGCCGACCTTGTTGTCATTACGGCAGGTTCCCCGCAAAA ACCGGGCGAAACAAGGCTTGATCTTGTTTCCAAAAACGCAAAAATTTTTAA AGGCATGATTAAGAGCATCATGGACAGCGGCTTTAACGGGATTTTTCTTGTT GCCAGCAACCCGGTTGACATTTTGACATATGTAACTTGGAAAGAGTCCGGC CTGCCGAAAGAACATGTTATCGGTTCGGGCACAGTGCTTGACTCCGCGCGT CTCCGCAACTCTTTGAGCGCCCAATTTGGAATTGACCCGCGCAATGTGCAT GCTGCGATTATCGGCGAACACGGCGATACGGAACTTCCGGTATGGAGCCAT ACAACTATCGGTTACGATACGATTGAAAGTTATCTACAAAAAGGAATTATTG ACGAAAAGACGTTAGATGACATTTTTGTCAATACGAGAGATGCGGCTTATC ATATTATTGAACGAAAAGGGGCCACATTTTACGGCATCGGGATGTCCCTGAC CCGGATTACAAGGGCAATCCTGAACAATGAAAACAGCGTATTGACGGTCTC TGCATTTCTTGAAGGCCAATACGGAAACAGCGATGTGTACGTTGGCGTTCC GGCCATCATCAATCGCCAGGGCATCCGTGAAGTGGTTGAAATCAAACTGAA CGAAAAAGAACAGGAACAGTTCAATCATTCTGTAAAAGTGCTAAAAGAAA CAATGGCACCGATATTGTAA (pfkandpykcodingsequence-BacilluscoagulansH-2strain) SEQIDNo.6 ATGAAGCGAATTGGAGTATTGACAAGCGGCGGCGATGCACCGGGGATGAA TGCGGCGGTCCGCGCGATTGCCCGTAAAGGGATTTATCACGGCCTGGAAGT TTACGGCATTCGCCAAGGTTATAACGGATTGATTCAAGGAAACATCCAAAA GCTCGAAGCAGGATCTGTTGGCGATATTCTCCAGCGGGGCGGCACGGTTTT GCAGTCGGCAAGAAGCGAAGAATTCAAAACGCCGGAAGGGCAGCAAAAA GCGATCAGGCAGCTGAAAGACCATGGCATTGAAGCGCTCGTTGTGATCGG CGGCGACGGTTCCTACCAAGGGGCCAAAAAGTTGACGGAACAGGGCTTTA ACTGCATTGGTGTGCCAGGGACAATCGATAACGACATCCCGGGGACGGATT TTACAATCGGTTTTGATACGGCATTGAACACAGTGCTTGATGCGATTGATAA AATTCGCGACACCGCTTCTTCCCACGAACGCACCTTTATTATTGAAGTCATG GGCAGAAATGCCGGGGATATCGCGCTCTGGTCCGGCCTGGCCGGCGGAGC CGAATCGATTATTATTCCGGAAGAAAAATATGACTTAAAAGATGTCGTGGAG CGTCTTGAACAGGGGAGAAAACGCGGCAAACGCCACAGCATCATCATTGT CGCGGAAGGCGTGATGAGCGGCAACGAGTTTGCTGAACAATTGAAAAAAA CCGGTGTGATCGGCGATACCCGCGTTTCTGTTCTCGGCCATATCCAGCGCGG CGGTTCTCCGACGGCATTTGACCGCGTGCTTGCAAGCCGCCTCGGCGCAA GGGCTGTTGAACTGCTGCTTGAAGGAAAAGGGGGCCGCGCTGTCGGCATT CAAAATAACCAGCTGGTTGACCACGATATCCTTGAGATTCTCGGAAAACCG CACGCCGTTAATAAAAACATGTACAAGCTGTCGAAAGAATTGTCGATCTAA CGTTTCTTAGGAGGAAATAAAATGAAAAAAACCAAAATTGTATGTACAATC GGACCTGCCAGTGAAAGTGTGGAAATGCTTGAAAGATTAATGGCAAACGG GATGGATGTTTGCCGCCTGAACTTCTCGCACGGCAGCCATGAGGAACATCT TGCCCGGATTAAAAATATCCGTGAAGCTGCAAAAAACCAAAACAAAACGA TCGGGCTTCTGCTCGATACAAAGGGCCCGGAAATCCGCACCCATGATATGA AAGACGGCGGATTCGAGCTCGTTGAAGGCATGACACCGGTCATTTCAATGA CAGAAGTGCTCGGGACACCGGAAAAATTTTCGGTCACATATGAAGGGCTG ATTGATGATGTGCACGTTGGCTCTAAAATTTTACTTGATGACGGTTTGATTG AACTGGAAGTGACGGCCATCGATAAAAACGCCGGTGAAATCCATACAAAA GTGCTGAACCGCGGCGTTTTGAAAAACAAAAAAGGTGTTAACGTCCCGGG TGTTTCCGTGAACCTTCCGGGCATCACCGAAAAAGACGTGAGCGATATCCT GTTCGGGCTTGAACAAGGCATTGACTTCATTGCGGCTTCGTTTGTACGCCG GCCGTCCGACGTTTTGGAAATCCGCCAGCTCCTTGAAGAACACGATGCTTT GCATGTGAAAATTTTCCCTAAAATTGAAAACCAGGAAGGCGTCGACAATAT CGATGAAATCCTTGCGGTATCAGACGGCTTAATGGTTGCCCGCGGCGACCT CGGCGTTGAAATTCCGACCGAAGCGGTGCCGCTCGTACAAAAAGAAATGA TCAGAAAATGTAATACGCTCGGCAAACCGGTGATTACCGCAACGCAAATGC TTGATTCGATGCAACGCAACCCGCGCCCGACCCGCGCGGAAGCAAGCGAC GTGGCCAACGCCATTTTTGACGGCACGGATGCGATCATGCTTTCCGGCGAA ACGGCAGCCGGGAAATATCCTGCTGAAGCGGTTAAGACGATGTACAATATT GCGGTTCATGTGGAAAAAGCAATTAACCATCGCGATATTCTGAACAAGCGC AGCAAGAGCACGGACCATAATATGACAGACGCTATCTGCCAGTCCGTTGCC CATACGGCTTTAAATCTTGATGTGAATGCCATTATTGCGCCGACTGAAAGCG GCTATACGGCACGCATGATCTCCAAATACCGCCCGGCGGCCCCAATCATTGC TGTCACGAGCGATCCGAAAGTACAACGCGGCTTAACTGTTGTGTCCGGCGT ATACCCACAATTGGGCACAAAGGCAAACAATACGGATGAAATGCTTGAAAT TGCAGTGGAGGAAGCGTTGAAATCCGAAATCGTCCATCACGGCGACCTTG TGATCATTACAGCAGGCGTCCCGGTTGGTGGGAAAGGCACCACCAACCTG ATGAAAGTGCACCTGATCGGTGATATATTGGCAAAAGGCCAGGGAATCGGC AGAAAATCGGCATTCGGCCCGGTCATCGTTGCTGAAAGCCCTGAAGAAGC AAACGCAAAGGCAACAGAAGGTTGTGTGCTCGTCACGAGAACGACCGAC AAAGAAATCATGCCGGCCATTGAAAAATGCGCCGCGCTGATTACGGAAGA AGGCGGCTTGACAAGCCATGCTGCAGTTGTTGGCATCAATGTCGGCATTCC GGTCATTGTTGGCGTTGAAAAAGCCGTTTCCATTTTTGAAGACGGGCAGGA AGTTACGGTAGATGCGGCAACCGGCTCGGTTTACAACGGCCATGCGACTGT ATTGTAA (P.sub.ldhpromoter-Geobacillusstearothermophilus): SEQIDNo.7 GCGGCCGCACTAGTGCGGGACGGGGAGCTGAGTGCTCCCGTTGTTTGCCG CGGCGTCTGTCATGAAATGGACAAACAATAGTCAAACAATCGCCACAATCG CGCATGCATTGCGGTGCGCCTTTCGCGTAAAATATTTATATGAAAGTGTTCG CATTATATTGAAGGAGGATGAATGCA (L-ldhcodingsequence-Geobacillusthermoglucosidasius DSM2542) SEQIDNo.8 ATGAAACAACAAGGCATGAATCGAGTAGCACTTATAGGAACGGGGTTCGTT GGGGCCAGCTATGCATTTGCCCTTATGAACCAAGGAATAGCAGATGAGTTA GTATTGATTGATGTAAATAAGAATAAGGCAGAGGGCGATGTGATGGATTTAA ATCACGGAAAAGTATTCGCGCCGAAGCCGATGAATATTTGGTTTGGAGATTA TCAAGATTGCCAAGACGCCGATTTGGTGGTGATTTGTGCAGGGGCTAACCA AAAGCCGGGAGAAACAAGACTGGATCTTGTTGACAAAAATATTAATATCTT CAAAACGATTGTCGATTCTGTGATGAAATCCGGATTTGATGGCGTTTTTCTT GTGGCAACGAACCCAGTGGATATTTTAACGTATGCTACTTGGAAATTTAGCG GGTTACCGAAAGAGCGGGTAATCGGCTCAGGAACGATTCTTGATACAGCAA GATTCCGCTTCTTGCTAAGTGAATATTTTCAAGTGGCTCCGACCAATGTACA TGCGTATATTATTGGCGAGCATGGGGATACAGAGCTGCCTGTTTGGAGCCAT GCGGAAATTGGAAGCATTCCAGTTGAGCAAATATTGATGCAAAACGATAAC TATAGAAAAGAGGATTTAGACAATATCTTTGTTAATGTTCGTGATGCGGCAT ATCAAATCATTGAGAAAAAAGGGGCAACGTATTACGGCATTGCAATGGGAT TAGTCCGTATCACTCGTGCTATTTTGCACAATGAAAATGCCATCTTAACCGT TTCTGCTCATTTGGACGGCCAATATGGCGAACGAAATGTTTATATTGGCGTG CCTGCCATTATCAACCGAAACGGTATTCGTGAAGTGATGGAATTGACGCTA AATGAAACAGAACAACAACAATTCCATCATAGTGTAACTGTATTAAAAGAC ATTCTTTCCCGTTATTTTGATGATGTAAAATAA

[0055] The amino acid sequences involved in the present invention are as follows.

TABLE-US-00002 (D-ldh.sup.P101Qaminoacidsequence): SEQIDNo.9 MKVIFFSMHPYEEEFLGPILPSDWDVEMTPDFLDETTVEKAKGAQVVSLF VSDKADGPVLEALHSYGVGLLALRSAGYDHIDIETAKRLGIKVVNVPAYS QHAIADHTLAIMLALIRRLHRAHDKVRLGDFDLDGLMGFDLNGKVAGVIG LGKIGRLVATRLKAFGCKVLGYDPYIQPEIVENVDLDTLITQADIISIHC PLTRENFHMFNEETFKRMKPGAILVNTARGGLIDTKALLEALKSGKLGGA ALDVYEYERGLFFKNHQKEGIKDPYLAQLLGLANVVLTGHQAFLTREAVK NIEETTVENILEWQKNPQAKLKNEI (D-ldh.sup.P101Naminoacidsequence): SEQIDNo.10 MKVIFFSMHPYEEEFLGPILPSDWDVEMTPDFLDETTVEKAKGAQVVSLF VSDKADGPVLEALHSYGVGLLALRSAGYDHIDIETAKRLGIKVVNVPAYS NHAIADHTLAIMLALIRRLHRAHDKVRLGDFDLDGLMGFDLNGKVAGVIG LGKIGRLVATRLKAFGCKVLGYDPYIQPEIVENVDLDTLITQADIISIHC PLTRENFHMFNEETFKRMKPGAILVNTARGGLIDTKALLEALKSGKLGGA ALDVYEYERGLFFKNHQKEGIKDPYLAQLLGLANVVLTGHQAFLTREAVK NIEETTVENILEWQKNPQAKLKNEI

[0056] The sequence shown in SEQ ID No. 1 is the sequence of the D-lactate dehydrogenase gene of Bacillus licheniformis.

[0057] The sequence shown in SEQ ID No. 2 is an encoding sequence of D-ldh.sup.P101Q. It results from mutation of the codon in the nucleotide sequence of SEQ ID No. 1 that encodes the amino acid at position 101 into a glutamine-coding codon.

[0058] The sequence shown in SEQ ID No. 3 is a sequence from codon-optimization of the coding sequence as shown in SEQ ID No. 2 for Geobacillus thermoglucosidasius as a host.

[0059] The sequence shown in SEQ ID No. 4 is an encoding sequence of D-ldh.sup.P101N It results from mutation of the codon in the nucleotide sequence of SEQ ID No. 1 that encodes the amino acid at position 101 into an asparagine-coding codon.

[0060] The sequence shown in SEQ ID No. 5 is an encoding sequence of L-lactate dehydrogenase in a Bacillus coagulans H-2 strain.

[0061] The sequence shown in SEQ ID No. 6 is an encoding sequence of 6-phosphofructokinase and pyruvate kinase in a Bacillus coagulans H-2 strain.

[0062] The sequence shown in SEQ ID No. 7 is a promoter sequence upstream of the L-lactate dehydrogenase gene of Geobacillus stearothermophilus.

[0063] The sequence shown in SEQ ID No. 8 is an encoding sequence of L-lactate dehydrogenase in Geobacillus thermoglucosidasius DSM 2542.

[0064] The sequence shown in SEQ ID No. 9 is the amino acid sequence of D-ldh.sup.P101Q.

[0065] The sequence shown in SEQ ID No. 10 is the amino acid sequence of D-ldh.sup.P101N.

[0066] Information of sequences of primers used in the present invention is as shown in Table 1 below.

TABLE-US-00003 TABLE1 InformationofSequencesofPrimersUsedinPresentInvention Primer Sequence(5-3) pUBTY-F AGGAATATTCAGCAATTTGCCC (SEQIDNo.11) pUBTY-R CAGCTGGCACGACAGGTTT (SEQIDNo.12) LDH-Up-F tagttagttagcccttagtgactcgGCTTGCATCTTTCGCTGCA (SEQIDNo.13) LDH-Up-R AAAAATTACTTTCATCGCTGTCTGTCATCCTTTCCA (SEQIDNo.14) LDH-DLDH-F GGATGACAGACAGCGATGAAAGTAATTTTTTTTTCTATGCAC (SEQIDNo.15) LDH-DLDH-R TATTCAAAGTCAGTATTAGATTTCGTTTTTCAGCTTTGC (SEQIDNo.16) LDH-Down-F AAAAACGAAATCTAATACTGACTTTGAATACAACAAGGTGA (SEQIDNo.17) LDH-Down-R aacagctatgaccatgattacgccaTTAATACCCTTCCACTTATCCAAGA (SEQIDNo.18) LDH-YZ-F TCCGGTGCACAGCTTGTTTC (SEQIDNo.19) LDH-YZ-R AGCTGTATTTGCATTTTTCTCCG (SEQIDNo.20) GPFYK-Up-F tagttagttagcccttagtgactcgGCAACGCGTATGCTGAATAGTAA (SEQIDNo.21) GLFYK-Up-R CACTAGTGCGGCCGCCCGCTATCAGCTTCTTAATGTTGT (SEQIDNo.22) GLFYK-Pl-F AGAAGCTGATAGCGGGCGGCCGCACTAGTGCG (SEQIDNo.23) GLFYK-Pl-R TCCAATTCGCTTCATTGCATTCATCCTCCTTCAATATAAT (SEQIDNo.24) GLFYK-F AGGAGGATGAATGCAATGAAGCGAATTGGAGTATTGACA (SEQIDNo.25) GPFYAK-R CTGATAGCAACTTCCTTACAATACAGTCGCATGGCC (SEQIDNo.26) GPFYAK-DOWN-F GCGACTGTATTGTAAGGAAGTTGCTATCAGCTTTTTCTTT (SEQIDNo.27) GPFYK-DOWN-R aacagctatgaccatgattacgccaGATTCATCTCAGCGGAAACTACTAA (SEQIDNo.28) GFYK-YZ-F CGATCGACGTTTGTGATTGACC (SEQIDNo.29) GFYK-YZ-R TATCCAATCAATCGCTTTCCG (SEQIDNo.30) AcDH1-Up-F tagttagttagcccttagtgactcgCACTTCTGTTGGAGCGTTTCC (SEQIDNo.31) AcDH1-Up-R AAAGGGGACAATTTCATGAAATTCCCAAGGTTAACAGATA (SEQIDNo.32) AcDH1-Down-F CCTTGGGAATTTCATGAAATTGTCCCCTTTCAGTCTGA (SEQIDNo.33) AcDH1-Down-R aacagctatgaccatgattacgccaGGCGCCTCGACCATGTTAA (SEQIDNo.34) AcDH1-YZ-F AAGAAAACTATTATACACACCCGCA (SEQIDNo.35) AcDH1-YZ-R CATGGTGAGGCCTATCAGTTTAC (SEQIDNo.36) AcDH2-Up-F tagttagttagcccttagtgactcgGCGCAGACTGCCGTCAAT (SEQIDNo.37) AcDH2-Up-R GGAAGGGGAATGGACTCGAAACGAAAGGAGCGG (SEQIDNo.38) AcDH2-Down-F CTCCTTTCGTTTCGAGTCCATTCCCCTTCCCATATAA (SEQIDNo.39) AcDH2-Down-R aacagctatgaccatgattacgccaCCATCGTTGGCAAAAAAATTG (SEQIDNo.40) AcDH2-YZ-F CTTAACACCTGTTTCATATCCAAGG (SEQIDNo.41) AcDH2-YZ-R GAACATTACAGAAATCGCCGC (SEQIDNo.42) AADH-Up-F tagttagttagcccttagtgactcgCAAGCTATATGCTCAAGACATCGA (SEQIDNo.43) AADH-Up-R GAGTGGTTTTTATTTACGCATTCTCCCTCCTGATTG (SEQIDNo.44) AADH-Down-F GGAGGGAGAATGCGTAAATAAAAACCACTCCCCCAAA (SEQIDNo.45) AADH-Down-R aacagctatgaccatgattacgccaCGTTTATCAACGCTTCCAAT (SEQIDNo.46) AADH-YZ-F CGACAAAACAGATTATCGCCC (SEQIDNo.47) AADH-YZ-R AACGCCGATACGTGGGAAA (SEQIDNo.48) DLDH-Up-R AAAAATTACTTTCATACGCATTCTCCCTCCTGATTG (SEQIDNo.49) DLDH-F GGAGGGAGAATGCGTATGAAAGTAATTTTTTTTAGCATGCA (SEQIDNo.50) DLDH-R GAGTGGTTTTTATTTTCAGATTTCATTTTTAAGTTTCGCT (SEQIDNo.51) DLDH-Down-F AAAAATGAAATCTGAAAATAAAAACCACTCCCCCAAA (SEQIDNo.52) pflB-Up-F tagttagttagcccttagtgactcgAATTCCAGGCGTCGCCC (SEQIDNo.53) pflB-Up-R AAAAATTACTTTCATAACAGTTTCCCTCCCATGCAT (SEQIDNo.54) DLDH-F1 GGGAGGGAAACTGTTATGAAAGTAATTTTTTTTAGCATGCA (SEQIDNo.55) DLDH-R1 GGAGGGGGGAGATTATCAGATTTCATTTTTAAGTTTCGCT (SEQIDNo.56) pflB-Down-F AAAAATGAAATCTGATAATCTCCCCCCTCCTTCTTAAG (SEQIDNo.57) pflB-Down-R aacagctatgaccatgattacgccaCATTCTTTAATGTGCGAATAAAAGC (SEQIDNo.58) pflB-YZ-F TTAACGGCCGCTTTGTCTTC (SEQIDNo.59) pflB-YZ-R GCTTTCTTCCGAAGGAGGCTC (SEQIDNo.60) pta-Up-F tagttagttagcccttagtgactcgTAGAAGCAAACGTGTTTCGTTTT (SEQIDNo.61) pta-Up-RI TCACCTTTTTCTGAAGAACGAATCCTCCCTAATGTTTG (SEQIDNo.62) pta-Down-F1 AGGGAGGATTCGTTCTTCAGAAAAAGGTGACGAAACG (SEQIDNo.63) pta-Down-R aacagctatgaccatgattacgccaAGCCGCTGCCGTTTATACATA (SEQIDNo.64) pta-YZ-F GCATATTTCCGTCCTGTCATGC (SEQIDNo.65) pta-YZ-R CTCGGAACAATCGTCGGGTAT (SEQIDNo.66) LLDH-Up-R1 GTTTACTTTTTTCATACGCATTCTCCCTCCTGATTG (SEQIDNo.67) LLDH-F1 GGAGGGAGAATGCGTATGAAAAAAGTAAACCGTGTTGCA (SEQIDNo.68) LLDH-R1 GAGTGGTTTTTATTTTTACAATATCGGTGCCATTGTTTC (SEQIDNo.69) LLDH-Down-F1 GCACCGATATTGTAAAAATAAAAACCACTCCCCCAAA (SEQIDNo.70)

[0067] The present invention has found that lactic acid-producing strains constructed by combinations of genetic modifications proposed in the present invention can maximumize lactic acid production. When a high-temperature fermentation strain is used as a starting strain, the advantages of high-temperature fermentation can be further combined.

[0068] On the basis of this concept, in a first aspect of the present invention, there is provided a method for constructing a lactic acid-producing strain, characterized by genetically engineering a starting strain to increase lactic acid production, which comprises: 1) introducing a lactic acid synthesis pathway; 2) optimizing the lactic acid synthesis pathway; 3) inhibiting by-product synthesis pathways.

[0069] Wherein, the lactic acid is L-lactic acid or D-lactic acid.

[0070] In some embodiments of the present invention, the starting strain is a lactic acid-and/or pyruvic acid-producing strain; preferably, the starting strain shows growth activity under a 37 C. to 70 C. temperature condition, can withstand a temperature of 37 C. to 70 C., and can produce lactic acid and/or pyruvic acid at a temperature of 37 C. to 70 C.

[0071] In some embodiments of the present invention, the starting strain includes microorganisms of genus Bacillus and Geobacillus. Preferably, the starting strain includes Bacillus lichenformis, Bacillus coagulans, Bacillus smithii, Bacillus pallidus and Geobacillus thermoglucosidasius.

[0072] In some specific preferred embodiments, the starting strain is Geobacillus thermoglucosidasius.

[0073] In the method of the present invention for constructing a lactic acid-producing strain, genetically engineering the starting strain comprises introducing a lactic acid synthesis pathway. In some embodiments, introducing the lactic acid synthesis pathway comprises introducing, into the starting strain, an L-lactate dehydrogenase gene L-ldh or a D-lactate dehydrogenase gene D-ldh.

[0074] In the method of the present invention for constructing a lactic acid-producing strain, genetically engineering the starting strain further comprises optimizing the lactic acid synthesis pathway. In some embodiments, optimizing the lactic acid synthesis pathway comprises introducing, into the starting strain, coding sequences of one or more genes selected from: a lactate dehydrogenase gene ldh, a 6-phosphofructokinase gene pfk and a pyruvate kinase gene pyk, wherein the lactate dehydrogenase gene ldh is an L-lactate dehydrogenase gene L-ldh or a D-lactate dehydrogenase gene D-ldh.

[0075] In some embodiments of the present invention, the L-lactate dehydrogenase gene L-ldh is derived from Bacillus coagulans and/or Geobacillus thermoglucosidasius, the D-lactate dehydrogenase gene D-ldh is derived from Bacillus lichenformis, the 6-phosphofructokinase gene pfk is derived from Bacillus coagulans and the pyruvate kinase gene pyk is derived from Bacillus coagulans.

[0076] According to the present invention, introducing the coding sequences of the target genes into the starting strain comprises integrating the coding sequences of the target genes into a genome of the starting strain, or expressing them in the form of a plasmid in the starting strain. Depending on required expression strength of each inserted gene, single-copy or multi-copy of its coding sequence may be introduced into the starting strain.

[0077] If the coding sequences of multiple target genes are introduced, the introduced coding sequences of the multiple target genes may be introduced into the starting strain as individual single-gene expression fragments, and may be introduced into the starting strain as tandem expression fragments.

[0078] In the method of the present invention for constructing a lactic acid-producing strain, genetically engineering the starting strain further comprises inhibiting by-product synthesis pathways. In some embodiments of the method for constructing an L-lactic acid-producing strain, the by-products include D-lactic acid, formic acid, acetic acid and ethanol. In some embodiments of the method for constructing a D-lactic acid-producing strain, the by-products include L-lactic acid, formic acid, acetic acid and ethanol.

[0079] In some embodiments, inhibiting the by-product synthesis pathways comprises knocking out or down one or more coding genes in the starting strain selected from: a lactate dehydrogenase gene ldh, a formate acetyltransferase gene pflB, an acetaldehyde dehydrogenase gene acdh, an aldehyde-alcohol dehydrogenase gene aadh and a phosphate acetyltransferase gene pta, wherein the lactate dehydrogenase gene ldh is an L-lactate dehydrogenase gene L-ldh or a D-lactate dehydrogenase gene D-ldh.

[0080] The genomes of some of the microorganisms contain do not contain only one acetaldehyde dehydrogenase gene acdh. In the present invention, one or more may be knocked out or knocked down, preferably all are knocked out or knocked down, of the acetaldehyde dehydrogenase genes acdh. Different acetaldehyde dehydrogenase genes acdh may be distinguished by acdh1, acdh2, etc.

[0081] According to the genetic engineering strategy of the present invention, if it is desired to obtain a D-lactic acid-producing strain, then into the starting strain, a D-lactic acid synthesis pathway is introduced and optimized, and an L-lactic acid synthesis pathway is inhibited; and vice versa.

[0082] For example, in some embodiments of the present invention, into the starting strain, a D-lactate dehydrogenase gene D-ldh, a 6-phosphofructokinase gene pfk and a pyruvate kinase gene pyk are introduced, an L-lactate dehydrogenase gene L-ldh, an acetaldehyde dehydrogenase gene acdh and an aldehyde-alcohol dehydrogenase gene aadh of the starting strain are knocked out or knocked down, and preferably, a formate acetyltransferase gene pflB and a phosphate acetyltransferase gene pta are knocked out or knocked down, thereby constructing a D-lactic acid-producing strain.

[0083] In some other embodiments of the present invention, into the starting strain, an L-lactate dehydrogenase gene L-ldh, a 6-phosphofructokinase gene pfk and a pyruvate kinase gene pyk are introduced, a D-lactate dehydrogenase gene D-ldh, an acetaldehyde dehydrogenase gene acdh and an aldehyde-alcohol dehydrogenase gene aadh of the starting strain are knocked out or knocked down, and preferably, a formate acetyltransferase gene pflB and a phosphate acetyltransferase gene pta are knocked out or knocked down, thereby constructing an L-lactic acid-producing strain.

[0084] The invention has further found that mutating position 101 of D-lactate dehydrogenase into glutamine or asparagine can significantly improve catalytic performance of D-lactate dehydrogenase and that introducing it into the starting strain can further significantly increase lactic acid production. Accordingly, in a preferred embodiment of the present invention, into the starting strain, a mutant D-lactate dehydrogenase gene is introduced. It may be introduced in the step of introducing the lactic acid synthesis pathway, may also be introduced in the step of optimizing the lactic acid synthesis pathway, and may also be introduced in both steps. The mutant D-lactate dehydrogenase gene encodes the amino acid sequence as shown in SEQ ID No. 9 or SEQ ID No. 10, or encodes an amino acid sequence exhibiting 30% or higher identity to the amino acid sequence shown in SEQ ID No. 9 or SEQ ID No. 10.

[0085] In some specific preferred embodiments, the method of the present invention for constructing a D-lactic acid-producing strain includes sequence-independent steps of: [0086] 1) selecting a starting strain, such as Geobacillus thermoglucosidasius, and using the polymerase chain reaction (PCR) technique and genetic manipulation methods to knock out an L-ldh gene and introduce a D-ldh gene at the same position; [0087] 2) overexpressing a pfk gene and a pyk gene; [0088] 3) knocking out an acdh gene; [0089] 4) knocking out an aadh gene; and [0090] 5) inserting a codon-optimized D-ldh gene (e.g., the gene as shown in SEQ ID No. 3).

[0091] In other preferred embodiments, the method of the present invention for constructing a D-lactic acid-producing strain further includes steps of: [0092] 6) knocking out a pflB gene and inserting a codon-optimized D-ldh gene (e.g., the gene as shown in SEQ ID No. 3); and [0093] 7) knocking out a pta gene.

[0094] In some specific preferred embodiments, the method of the present invention for constructing an L-lactic acid-producing strain includes sequence-independent steps of: [0095] 1) selecting a starting strain, such as Geobacillus thermoglucosidasius, and using the polymerase chain reaction (PCR) technique and genetic manipulation methods to knock out a D-ldh gene and introduce an L-ldh gene at the same position; [0096] 2) overexpressing a pfk gene and a pyk gene; [0097] 3) knocking out an acdh gene; [0098] 4) knocking out an aadh gene; [0099] 5) inserting an L-ldh gene; and [0100] 6) preferably, knocking out a pflB gene and a pta gene.

[0101] In some specific preferred embodiments, the method of the present invention for constructing an L-lactic acid-producing strain may also, directly with a constructed efficient D-lactic acid-producing strain as a basis, knock out a D-ldh gene and introduce an L-ldh gene, thereby easily constructing an efficient L-lactic acid-producing strain.

[0102] In a second aspect of the present invention, there is provided a lactic acid-producing strain characterized by being constructed according to a method of the present invention as described in the present invention.

[0103] In some embodiments of the present invention, the lactic acid-producing strain is Geobacillus thermoglucosidasius.

[0104] In a preferred embodiment of the present invention, the lactic acid-producing strain is selected from D-lactic acid-producing strains GT6, GT7 and GT8 and L-lactic acid-producing strains GT9 and GT10. The lactic acid-producing strains according to the present invention have been deposited with the China Center for Type Culture Collection (CCTCC). The deposition numbers of the D-lactic acid-producing strains GT6 and GT8 and the L-lactic acid-producing strains GT9 and GT10 are CCTCC M 20221822, CCTCC M 20221823, CCTCC M 20221824 and CCTCC M 20221825 respectively.

[0105] In some embodiments of the present invention, the constructed lactic acid-producing strain shows growth activity under a 37 C. to 70 C. temperature condition, can withstand a temperature of 37 C. to 70 C., and can produce lactic acid and/or pyruvic acid at a temperature of 37 C. to 70 C.

[0106] In some embodiments of the present invention, the constructed lactic acid-producing strain includes microorganisms of genus Bacillus and Geobacillus, and is preferably selected from Bacillus lichenformis, Bacillus coagulans, Bacillus smithii, Bacillus pallidus and Geobacillus thermoglucosidasius.

[0107] In some specific preferred embodiments, the constructed lactic acid-producing strain is Geobacillus thermoglucosidasius.

[0108] In a third aspect of the present invention, there is provided use of a lactic acid-producing strain according to the present invention in lactic acid production. In embodiments of the present invention, the lactic acid is L-lactic acid or D-lactic acid.

[0109] In a preferred embodiment of the present invention, using a lactic acid-producing strain according to the present invention, for lactic acid, can be achieved a titer of at least 80 g L.sup.1, such as 90 g L.sup.1, 100 g L.sup.1, 110 g L.sup.1, 120 g L.sup.1, 130 g L.sup.1, 140 g L.sup.1 or 150 g L.sup.1, and a yield of at least 80%, such as 90%, 91%, 92%, 93%, 94% or 95%. Preferably, chiral purity of the lactic acid is at least 99%, such as 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%.

[0110] In a preferred embodiment of the present invention, using a D-lactic acid-producing strain according to the present invention, for D-lactic acid, can be achieved a titer of 153.07 g L.sup.1, a yield of 93.04% and chiral purity as high as 99.63%. Using an L-lactic acid-producing strain according to the present invention, for L-lactic acid, can be achieved a titer of 151.12 g L.sup.1, a yield of 98.68% and chiral purity as high as 99.04%, or even higher.

[0111] In a fourth aspect of the present invention, there is provided a method for producing lactic acid, characterized by comprising steps of: [0112] 1) providing a lactic acid-producing strain according to the present invention; [0113] 2) at 37 C. to 70 C., culturing the strain for 6 hours to 15 hours, thereby providing a seed; and [0114] 3) at 37 C. to 70 C. in a presence of a carbon source, subjecting the seed to fermentation culture, thereby obtaining lactic acid.

[0115] In some embodiments of the present invention, the lactic acid is L-lactic acid or D-lactic acid.

[0116] In some embodiments of the present invention, the carbon source is selected from one or more of glucose, xylose, sucrose, glycerol, arabinose and mannitol.

[0117] In some embodiments of the present invention, in step 3), an inoculum volume of the seed has an OD.sub.620 nm value of 0.2 to 0.8. For example, the inoculum volume is such that the OD.sub.620 nm value reaches 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8.

[0118] In some embodiments of the present invention, an initial concentration of the carbon source may be 40 g/L to 100 g/L. In preferred embodiments, a fed-batch fermentation strategy is employed. In the process of fermentation, when the concentration of the carbon source drops blow 40.0 g/L, the carbon source is fed to maintain the concentration in the range of 0 to 100 g/L.

[0119] In some embodiments of the present invention, the fermentation temperature may be 37 C. to 70 C. Those skilled in the art may adjust the fermentation temperature depending on the fermentative bacterium used. Fermentation temperatures include, but are not limited to, 50 C., 55 C., 56 C., 57 C., 58 C., 59 C., 60 C., 61 C., 62 C., 63 C., 64 C., 65 C. and 70 C. Fermentation temperature ranges include, but are not limited to, 40 C. to 70 C., 50 C. to 65 C., 55 C. to 65 C. and 60 C. to 65 C.

[0120] In some embodiments of the present invention, the fermentation culture may be carried out at a pH of 6.5 to 7.5. For example, the fermentation culture may be carried out at a pH of 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5.

[0121] In some embodiments of the present invention, the fermentation culture is carried out for a period of up to 50 hours, at least 1 hour. Preferably, it is from 48 hours to 50 hours, such as 48 hours, 48.5 hours, 49 hours, 49.25 hours, 49.67 hours or 50 hours.

[0122] In some embodiments of the present invention, the fermentation culture is carried out under agitation at a speed of 50 rpm to 150 rpm.

[0123] In some embodiments of the present invention, a fermentation vessel known in the art (e.g., a fermenter) may be used to ferment the carbon source.

[0124] In some embodiments of the present invention, a fermentation product may be separated and purified. Methods known in the art may be used to separate and purify the fermentation product.

[0125] Below, by way of a few examples, the present invention will be described in greater detail. The following examples are illustrative, but not limiting. The following examples should not be construed to limit the scope of protection of the present invention. The examples of the present invention are implemented on the basis of the technical solutions of the present invention. Although specific implementation and operation details are set forth, the scope of protection of the invention is not limited to the following examples.

EXAMPLES

[0126] Experimental methods in the following examples, for which particular conditions are not specified (e.g., PCR amplification, transformation, gene insertion, protein purification, etc.), are generally carried out in a conventional manner and conditions, for example, using the methods and conditions as taught in Molecular Cloning: A Laboratory Manual by Sambrook et al. (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions suggested by the manufacturers.

[0127] A method for genetic engineering of Geobacillus thermoglucosidasius DSM 2542 is as follows: a constructed recombinant plasmid is introduced by electroporation into Geobacillus thermoglucosidasius DSM 2542 (25 kV/cm, 10 F, 600 ); the resulting transformants are cultured and then incubated on a kanamycin-resistant plate at 68 C. for 12 hours, obtaining single-crossover strains; the single-crossover strains are incubated at 60 C. for 10 hours and subcultured for 2-5 generations, followed by green fluorescence-based on-plate screening for double-crossover strains; finally, corresponding primers are used to perform PCR verification, obtaining a correct engineered strain.

[0128] A method for handling a fermentation sample is as follows: the sample is boiled at 100 C. for 10 minutes; subsequently, 2 mL of the sample is taken and added to a 100 mL volumetric flask, and 2 mL of 2M sulfuric acid is used for acid-hydrolysis for 10 minutes; the volume is then brought to 100 mL; finally, it is centrifuged at 8,000 rpm for 10 minutes, filtered using a 0.22-micron syringe membrane filter for aqueous filtration, and then can be used for subsequent detection. For chiral purity determination of lactic acid, the above prepared sample is additionally diluted 4 times and then can be used.

[0129] Conditions and a method for detection using a high-performance liquid chromatography system are as follows: the high-performance liquid chromatography system (Agilent 1260 Series, Hewlett-Packard, USA) is equipped with a Bio-Rad Aminex HPX-87H column (3007.8 mm) and a differential refractive index detector (RID); the column temperature is 55 C.; the flow rate is 0.5 mL/min; the mobile phase is 5 mM sulfuric acid; the injection volume is 10 L.

[0130] Conditions and a method for detecting chiral purity of lactic acid using a high-performance liquid chromatography system are as follows: the high-performance liquid chromatography system (Agilent 1260 Series, Hewlett-Packard, USA) is equipped with an SCAS Sumichiral OA-5000 column (1504.6 mm) and a diode array detector (DAD); the column temperature is 30 C.; the flow rate is 0.8 mL/min; the mobile phase is 2 mM copper sulfate; the injection volume is 10 L; an ultraviolet absorbance wavelength is set to 254 nm.

[0131] The materials and reagents used in the examples, unless otherwise specified, can be all obtained from commercial sources.

Example 1: Construction of D-Lactic Acid-Producing Strain According to Present Invention

1.1 Knockout of L-ldh Gene and Insertion of D-ldh Gene in Geobacillus thermoglucosidasius

[0132] By double digestion of the pUB-sfGFP plasmid with BamHI and HindIII, a linearized vector was obtained. The primer pairs LDH-Up-F/LDH-Up-R and LDH-Down-F/LDH-Down-R were used to amplify, by the polymerase chain reaction (PCR) technique, from the genome of Geobacillus thermoglucosidasius DSM 2542, the upstream and downstream homology arms respectively. The primer pair LDH-DLDH-F/LDH-DLDH-R was used to amplify by the PCR technique, from the genome of Bacillus lichenformis BJQ, the D-ldh gene. Its codon encoding the amino acid at position 101 was mutated into a glutamine-coding codon (SEQ ID NO. 2). Specifically, the mutation process was as described in Example 6. The primer pair LDH-Up-F/LDH-Down-R was used to amplify, by overlap extension polymerase chain reaction (SOE-PCR), via splicing from the templates of upstream homology arm, D-ldh gene and downstream homology arm, the LDHDLDH-UD fragment. By ligating the LDH-DLDH-UD fragment into the linearized pUB-sfGFP vector through seamless cloning using the ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China), a pUB-1 plasmid was obtained. The primer pair pUBTY-F/pUBTY-R was used to verify insertion of the fragment in the MCS region.

[0133] Using the pUB-1 plasmid, through performing genetic manipulations on Geobacillus thermoglucosidasius DSM 2542, the native L-ldh gene was knocked out, and the D-ldh gene was successfully introduced at the same position, obtaining a strain GT1. The primer pair LDH-YZ-F/LDH-YZ-R was used to verify the double-crossover results.

1.2 Overexpression of pk and pvyk Genes in Strain GT1

[0134] By double digestion of the pUB-sfGFP plasmid with BamHI and HindIII, a linearized vector was obtained. The primer pairs GPFYK-Up-F/GLFYK-Up-R and GPFYAK-DOWN-F/GPFYK-DOWN-R were used to amplify, by the polymerase chain reaction (PCR) technique, from the genome of Geobacillus thermoglucosidasius DSM 2542, the upstream and downstream homology arms, respectively. The primer pair GLFYK-Pl-F/GLFYK-Pl-R was used to amplify, by the PCR technique, from the genome of Geobacillus stearothermophilus, the P.sub.ldh promoter (SEQ ID NO. 7). The primer pair GLFYK-F/GPFYAK-R was used to amplify, by the PCR technique, from the genome of Bacillus coagulans H-2, the pfk and pyk genes (SEQ ID NO. 6). The primer pair GPFYK-Up-F/GPFYK-DOWN-R was used to amplify, by overlap extension polymerase chain reaction (SOE-PCR), from the templates of upstream homology arm, P.sub.ldh, pfk and pyk genes and downstream homology arm, the GLFYK-UD fragment. By ligating the GLFYK-UD fragment into the linearized pUB-sfGFP vector through seamless cloning using the ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China), a pUB-2 plasmid was obtained. The primer pair pUBTY-F/pUBTY-R was used to verify insertion of the fragment in the MCS region.

[0135] Using the pUB-2 plasmid, through performing genetic manipulations on Geobacillus thermoglucosidasius DSM 2542, in the strain GT1, the pfk and pyk genes were successfully overexpressed, obtaining a strain GT2. The primer pair GFYKYZ-F/GFYKYZ-R was used to verify the double-crossover results.

1.3 Knockout of acdh1 Gene in Strain GT2

[0136] By double digestion of the pUB-sfGFP plasmid with BamHI and HindIII, a linearized vector was obtained. The primer pairs AcDH1-Up-F/AcDH1-Up-R and AcDH1-Down-F/AcDH1-Down-R were used to amplify, by the polymerase chain reaction (PCR) technique, from the genome of Geobacillus thermoglucosidasius DSM 2542, the upstream and downstream homology arms, respectively. The primer pair AcDH1-Up-F/AcDH1-Down-R was used to amplify, by overlap extension polymerase chain reaction (SOE-PCR), from the templates of upstream and downstream homology arms, the AcDH1-UD fragment. By ligating the AcDH1-UD fragment into the linearized pUB-sfGFP vector through seamless cloning using the ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China), a pUB-3 plasmid was obtained. The primer pair pUBTY-F/pUBTY-R was used to verify insertion of the fragment in the MCS region.

[0137] Using the pUB-3 plasmid, through performing genetic manipulations on Geobacillus thermoglucosidasius DSM 2542, in the strain GT2, the acdh1 gene was successfully knocked out, obtaining a strain GT3. The primer pair AcDH1-YZ-F/AcDH1-YZ-R was used to verify the double-crossover results.

1.4 Knockout of acdh2 Gene in Strain GT3

[0138] By double digestion of the pUB-sfGFP plasmid with BamHI and HindIII, a linearized vector was obtained. The primer pairs AcDH2-Up-F/AcDH2-Up-R and AcDH2-Down-F/AcDH2-Down-R were used to amplify, by the polymerase chain reaction (PCR) technique, from the genome of Geobacillus thermoglucosidasius DSM 2542, the upstream and downstream homology arms, respectively. The primer pair AcDH2-Up-F/AcDH2-Down-R was used to amplify, by overlap extension polymerase chain reaction (SOE-PCR), from the templates of upstream and downstream homology arms, the AcDH2-UD fragment. By ligating the AcDH2-UD fragment into the linearized pUB-sfGFP vector through seamless cloning using the ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China), a pUB-4 plasmid was obtained. The primer pair pUBTY-F/pUBTY-R was used to verify insertion of the fragment in the MCS region.

[0139] Using the pUB-4 plasmid, through performing genetic manipulations on Geobacillus thermoglucosidasius DSM 2542, in the strain GT3, the acdh2 gene was successfully knocked out, obtaining a strain GT4. The primer pair AcDH2-YZ-F/AcDH2-YZ-R was used to verify the double-crossover results.

1.5 Knockout of aadh Gene in Strain GT4

[0140] By double digestion of the pUB-sfGFP plasmid with BamHI and HindIII, a linearized vector was obtained. The primer pairs AADH-Up-F/AADH-Up-R and AADH-Down-F/AADH-Down-R were used to amplify, by the polymerase chain reaction (PCR) technique, from the genome of Geobacillus thermoglucosidasius DSM 2542, the upstream and downstream homology arms, respectively. The primer pair AADH-Up-F/AADH-Down-R was used to amplify, by overlap extension polymerase chain reaction (SOE-PCR), from the templates of upstream and downstream homology arms, the AADH-UD fragment. By ligating the AADH-UD fragment into the linearized pUB-sfGFP vector through seamless cloning using the ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China), a pUB-5 plasmid was obtained. The primer pair pUBTY-F/pUBTY-R was used to verify insertion of the fragment in the MCS region.

[0141] Using the pUB-5 plasmid, through performing genetic manipulations on the Geobacillus thermoglucosidasius DSM 2542, in the strain GT4, the aadh gene was successfully knocked out, obtaining a strain GT5. The primer pair AADH-YZ-F/AADH-YZ-R was used to verify the double-crossover results.

1.6 Insertion of Codon-Optimized D-ldh Gene in Strain GT5

[0142] By double digestion of the pUB-sfGFP plasmid with BamHI and HindIII, a linearized vector was obtained. The primer pairs AADH-Up-F/DLDH-Up-R and DLDH-Down-F/AADH-Down-R were used to amplify, by the polymerase chain reaction (PCR) technique, from the genome of Geobacillus thermoglucosidasius DSM 2542, the upstream and downstream homology arms, respectively. The primer pair DLDH-F/DLDH-R was used to amplify by the PCR technique the codon-optimized D-ldh gene (SEQ ID NO. 3) based on the synthesized sequence thereof. The primer pair AADH-Up-F/AADH-Down-R was used to amplify, by overlap extension polymerase chain reaction (SOE-PCR), from the templates of upstream homology arm, codon-optimized D-ldh gene and downstream homology arm, the DLDH1-UD fragment. By ligating the DLDH1-UD fragment into the linearized pUB-sfGFP vector through seamless cloning using the ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China), a pUB-6 plasmid was obtained. The primer pair pUBTY-F/pUBTY-R was used to verify insertion of the fragment in the MCS region.

[0143] Using the pUB-6 plasmid, through performing genetic manipulations on Geobacillus thermoglucosidasius DSM 2542, into the strain GT5, the codon-optimized D-ldh gene was successfully inserted, obtaining a strain GT6. The primer pair AADH-YZ-F/AADH-YZ-R was used to verify the double-crossover results.

Example 2: Inventive Construction of D-Lactic Acid-Producing Strain

[0144] Using the same methods as in Example 1, a strain GT6 was constructed, and the difference is that this example additionally includes the following manipulations.

2.1 Knockout of pflB Gene and Insertion of Codon-Optimized D-ldh Gene in Strain GT6

[0145] By double digestion of the pUB-sfGFP plasmid with BamHI and HindIII, a linearized vector was obtained. The primer pairs pflB-Up-F/pflB-Up-R and pflB-Down-F/pflB-Down-R were used to amplify, by the polymerase chain reaction (PCR) technique, from the genome of Geobacillus thermoglucosidasius DSM 2542, the upstream and downstream homology arms, respectively. The primer pair DLDH-F1/DLDH-R1 was used to amplify, by the PCR technique, based on the synthesized sequence of the codon-optimized D-ldh gene, the codon-optimized D-ldh gene (SEQ ID NO. 3). The primer pair pflB-Up-F/pflB-Down-R was used to amplify, by overlap extension polymerase chain reaction (SOE-PCR), from the templates of upstream homology arm, codon-optimized D-ldh gene and downstream homology arm, the pflB-DLDH1-UD fragment. By ligating the pflB-DLDH1-UD fragment into the linearized pUB-sfGFP vector through seamless cloning using the ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China), a pUB-7 plasmid was obtained. The primer pair pUBTY-F/pUBTY-R was used to verify insertion of the fragment in the MCS region.

[0146] Using the pUB-7 plasmid, through performing genetic manipulations on Geobacillus thermoglucosidasius DSM 2542, in the strain GT6, the pflB gene was successfully knocked out and the codon-optimized D-ldh gene was inserted, obtaining a strain GT7. The primer pair pflB-YZ-F/pflB-YZ-R was used to verify the double-crossover results.

2.2 Knockout of pta Gene in Strain GT7

[0147] By double digestion of the pUB-sfGFP plasmid with BamHI and HindIII, a linearized vector was obtained. The primer pairs pta-Up-F/pta-Up-R1 and pta-Down-F1/pta-Down-R were used to amplify, by the polymerase chain reaction (PCR) technique, from the genome of Geobacillus thermoglucosidasius DSM 2542, the upstream and downstream homology arms, respectively. The primer pair pta-Up-F/pta-Down-R was used to amplify, by overlap extension polymerase chain reaction (SOE-PCR), from the templates of upstream and downstream homology arms, the pta-UD fragment. By ligating the pta-UD fragment into the linearized pUB-sfGFP vector through seamless cloning using the ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China), a pUB-8 plasmid was obtained. The primer pair pUBTY-F/pUBTY-R was used to verify insertion of the fragment in the MCS region.

[0148] Using the pUB-8 plasmid, through performing genetic manipulations on Geobacillus thermoglucosidasius DSM 2542, in the strain GT7, the pta gene was successfully knocked out, obtaining a strain GT8. The primer pair pta-YZ-F/pta-YZ-R was used to verify the double-crossover results.

Example 3: Inventive Construction of L-Lactic Acid-Producing Strain

[0149] Using the same methods as in Example 1, a strain GT6 was constructed, and the difference is that this example additionally includes the following manipulations.

3.1 Knockout of D-ldh Gene, Insertion of L-ldh Gene and Knockout of Codon-Optimized D-ldh Gene in Strain GT6

[0150] By double digestion of the pUB-sfGFP plasmid with BamHI and HindIII, a linearized vector was obtained. The primer pair LDH-Up-F/LDH-Down-R was used to amplify, by the polymerase chain reaction (PCR) technique, from the genome of Geobacillus thermoglucosidasius DSM 2542, the LLDH-UD fragment. By ligating the LLDH-UD fragment into the linearized pUB-sfGFP vector through seamless cloning using the ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China), a pUB-9 plasmid was obtained. The primer pair pUBTY-F/pUBTY-R was used to verify insertion of the fragment in the MCS region.

[0151] Using the pUB-9 and pUB-5 plasmids, through performing genetic manipulations on Geobacillus thermoglucosidasius DSM 2542, in the strain GT6, the D-ldh gene was successfully knocked out, the L-ldh gene (SEQ ID No. 8) was inserted and the codon-optimized D-ldh gene was knocked out, obtaining a strain GT9. The primer pairs LDH-YZ-F/LDH-YZ-R and AADH-YZ-F/AADH-YZ-R were used to verify the double-crossover results.

3.2 Insertion of L-ldh Gene Derived from Bacillus coagulans H-2 in Strain GT9

[0152] By double digestion of the pUB-sfGFP plasmid with BamHI and HindIII, a linearized vector was obtained. The primer pairs AADH-Up-F/LLDH-Up-R1 and LLDH-Down-F1/AADH-Down-R were used to amplify by the polymerase chain reaction (PCR) technique, from the genome of Geobacillus thermoglucosidasius DSM 2542, the upstream and downstream homology arms, respectively. The primer pair LLDH-F1/LLDH-R1 was used to amplify, by the PCR technique, from the genome of Bacillus coagulans H-2, the L-ldh gene (SEQ ID NO. 5). The primer pair AADH-Up-F/AADH-Down-R was used to amplify, by overlap extension polymerase chain reaction (SOE-PCR), from the templates of upstream homology arm, L-ldh gene derived from Bacillus coagulans H-2 and downstream homology arm, the LLDH1-UD fragment. By ligating the LLDH1-UD fragment into the linearized pUB-sfGFP vector through seamless cloning using the ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China), a pUB-10 plasmid was obtained. The primer pair pUBTY-F/pUBTY-R was used to verify insertion of the fragment in the MCS region.

[0153] Using the pUB-10 plasmid, through performing genetic manipulations on Geobacillus thermoglucosidasius DSM 2542, into the strain GT9, the L-ldh gene derived from Bacillus coagulans H-2 was successfully inserted, obtaining a strain GT10. The primer pair AADH-YZ-F/AADH-YZ-R was used to verify the double-crossover results.

Example 4: D-Lactic Acid Production According to Present Invention

[0154] D-lactic acid-producing strains constructed in accordance with the present invention were subjected to, in a 5-L fermenter at 60 C., fed-batch fermentation.

[0155] Strain culture conditions: 37 C., 200 rpm, a fermentation culture period was up to 50 hours.

[0156] A strain culture medium contained the following components: 10 g L.sup.1 NaCl, 5 g L.sup.1 yeast extract, 10 g L.sup.1 peptone.

[0157] After the cultured strains were incubated in a seed culture medium for fermentation overnight, they were used to be inoculated into a fermentation medium for fermentation production.

[0158] The seed culture medium for fermentation contained the following components: 5 g L.sup.1 soya peptone, 15 g L.sup.1 peptone, 5 g L.sup.1 NaCl.

[0159] The fermentation medium contained the following components: 5 g L.sup.1 yeast extract, 3 g L.sup.1 Na.sub.2HPO.sub.4, 3 g L.sup.1 KH.sub.2PO.sub.4, 1 g L.sup.1 NH.sub.4Cl, 0.48 g L.sup.1 MgSO.sub.4, 0.5 g L.sup.1 NaCl, 0.42 g L.sup.1 citric acid, 0.028 g L.sup.1 FeSO.sub.4.Math.7H.sub.2O, 0.01 g L.sup.1 thiamin, Trace Metal Mix (4.4 mg L.sup.1 NiSO.sub.4.Math.6H.sub.2O, 2.86 mg L.sup.1 H.sub.3BO.sub.3, 1.81 mg L.sup.1 MnCl.sub.2-4H.sub.2O, 0.39 mg L.sup.1 Na.sub.2MoO.sub.4.Math.2H.sub.2O, 0.222 mg L.sup.1 ZnSO.sub.4-7H.sub.2O, 0.079 mg L.sup.1 CuSO.sub.4-5H.sub.2O, 0.049 mg L.sup.1 Co(NO.sub.3).sub.2-6H.sub.2O), 3.1 mg L.sup.1 biotin, 0.1 g L.sup.1 betaine.

[0160] The seed for fermentation was inoculated at an inoculum volume of 0.3% (v/v) to 10% (v/v).

4.1 D-Lactic Acid Production by Strain GT1 from Glucose as Carbon Source

[0161] The strain GT1 was cultured in the seed culture medium for fermentation at 60 C. and 200 rpm overnight, the seed was inoculated into the fermentation medium at 10% (v/v) and subjected to fermentation culture; fermentation conditions: 25% (w/v) Ca(OH).sub.2 was used to maintain a pH at 7.0, an agitation speed was 80 rpm, a culture temperature was 60 C., an initial glucose concentration was 40 g L.sup.1, in the process of fermentation, when the glucose concentration was 0.0 g/L to 40.0 g/L, glucose was added to the fermenter to maintain the glucose concentration within the range of 0 to 100.0 g/L, a fermentation period was 48 hours; samples were taken and subjected to high-performance liquid chromatography detection; according to the results of detection, after fermentation for 48 hours, in the fermentation broth, a D-lactic acid titer was 37.46 g L.sup.1, a D-lactic acid yield was 5.sup.20.40%, a D-lactic acid titer production rate was 0.780 g L.sup.1 h.sup.1.

4.2 D-Lactic Acid Production by Strain GT2 from Glucose as Carbon Source

[0162] The strain GT2 was cultured in the seed culture medium for fermentation at 60 C. and 200 rpm overnight, the seed was inoculated into the fermentation medium at 10% (v/v) and subjected to fermentation culture; fermentation conditions: 25% (w/v) Ca(OH).sub.2 was used to maintain a pH at 7.0, an agitation speed was 80 rpm, a culture temperature was 60 C., an initial glucose concentration was 40 g L.sup.1, in the process of fermentation, when the glucose concentration was 0.0 g/L to 40.0 g/L, glucose was added to the fermenter to maintain the glucose concentration within the range of 0 to 100.0 g/L, a fermentation period was 48 hours; samples were taken and subjected to high-performance liquid chromatography detection; according to the results of detection, after fermentation for 48 hours, in the fermentation broth, a D-lactic acid titer was 42.96 g L.sup.1, a D-lactic acid yield was 49.50%, a D-lactic acid production rate was 0.895 g L.sup.1 h.sup.1.

4.3 D-Lactic Acid Production by Strain GT3 from Glucose as Carbon Source

[0163] The strain GT3 was cultured in the seed culture medium for fermentation at 60 C. and 200 rpm overnight, the seed was inoculated into the fermentation medium at 10% (v/v) and subjected to fermentation culture; fermentation conditions: 25% (w/v) Ca(OH).sub.2 was used to maintain a pH at 7.0, an agitation speed was 80 rpm, a culture temperature was 60 C., an initial glucose concentration was 40 g L.sup.1, in the process of fermentation, when the glucose concentration was 0.0 g/L to 40.0 g/L, glucose was added to the fermenter to maintain the glucose concentration within the range of 0 to 100.0 g/L, a fermentation period was 48 hours; samples were taken and subjected to high-performance liquid chromatography detection; according to the results of detection, after fermentation for 48 hours, in the fermentation broth, a D-lactic acid titer was 33.43 g L.sup.1, a D-lactic acid yield was 58.48%, a D-lactic acid production rate was 0.696 g L.sup.1 h.sup.1.

4.4 D-Lactic Acid Production by Strain GT4 from Glucose as Carbon Source

[0164] The strain GT4 was cultured in the seed culture medium for fermentation at 60 C. and 200 rpm overnight, the seed was inoculated into the fermentation medium at 10% (v/v) and subjected to fermentation culture; fermentation conditions: 25% (w/v) Ca(OH).sub.2 was used to maintain a pH at 7.0, an agitation speed was 80 rpm, a culture temperature was 60 C., an initial glucose concentration was 40 g L.sup.1, in the process of fermentation, when the glucose concentration was 0.0 g/L to 40.0 g/L, glucose was added to the fermenter to maintain the glucose concentration within the range of 0 to 100.0 g/L, a fermentation period was 49 hours; samples were taken and subjected to high-performance liquid chromatography detection; according to the results of detection, after fermentation for 49 hours, in the fermentation broth, a D-lactic acid titer was 27.42 g L.sup.1, a D-lactic acid yield was 49.66%, a D-lactic acid production rate was 0.560 g L.sup.1 h.sup.1.

4.5 D-Lactic Acid Production by Strain GT5 from Glucose as Carbon Source

[0165] The strain GT5 was cultured in the seed culture medium for fermentation at 60 C. and 200 rpm overnight, the seed was inoculated into the fermentation medium at 10% (v/v) and subjected to fermentation culture; fermentation conditions: 25% (w/v) Ca(OH).sub.2 was used to maintain a pH at 7.0, an agitation speed was 80 rpm, a culture temperature was 60 C., an initial glucose concentration was 40 g L.sup.1, in the process of fermentation, when the glucose concentration was 0.0 g/L to 40.0 g/L, glucose was added to the fermenter to maintain the glucose concentration within the range of 0 to 100.0 g/L, a fermentation period was 48 hours; samples were taken and subjected to high-performance liquid chromatography detection; according to the results of detection, after fermentation for 48 hours, in the fermentation broth, a D-lactic acid titer was 57.38 g L.sup.1, a D-lactic acid yield was 76.79%, a D-lactic acid production rate was 1.148 g L.sup.1 h.sup.1.

4.6 D-Lactic Acid Production by Strain GT6 from Glucose as Carbon Source

[0166] The strain GT6 was cultured in the seed culture medium for fermentation at 60 C. and 200 rpm overnight, the seed was inoculated into the fermentation medium at 10% (v/v) and subjected to fermentation culture; fermentation conditions: 25% (w/v) Ca(OH).sub.2 was used to maintain a pH at 7.0, an agitation speed was 80 rpm, a culture temperature was 60 C., an initial glucose concentration was 40 g L.sup.1, in the process of fermentation, when the glucose concentration was 0.0 g/L to 40.0 g/L, glucose was added to the fermenter to maintain the glucose concentration within the range of 0 to 100.0 g/L, a fermentation period was 48.5 hours; samples were taken and subjected to high-performance liquid chromatography detection; according to the results of detection, after fermentation for 48.5 hours, in the fermentation broth, a D-lactic acid titer was 86.06 g L.sup.1, a D-lactic acid yield was 92.05%, a D-lactic acid production rate was 1.774 g L.sup.1 h.sup.1.

4.7 D-Lactic Acid Production by Strain GT6 from Glucose as Carbon Source

[0167] The strain GT6 was cultured in the seed culture medium for fermentation at 60 C. and 200 rpm overnight, the seed was inoculated into the fermentation medium at 10% (v/v) and subjected to fermentation culture; fermentation conditions: 25% (w/v) Ca(OH).sub.2 was used to maintain a pH at 7.0, an agitation speed was 80 rpm, a culture temperature was 60 C., an initial glucose concentration was 100 g L.sup.1, in the process of fermentation, when the glucose concentration was 0.0 g/L to 40.0 g/L, glucose was added to the fermenter to maintain the glucose concentration within the range of 0 to 100.0 g/L, a fermentation period was 48 hours; samples were taken and subjected to high-performance liquid chromatography detection; according to the results of detection, after fermentation for 48 hours, in the fermentation broth, a D-lactic acid titer was 153.07 g L.sup.1, a D-lactic acid yield was 93.04%, a D-lactic acid production rate was 3.189 g L.sup.1 h.sup.1, chiral purity ofD-lactic acid was 99.63%.

4.8 D-Lactic Acid Production by Strain GT6 from Xylose as Carbon Source

[0168] The strain GT6 was cultured in the seed culture medium for fermentation at 60 C. and 200 rpm overnight, the seed was inoculated into the fermentation medium at 10% (v/v) and subjected to fermentation culture; fermentation conditions: 25% (w/v) Ca(OH).sub.2 was used to maintain a pH at 7.0, an agitation speed was 80 rpm, a culture temperature was 60 C., an initial xylose concentration was 40 g L.sup.1, in the process of fermentation, when the xylose concentration was 0.0 g/L to 40.0 g/L, xylose was added to the fermenter to maintain the xylose concentration within the range of 0 to 100.0 g/L, a fermentation period was 48 hours; samples were taken and subjected to high-performance liquid chromatography detection; according to the results of detection, after fermentation for 48 hours, in the fermentation broth, a D-lactic acid titer was 30.81 g L.sup.1, a D-lactic acid yield was 88.31%, a D-lactic acid production rate was 0.642 g L.sup.1 h.sup.1.

4.9 D-Lactic Acid Production by Strain GT7 from Glucose as Carbon Source

[0169] The strain GT7 was cultured in the seed culture medium for fermentation at 60 C. and 200 rpm overnight, the seed was inoculated into the fermentation medium at 10% (v/v) and subjected to fermentation culture; fermentation conditions: 25% (w/v) Ca(OH).sub.2 was used to maintain a pH at 7.0, an agitation speed was 80 rpm, a culture temperature was 60 C., an initial glucose concentration was 40 g L.sup.1, in the process of fermentation, when the glucose concentration was 0.0 g/L to 40.0 g/L, glucose was added to the fermenter to maintain the glucose concentration within the range of 0 to 100.0 g/L, a fermentation period was 48 hours; samples were taken and subjected to high-performance liquid chromatography detection; according to the results of detection, after fermentation for 48 hours, in the fermentation broth, a D-lactic acid titer was 93.82 g L.sup.1, a D-lactic acid yield was 93.27%, a D-lactic acid production rate was 1.955 g L.sup.1 h.sup.1.

4.10 D-Lactic Acid Production by Strain GT8 from Glucose as Carbon Source

[0170] The strain GT8 was cultured in the seed culture medium for fermentation at 60 C. and 200 rpm overnight, the seed was inoculated into the fermentation medium at 10% (v/v) and subjected to fermentation culture; fermentation conditions: 25% (w/v) Ca(OH).sub.2 was used to maintain a pH at 7.0, an agitation speed was 80 rpm, a culture temperature was 60 C., an initial glucose concentration was 40 g L.sup.1, in the process of fermentation, when the glucose concentration was 0.0 g/L to 40.0 g/L, glucose was added to the fermenter to maintain the glucose concentration within the range of 0 to 100.0 g/L, a fermentation period was 48 hours; samples were taken and subjected to high-performance liquid chromatography detection; according to the results of detection, after fermentation for 48 hours, in the fermentation broth, a D-lactic acid titer was 88.82 g L.sup.1, a D-lactic acid yield was 92.21%, a D-lactic acid production rate was 1.850 g L.sup.1 h.sup.1.

Example 5: Production of L-Lactic Acid According to Present Invention

[0171] L-lactic acid-producing strains constructed in accordance with the present invention were subjected to, in a 5-L fermenter at 60 C., fed-batch fermentation.

[0172] Components of the seed culture medium, seed culture medium for fermentation and fermentation medium were the same as in Example 4.

5.1 L-Lactic Acid Production by Strain GT9 from Glucose as Carbon Source

[0173] The strain GT9 was cultured in the seed culture medium for fermentation at 60 C. and 200 rpm overnight, the seed was inoculated into the fermentation medium at 10% (v/v) and subjected to fermentation culture; fermentation conditions: 25% (w/v) Ca(OH).sub.2 was used to maintain a pH at 7.0, an agitation speed was 80 rpm, a culture temperature was 60 C., an initial glucose concentration was 40 g L.sup.1, in the process of fermentation, when the glucose concentration was 0.0 g/L to 40.0 g/L, glucose was added to the fermenter to maintain the glucose concentration within the range of 0 to 100.0 g/L, a fermentation period was 48 hours; samples were taken and subjected to high-performance liquid chromatography detection; according to the results of detection, after fermentation for 48 hours, in the fermentation broth, an L-lactic acid titer was 92.34 g L.sup.1, an L-lactic acid yield was 95.29%, an L-lactic acid production rate was 1.924 g L.sup.1 h.sup.1, and chiral purity of L-lactic acid was 99.46%.

5.2 L-Lactic Acid Production by Strain GT10 from Glucose as Carbon Source

[0174] The strain GT10 was cultured in the seed culture medium for fermentation at 60 C. and 200 rpm overnight, the seed was inoculated into the fermentation medium at 10% (v/v) and subjected to fermentation culture; fermentation conditions: 25% (w/v) Ca(OH).sub.2 was used to maintain a pH at 7.0, an agitation speed was 80 rpm, a culture temperature was 60 C., an initial glucose concentration was 40 g L.sup.1, in the process of fermentation, when the glucose concentration was 0.0 g/L to 40.0 g/L, glucose was added to the fermenter to maintain the glucose concentration within the range of 0 to 100.0 g/L, a fermentation period was 48 hours; samples were taken and subjected to high-performance liquid chromatography detection; according to the results of detection, after fermentation for 48 hours, in the fermentation broth, an L-lactic acid titer was 94.15 g L.sup.1, an L-lactic acid yield was 91.52%, an L-lactic acid production rate was 1.961 g L.sup.1 h.sup.1, and chiral purity of L-lactic acid was 99.50%.

5.3 L-Lactic Acid Production by Strain GT10 from Glucose as Carbon Source

[0175] The strain GT10 was cultured in the seed culture medium for fermentation at 60 C. and 200 rpm overnight, the seed was inoculated into the fermentation medium at 10% (v/v) and subjected to fermentation culture; fermentation conditions: 25% (w/v) Ca(OH).sub.2 was used to maintain a pH at 7.0, an agitation speed was 80 rpm, a culture temperature was 60 C., an initial glucose concentration was 100 g L.sup.1, in the process of fermentation, when the glucose concentration was 0.0 g/L to 40.0 g/L, glucose was added to the fermenter to maintain the glucose concentration within the range of 0 to 100.0 g/L, a fermentation period was 48 hours; samples were taken and subjected to high-performance liquid chromatography detection; according to the results of detection, after fermentation for 48 hours, in the fermentation broth, an L-lactic acid titer was 151.12 g L.sup.1, an L-lactic acid yield was 98.68%, an L-lactic acid production rate was 3.148 g L.sup.1 h.sup.1, and chiral purity of L-lactic acid was 99.04%.

Comparative Example 1: Fed-Batch Fermentation by Geobacillus thermoglucosidasius DSM 2542 in 5-L Fermenter at 60 C.

[0176] Non-genetically engineered Geobacillus thermoglucosidasius DSM 2542 was used to produce L-lactic acid. In this example, components of the seed culture medium, seed culture medium for fermentation and fermentation medium were the same as in Example 5.

[0177] Geobacillus thermoglucosidasius DSM 2542 was cultured in the seed culture medium for fermentation at 60 C. and 200 rpm overnight, the seed was inoculated into the fermentation medium at 10% (v/v) and subjected to fermentation culture; fermentation conditions: 25% (w/v) Ca(OH).sub.2 was used to maintain a pH at 7.0, an agitation speed was 80 rpm, a culture temperature was 60 C., an initial glucose concentration was 40 g L.sup.1, in the process of fermentation, when the glucose concentration was 0.0 g/L to 40.0 g/L, glucose was added to the fermenter to maintain the glucose concentration within the range of 0 to 100.0 g/L, a fermentation period was 49.67 hours; samples were taken and subjected to high-performance liquid chromatography detection; according to the results of detection, after fermentation for 49.67 hours, in the fermentation broth, an L-lactic acid titer was 69.17 g L.sup.1, an L-lactic acid yield was 88.14%, an L-lactic acid production rate was 1.393 g L.sup.1 h.sup.1.

Comparative Example 2: Fed-Batch Fermentation by Bacillus smithii in 5-L Fermenter at 60 C.

[0178] Non-genetically engineered Bacillus smithii was used to produce L-lactic acid, as well as the same seed culture medium. In this example, components of the seed culture medium, seed culture medium for fermentation and fermentation medium were the same as in Example 5.

[0179] Bacillus smithii was cultured in the seed culture medium for fermentation at 60 C. and 200 rpm overnight, the seed was inoculated into the fermentation medium at 10% (v/v) and subjected to fermentation culture; fermentation conditions: 25% (w/v) Ca(OH).sub.2 was used to maintain a pH at 7.0, an agitation speed was 80 rpm, a culture temperature was 60 C., an initial glucose concentration was 40 g L.sup.1, in the process of fermentation, when the glucose concentration was 0.0 g/L to 40.0 g/L, glucose was added to the fermenter to maintain the glucose concentration within the range of 0 to 100.0 g/L, a fermentation period was 49.25 hours; samples were taken and subjected to high-performance liquid chromatography detection; according to the results of detection, after fermentation for 49.25 hours, in the fermentation broth, an L-lactic acid titer was 43.50 g L.sup.1, an L-lactic acid yield was 83.60%, an L-lactic acid production rate was 0.883 g L.sup.1 h.sup.1.

Comparative Example 3: Fed-Batch Fermentation by Bacillus pallidus in 5-L Fermenter at 60 C.

[0180] Non-genetically engineered Bacillus pallidus was used to produce L-lactic acid, as well as the same seed culture medium. In this example, components of the seed culture medium, seed culture medium for fermentation and fermentation medium were the same as in Example 5.

[0181] Bacillus pallidus was cultured in the seed culture medium for fermentation at 60 C. and 200 rpm overnight, the seed was inoculated into the fermentation medium at 10% (v/v) and subjected to fermentation culture; fermentation conditions: 25% (w/v) Ca(OH).sub.2 was used to maintain a pH at 7.0, an agitation speed was 80 rpm, a culture temperature was 60 C., an initial glucose concentration was 40 g L.sup.1, in the process of fermentation, when the glucose concentration was 0.0 g/L to 40.0 g/L, glucose was added to the fermenter to maintain the glucose concentration within the range of 0 to 100.0 g/L, a fermentation period was 48 hours; samples were taken and subjected to high-performance liquid chromatography detection; according to the results of detection, after fermentation for 48 hours, in the fermentation broth, an L-lactic acid titer was 13.80 g L.sup.1, an L-lactic acid production rate was 0.288 g L.sup.1 h.sup.1.

[0182] The results of D-lactic acid production in Example 4 are summarized in Table 2 below.

TABLE-US-00004 TABLE 2 D-Lactic Acid Production Production Carbon Source Titer of Rate of Chiral and Initial D-Lactic Yield of D-Lactic Purity of Concentration Acid D-Lactic Acid D-lactic Strain (g L.sup.1) (g L.sup.1) Acid (%) (g L.sup.1 h.sup.1) acid (%) GT1 glucose 40 37.46 52.40 0.780 GT2 glucose 40 42.96 49.50 0.895 GT3 glucose 40 33.43 58.48 0.696 GT4 glucose 40 27.42 49.66 0.560 GT5 glucose 40 57.38 76.79 1.148 GT6 glucose 40 86.06 92.05 1.774 GT6 glucose 100 153.07 93.04 3.189 99.63 GT6 xylose 40 30.81 88.31 0.642 GT7 glucose 40 93.82 93.27 1.955 GT8 glucose 40 88.82 92.21 1.850 means not determined.

[0183] As can be seen from the results of Table 2, after introducing and optimizing the D-lactic acid synthesis pathway and inhibiting the by-product synthesis pathways through the efficient D-lactic acid-producing strain construction stratery of the present invention, the obtained strain GT6, when used for D-lactic acid production by fermentation of glucose as a carbon source, can achieve a D-lactic acid titer of up to 153.07 g L.sup.1, a D-lactic acid yield of up to 93.04%, a D-lactic acid production rate of up to 3.189 g L.sup.1 h.sup.1, and chiral purity of D-lactic acid was up to 99.63%.

[0184] Under the same production conditions, compared with the strain GT1 with only the introduction of the D-lactic acid synthesis pathway, GT6 achieved a titer and a production rate each more than 4 times that of GT1, and a D-lactic acid yield increased by about 78%. Compared with the strain GT2 with only the introduction and partially optimization of the D-lactic acid synthesis pathway and the strains GT3, GT4 and GT5 with only the introduction and partially optimization of the D-lactic acid synthesis pathway and partially inhibition of by-product synthesis pathways, GT6 that has experienced all the three engineering pathways exhibited significantly enhanced ability to produce D-lactic acid and is remarkably improved in terms of D-lactic acid titer, D-lactic acid yield and D-lactic acid production rate.

[0185] Additionally, the efficient D-lactic acid-producing strains according to the present invention can use different carbon sources. For example, when using xylose as a carbon source, GT6 can also obtain a D-lactic acid yield of 88.31%.

[0186] On the basis of GT6, the D-lactic acid synthesis pathway is further optimized and by-product synthesis pathways are inhibited, obtaining the strains GT7 and GT8. When using glucose as a carbon source, GT7 and GT8 maintain a high D-lactic acid titer of 88.82 g L.sup.1 or more and a high D-lactic acid yield of 92.21% or above.

[0187] The results of L-lactic acid production in Example 5 and Comparative Examples 1 to 3 are summarized in Table 3 below.

TABLE-US-00005 TABLE 3 L-Lactic Acid Production Carbon Source Titer of Production Chiral and Initial L-Lactic Yield of Rate of Purity of Concentration Acid L-Lactic L-Lactic Acid L-lactic Strain (g L.sup.1) (g L.sup.1) Acid (%) (g L.sup.1 h.sup.1) acid (%) Inventive GT9 glucose 40 92.34 95.29 1.924 99.46 Inventive GT10 glucose 40 94.15 91.52 1.961 99.50 Inventive GT10 glucose 100 151.12 98.68 3.148 99.04 Comparative glucose 40 69.17 88.14 1.393 Example 1 (Geobacillus thermoglucosidasius DSM 2542) Comparative glucose 40 43.50 83.60 0.883 Example 2 (Bacillus smithii) Comparative glucose 40 13.80 0.288 Example 3 (Bacillus pallidus) means not determined.

[0188] As can be seen from Table 3, under the same production conditions, or even within a shorter fermentation time, the engineered strains GT9 and GT10 according to the present invention can obtain an L-lactic acid titer of 92.34 g L.sup.1 or above, an L-lactic acid yield of up to 95.29% and an L-lactic acid production rate higher than 1.924 g L.sup.1 h.sup.1, and chiral purity of L-lactic acid is 99.46% or higher. When an initial glucose concentration reaches 100 g L.sup.1, the engineered strain GT10 according to the present invention can obtain an L-lactic acid titer of up to 151.12 g L.sup.1, an L-lactic acid yield of up to 98.68% and an L-lactic acid production rate of up to 3.148 g L.sup.1 h.sup.1, and chiral purity of L-lactic acid is up to 99.04%. In contrast, the non-engineered starting Geobacillus thermoglucosidasius DSM 2542 strain only obtains an L-lactic acid titer of 69.17 g L.sup.1, an L-lactic acid yield of 88.14% and an L-lactic acid production rate of 1.393 g L.sup.1 h.sup.1; the non-engineered Bacillus smithii strain produced L-lactic acid can only obtain an L-lactic acid titer of 43.50 g L.sup.1, an L-lactic acid yield of 83.60% and an L-lactic acid production rate of only 0.883 g L.sup.1 h.sup.1; with the non-engineered Bacillus pallidus strain, an obtained L-lactic acid titer is as low as 13.80 g L.sup.1, an L-lactic acid production rate is only 0.288 g L.sup.1 h.sup.1. These indicate that, compared with the starting strains, the L-lactic acid-producing strains constructed through combined manipulations according to the present invention significantly increase the L-lactic acid titer and L-lactic acid production rate in L-lactic acid production. Moreover, compared with other non-engineered strains, the L-lactic acid-producing strains of the present invention are also advantageous in producing L-lactic acid with higher chiral purity and higher efficiency.

Example 6: Directed Engineering, Expression and Purification of D-Lactate Dehydrogenase

[0189] The amino acid sequences and nucleotide sequences involved in Example 6 are as follows:

TABLE-US-00006 (D-LDH.sup.P101G): SEQIDNo.71 MKVIFFSMHPYEEEFLGPILPSDWDVEMTPDFLDETTVEKAKGAQVVSLF VSDKADGPVLEALHSYGVGLLALRSAGYDHIDIETAKRLGIKVVNVPAYS GHAIADHTLAIMLALIRRLHRAHDKVRLGDFDLDGLMGFDLNGKVAGVIG LGKIGRLVATRLKAFGCKVLGYDPYIQPEIVENVDLDTLITQADIISIHC PLTRENFHMFNEETFKRMKPGAILVNTARGGLIDTKALLEALKSGKLGGA ALDVYEYERGLFFKNHQKEGIKDPYLAQLLGLANVVLTGHQAFLTREAVK NIEETTVENILEWQKNPQAKLKNEI (D-LDH): SEQIDNo.72 MKVIFFSMHPYEEEFLGPILPSDWDVEMTPDFLDETTVEKAKGAQVVSLF VSDKADGPVLEALHSYGVGLLALRSAGYDHIDIETAKRLGIKVVNVPAYS PHAIADHTLAIMLALIRRLHRAHDKVRLGDFDLDGLMGFDLNGKVAGVIG LGKIGRLVATRLKAFGCKVLGYDPYIQPEIVENVDLDTLITQADIISIHC PLTRENFHMFNEETFKRMKPGAILVNTARGGLIDTKALLEALKSGKLGGA ALDVYEYERGLFFKNHQKEGIKDPYLAQLLGLANVVLTGHQAFLTREAVK NIEETTVENILEWQKNPQAKLKNEI (codon-optimizedsequenceofD-LDHcoding sequenceforBacillus): SEQIDNo.73 ATGAAAGTAATTTTTTTTTCTATGCACCCGTATGAAGAGGAATTTCTGGG TCCGATTCTGCCGTCTGACTGGGACGTAGAAATGACCCCGGACTTTCTGG ACGAAACCACCGTGGAAAAGGCTAAAGGTGCCCAGGTAGTAAGCCTGTTT GTTTCTGACAAAGCTGATGGTCCGGTACTGGAAGCGCTGCATTCTTACGG TGTGGGCCTGCTGGCCCTGCGTTCTGCTGGCTATGATCACATCGATATTG AGACCGCAAAACGCCTGGGTATCAAAGTAGTTAACGTGCCAGCCTATTCT CCGCACGCTATCGCTGACCATACTCTGGCTATCATGCTGGCTCTGATTCG TCGTCTGCACCGTGCCCATGATAAAGTGCGCCTGGGTGATTTTGATCTGG ATGGTCTGATGGGCTTTGATCTGAACGGCAAAGTTGCTGGTGTAATTGGT CTGGGTAAAATCGGTCGCCTGGTAGCTACCCGCCTGAAAGCGTTTGGTTG CAAAGTTCTGGGCTATGATCCATACATTCAGCCGGAAATCGTAGAAAACG TTGATCTGGATACCCTGATCACTCAGGCTGATATCATTTCTATTCATTGT CCGCTGACCCGTGAAAACTTTCATATGTTTAACGAAGAGACTTTTAAGCG TATGAAACCGGGTGCTATTCTGGTTAACACCGCGCGTGGTGGTCTGATCG ATACCAAGGCCCTGCTGGAGGCCCTGAAGTCTGGTAAACTGGGCGGCGCA GCCCTGGATGTGTATGAATATGAACGTGGCCTGTTTTTTAAAAACCACCA AAAAGAAGGTATCAAAGACCCGTATCTGGCCCAGCTGCTGGGTCTGGCCA ACGTAGTGCTGACCGGTCATCAGGCCTTTCTGACCCGTGAGGCTGTAAAA AACATCGAAGAAACTACCGTAGAAAACATTCTGGAATGGCAAAAGAACCC GCAGGCAAAGCTGAAAAACGAAATCTAA

[0190] The sequences of the primers used in Example 6 are as follows:

TABLE-US-00007 ldh-F: (SEQIDNo.74) CGCGGATCCGATGAAAGTAATTTTTTTTTCTATGCAC ldh-R: (SEQIDNo.75) CCCAAGCTTTTAGATTTCGTTTTTCAGCTTTG ldh1-R: (SEQIDNo.76) TAGCGTGCTGAGAATAGG ldh1-F: (SEQIDNo.77) CCTATTCTCAGCACGCTA ldh2-R: (SEQIDNo.78) TAGCGTGCCCAGAATAGG ldh2-F: (SEQIDNo.79) CCTATTCTGGGCACGCTA ldh3-R: (SEQIDNo.80) TAGCGTGGTTAGAATAGGCTG ldh3-F: (SEQIDNO.81) CAGCCTATTCTAACCACGCTA

[0191] In this example, prepared were wild-type D-lactate dehydrogenase D-LDH, D-lactate dehydrogenase mutants D-LDH.sup.P101Q and D-LDH.sup.P101N and a comparative D-lactate dehydrogenase mutant D-LDH.sup.P101G. Wherein, the amino acid sequence of D-LDH is as shown in SEQ ID No. 72; D-LDH.sup.P101Q is from a proline-to-glutamine mutation of D-LDH at position 101, and its amino acid sequence is as shown in SEQ ID No. 9; D-LDH.sup.P101N is from a proline-to-asparagine mutation of D-LDH at position 101, and its amino acid sequence is as shown in SEQ ID No. 10; D-LDH.sup.P101G is from a proline-to-glycine mutation of D-LDH at position 101, and its amino acid sequence is as shown in SEQ ID No. 71.

[0192] A specific preparation process is as follows:

[0193] The primer pair ldh-F (BamHI)/ldh-R (HindIII) was used to amplify, by the polymerase chain reaction (PCR) technique, from the genome of Bacillus lichenformis BJQ, the original D-ldh gene, and the BamHI and HindIII restriction sites were introduced. The vector pETDuet-1 was double digested with BamHI and HindIII, and the D-ldh gene was then cloned into the BamHI-HindIII site of the pETDuet-1 vector's multiple cloning site (MCS) I, producing pETDuet-ldh for D-LDH expression and purification.

[0194] The primer pairs ldh-F/ldh1-R and ldh1F/ldh-R were used to amplify, from the template of D-ldh gene, by the overlap extension polymerase chain reaction (SOE-PCR) technique, obtaining the mutated gene D-ldh1 (CCG/CAG; encoding the protein D-LDH.sup.P101Q), and the BamHI and HindIII restriction sites were introduced. The vector pETDuet-1 was double digested with BamHI and HindIII, and the D-ldhI gene was then cloned into the BamHI-HindIII site of the MCS I of the pETDuet-1 vector, producing pETDuet-ldhI for D-LDH.sup.P101Q expression and purification.

[0195] The primer pairs ldh-F/ldh2-R and ldh2-F/ldh-R were used to amplify, from the template of D-ldh gene, by the overlap extension polymerase chain reaction (SOE-PCR) technique, obtaining the mutated gene D-ldh2 (CCG/GGG; encoding the protein D-LDH.sup.P101G), and the BamHI and HindIII restriction sites were introduced. The vector pETDuet-1 was double digested with BamHI and HindIII, and the D-ldh2 gene was then cloned into the BamHI-HindIII site of the MCS I of the pETDuet-1 vector, producing pETDuet-ldh2 for D-LDH.sup.P101G expression and purification.

[0196] The primer pairs ldh-F/ldh3-R and ldh3-F/ldh-R were used to amplify, from the template of D-ldh gene, by the overlap extension polymerase chain reaction (SOE-PCR) technique, obtaining the mutated gene D-ldh3 (CCG/AAC; encoding the protein D-LDH.sup.P101N), and the BamHI and HindIII restriction sites were introduced. The vector pETDuet-1 was double digested with BamHI and HindIII, and the D-ldh3 gene was then cloned into the BamHI-HindIII site of the MCS I of the pETDuet-1 vector, producing pETDuet-ldh3 for D-LDH.sup.P101N expression and purification.

[0197] pETDuet-ldh, pETDuet-ldh1, pETDuet-ldh2 and pETDuet-ldh3 were separately introduced into Escherichia coli BL21 (DE3) strains (purchased from Novagen), obtaining four recombinant Escherichia coli strains; the four recombinant Escherichia coli strains were then each cultured in a seed culture medium (containing 10 g L.sup.1 NaCl, 5 g L.sup.1 yeast extract and 10 g L.sup.1 peptone) supplemented with ampicillin (100 g mL.sup.1) at 37 C. and 200 rpm. When the cell density OD.sub.620 nm reached 0.6-0.8, through adding 0.4 mM isopropyl--D-thiogalactoside and continuing incubation at 16 C. for about 20 hours, recombinant D-LDH expression was induced. The collected cells were washed twice with a 10 mM phosphate buffer, and the cells were then resuspended in an equilibration buffer (25 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 500 mM NaCl, 25 mM imidazole, pH 8.0), and disrupted at 4 C. by high pressure treatment. The lysed cells were centrifuged at 4 C. and 12,000 rpm for 40 minutes, and the supernatant was then used for subsequent purification.

[0198] A nickel column method was used to purify D-lactate dehydrogenase and the mutants. Specifically, Ni.sup.2+-NTA columns were used to purify, via affinity chromatography, from the crude extracts obtained as described above, D-lactate dehydrogenase and the mutants. The columns had been each equilibrated with 30 mL of the equilibration buffer, and with a wash buffer (25 mM Tris-HCl, 500 mM NaCl, 80 mM imidazole, pH 8.0) and an eluting/dissolving buffer (25 mM Tris-HCl, 500 mM NaCl, 300 mM imidazole, pH 8.0), D-LDH was washed and eluted; then, the target protein solutions were transferred to Amicon Ultra-15 30 K centrifugal ultrafilters for further protein concentration, and finally they were desalted by gel filtration on an AKTA Purifier equipped with a Superdex 200 10/300 GL column. The gel-filtration buffer was a 10 mM phosphate buffer (pH 7.0). Through sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the expressed and purified enzymes were measured. All the purification processes were carried out at 4 C. to ensure activity of the enzymes, and the purified enzymes were quickly frozen in liquid nitrogen and then stored at 80 C. for investigative experimentation.

[0199] According to the results of SDS-PAGE and gel filtration chromatography on the KTA Purifier, the molecular weights of D-LDH, D-LDH.sup.P101Q, D-LDH.sup.P101N and D-LDH.sup.P101G were all obtained as to be approximately 35 kD, and all of them were of tetramer structures.

Example 7: Enzymatic Kinetics of D-Lactate Dehydrogenase and Mutants

[0200] Enzyme catalysis testing method for D-lactate dehydrogenase and the mutants: a reaction mixture had a total volume of 0.8 mL, in which were contained: 10 mM phosphate buffer (pH 7.0), an appropriate amount (0.0001 mg/L to 0.0035 mg/L) of purified D-lactate dehydrogenase or a mutant thereof, 0.25 mM NADH, sodium pyruvate at a cencontration of 0.01-0.4 mM or sodium pyruvate at a cencontration of 0.625 mM, NADH at a cencontration of 0.005-0.2 mM. In this testing method, wild-type D-lactate dehydrogenase and the mutants use the same reaction conditions. The testing temperature was 50 C. Based on the initial rate of change in absorbance at 340 nm due to the oxidation of NADH (340=6,220 M.sup.1 cm.sup.1), catalytic performance of D-lactate dehydrogenase and the mutants was measured.

[0201] According to the results of the above tests, catalytic activity (k.sub.cat), specific enzyme activity and catalytic efficiency were calculated. The calculation method is the same as a method commonly used in the art.

[0202] Through the above tests, enzymatic kinetics of D-lactate dehydrogenase and the mutants were characterized, and the test data is summarized in Table 4 below.

TABLE-US-00008 TABLE 4 Enzymatic Kinetics of D-Lactate Dehydrogenase and Mutants Enzymatic D-LDH.sup.P101Q D-LDH.sup.P101N D-LDH.sup.P101G Substrate Kinetics D-LDH (inventive) (inventive) (comparative) Sodium k.sub.cat (s.sup.1) 6.67 0.16 145.00 4.62 106.60 2.82 12.56 0.27 Pyruvate Specific 11.11 0.26 241.70 7.71 177.70 4.70 20.94 0.46 Enzymatic Activity (U mg.sup.1) k.sub.cat/K.sub.m (s.sup.1 2.14 10.sup.5 1.07 10.sup.6 7.47 10.sup.5 1.81 10.sup.5 M.sup.1) NADH k.sub.cat (s.sup.1) 13.21 0.17 145.10 1.71 102.60 1.23 22.60 0.50 Specific 22.02 0.28 241.90 2.85 171.00 2.06 37.67 0.83 Enzymatic Activity (U mg.sup.1) k.sub.cat/K.sub.m (s.sup.1 9.38 10.sup.5 5.44 10.sup.6 7.50 10.sup.6 7.66 10.sup.5 M.sup.1)

[0203] As can be seen from the results of Table 4, when the mutant D-LDH.sup.P101Q is obtained by a proline-to-glutamine mutation at position 101 of D-LDH, catalytic performance is significantly improved. Specifically, in the case of sodium pyruvate being a substrate, catalytic activity (k.sub.cat) of D-LDH.sup.P101Q is about 22 times that of D-LDH, specific enzyme activity is about 22 times that of D-LDH, and catalytic efficiency (k.sub.cat/K.sub.m) is about 5 times that of D-LDH. In the case of NADH being a substrate, catalytic activity (k.sub.cat) of D-LDH.sup.P101Q is about 11 times that of D-LDH, specific enzyme activity is about 11 times that of D-LDH, and catalytic efficiency (k.sub.cat/K.sub.m) is about 6 times that of D-LDH.

[0204] Similarly, when the mutant D-LDH.sup.P101N is obtained by a proline-to-asparagine mutation at position 101 of D-LDH, catalytic performance is significantly improved. Specifically, in the case of sodium pyruvate being a substrate, catalytic activity (k.sub.cat) of D-LDH.sup.P101N is about 16 times that of D-LDH, specific enzyme activity is about 16 times that of D-LDH, and catalytic efficiency (k.sub.cat/K.sub.m) is about 3.5 times that of D-LDH. In the case of NADH being a substrate, catalytic activity (k.sub.cat) of D-LDH.sup.P101N is about 8 times that of D-LDH, specific enzyme activity is about 8 times that of D-LDH, and catalytic efficiency (k.sub.cat/K.sub.m) is about 8 times that of D-LDH.

[0205] However, when the mutant D-LDH.sup.P101G is obtained by a proline-to-glycine mutation at position 101 of D-LDH, catalytic activity (k.sub.cat) and specific enzyme activity are only slightly improved over D-LDH (less than twice), and catalytic efficiency (k.sub.cat/K.sub.m) is even lower than that of D-LDH.

[0206] This indicates that the mutation position and the species of target amino acids for mutation selected by the present invention can make the obtained D-lactate dehydrogenase mutants' catalytic performance significantly improved. This is a result of creative effort. However, when the amino acid is mutated into glycine, ideal catalytic performance improving results are not obtained.

Example 8: Thermostability of D-Lactate Dehydrogenase and Mutants

[0207] Thermostability testing method for D-lactate dehydrogenase or the mutant: D-lactate dehydrogenase and the mutants were subjected to water bath at 50 C., within the time period from 0 to 70 hours, at different time points, samples were taken and used for enzyme activity testing.

[0208] Specific method: 0.004 mg/L of D-lactate dehydrogenase or the mutant (ensuring final concentrations of the different D-lactate dehydrogenase and mutants in the reaction system were consistent) was added to a reaction system, the reaction mixture had a total volume of 0.8 mL, in which were contained 10 mM PBS (pH 7.0), 0.25 mM NADH, 0.625 mM sodium pyruvate, and when at 50 C., testing was performed. The enzyme activity measured at 0 hours of incubation of D-lactate dehydrogenase or the mutant was taken as 100% (initial enzyme activity), and the enzyme activity measured at other time points was represented by a percentage relative to the initial enzyme activity.

[0209] Thermostability data of D-lactate dehydrogenase and the mutants is summarized in Table 5 below.

TABLE-US-00009 TABLE 5 Thermostability of D-lactate Dehydrogenase and Mutants at 50 C. Incubation D-LDH.sup.P101Q D-LDH.sup.P101N D-LDH.sup.P101G Time at 50 C. D-LDH (inventive) (inventive) (comparative) 0 hours 100% 100% 100% 100% 0-9 hours gradual increase slight increase relatively high gradual increase 9-44 hours up to 1194% slight decrease relatively high up to 288% 44-67 hours slight decrease slight decrease relatively high gradual decrease 67 hours 968% 60.53% 100.73% 89.07%

[0210] As can be seen from the results of Table 5, for the D-lactate dehydrogenase mutant D-LDH.sup.P101Q according to the present invention, thermostability when at 50 C. is very good, the D-LDH.sup.P101Q protein, within 0 hours to 9 hours of incubation, has enzyme activity slightly higher compared with at the beginning, within 9 hours to 67 hours, has a slight decrease, finally, it is 60.53% of the initial enzyme activity, at this time, this enzyme still maintains a relativel high level of enzyme activity, This indicates that D-LDH.sup.P101Q has good thermostability.

[0211] For the D-lactate dehydrogenase mutant D-LDH.sup.P101N according to the present invention, when at 50 C., thermostability is very good, within the heating period from 0 to 67 hours, its enzyme activity always remains at a relatively high level, the final enzyme activity is still maintained at 100.73% of the initial enzyme activity. This indicates that D-LDH.sup.P101N has excellent thermostability.

[0212] It can be understood that, during the performance of the enzymatic thermostability comparison experiments, comparing each enzyme with itself at different time points is of biological significance, but comparisons between different enzymes are not of biological significance, because initial enzyme activity of each enzyme is different, at all the other time points, according to the initial enzyme activity of each enzyme, relative enzyme activity is calculated and obtained.

Example 9: D-Lactic Acid Production by Recombinant Geobacillus thermoglucosidasius with D-LDH.SUP.P101Q .Gene through Fed-Batch Fermentation

[0213] The nucleotide sequence encoding the mutant protein D-LDH.sup.P101Q (SEQ ID No. 3) was integrated into Geobacillus thermoglucosidasius, thereby obtaining recombinant Geobacillus thermoglucosidasius. The recombinant Geobacillus thermoglucosidasius was incubated in a seed culture medium A for fermentation (components: 5 g L.sup.1 soya peptone, 15 g L.sup.1 peptone, 5 g L.sup.1 NaCl) at 60 C. and 200 rpm overnight, making an OD.sub.620 nm value reach 2.0 to 8.0.

[0214] An inoculum volume of the fermentation seed may be 0.3% (v/v) to 10% (v/v). In this example, the fermentation seed was inoculated at 10% (v/v) into a fermentation medium A (components: 5 g L.sup.1 yeast extract, 3 g L.sup.1 Na.sub.2HPO.sub.4, 3 g L.sup.1 KH.sub.2PO.sub.4, 1 g L.sup.1 NH.sub.4Cl, 0.48 g L.sup.1 MgSO.sub.4, 0.5 g L.sup.1 NaCl, 0.42 g L.sup.1 citric acid, 0.028 g L.sup.1 FeSO.sub.4.Math.7H.sub.2O, 0.01 g L.sup.1 thiamin, Trace Metal Mix (4.4 mg L.sup.1 NiSO.sub.4.Math.6H.sub.2O, 2.86 mg L.sup.1 H.sub.3BO.sub.3, 1.81 mg L.sup.1 MnCl.sub.2.Math.4H.sub.2O, 0.39 mg L.sup.1 Na.sub.2MoO.sub.4.Math.2H.sub.2O, 0.222 mg L.sup.1 ZnSO.sub.4.Math.7H.sub.2O, 0.079 mg L.sup.1 CuSO.sub.4.Math.5H.sub.2O, 0.049 mg L.sup.1 Co(NO.sub.3).sub.2-6H.sub.2O), 3.1 mg L.sup.1 biotin, 0.1 g L.sup.1 betaine) for fermentation culture. Fermentation conditions: a 5-L fermenter was used, 25% (w/v) Ca(OH).sub.2 was used to control a pH at 7.0, a speed of agitation was 80 rpm, a fermentation temperature was 60 C. An initial glucose concentration was 100 g L.sup.1, in the process of fermentation, when the glucose concentration was 0.0 g/L to 40.0 g/L, to the fermenter, glucose was added to maintain the glucose concentration in the range of 0 to 70 g/L, a fermentation period was 48 hours.

[0215] The fermentation broth was boiled at 100 C. for 10 minutes, then 2 mL was taken and placed in a 100 mL volumetric flask, and 2 mL of 2M sulfuric acid was used for acid-hydrolysis for 10 minutes, the volume was then brought to 100 mL, finally, the above solution was centrifuged at 8,000 rpm for 10 minutes, and after being filtered through a 0.22-micron syringe membrane filter for aqueous filtration, subjected to high-performance liquid chromatography for detecting D-lactic acid.

[0216] According to the results of high-performance liquid chromatography, in the fermentation broth resulting from fermentation for 48 hours, a D-lactic acid titer was 153.07 g L.sup.1, a D-lactic acid yield was 93.04%, a D-lactic acid production rate was 3.189 g L.sup.1 h.sup.1.

Example 10: D-Lactic Acid Production by Recombinant Bacillus licheniformis with D-LDH.SUP.P101Q .Gene through Fed-Batch Fermentation

[0217] The nucleotide sequence encoding the mutant protein D-LDH.sup.P101Q (SEQ ID No. 3) was integrated into Bacillus licheniformis, thereby obtaining recombinant Bacillus lichenformis. The recombinant Bacillus lichenformis was incubated in a seed culture medium B for fermentation (components: 100 g L.sup.1glucose, 10 g L.sup.1 yeast extract, 5 g L.sup.1 peptone, 50 g L.sup.1 CaCO.sub.3) at 50 C. without agitation overnight, making an OD.sub.620 nm value reach 2.0 to 8.0.

[0218] The fermentation seed was inoculated at 10% (v/v) into a fermentation medium B (components: 40 g L.sup.1 peanut meal, 100 g L.sup.1 glucose and 0.3 g L.sup.1 neutral protease) for fermentation culture. Fermentation conditions: a 5-L fermenter was used, 25% (w/v) Ca(OH).sub.2 was used to control a pH at 7.0, a speed of agitation was 80 rpm, a fermentation temperature was 50 C. An initial glucose concentration was 100 g L.sup.1, in the process of fermentation, when the glucose concentration was 0.0 g/L to 40.0 g/L, to the fermenter, glucose was added to maintain the glucose concentration in the range of 0 to 100 g/L, a fermentation period was 60 hours.

[0219] The fermentation broth was boiled at 100 C. for 10 minutes, then 2 mL was taken and placed in a 100 mL volumetric flask, and 2 mL of 2M sulfuric acid was used for acid-hydrolysis for 10 minutes, the volume was then brought to 100 mL, finally, the above solution was centrifuged at 8,000 rpm for 10 minutes, and after being filtered through a 0.22-micron syringe membrane filter for aqueous filtration, subjected to high-performance liquid chromatography for detecting D-lactic acid.

[0220] According to the results of high-performance liquid chromatography, in the fermentation broth resulting from fermentation for 60 hours, a D-lactic acid titer was 145.91 g L.sup.1, a D-lactic acid yield was 86.00%, a D-lactic acid production rate was 2.43 g L.sup.1 h.sup.1.

Example 11: D-Lactic Acid Production by Recombinant Bacillus licheniformis with D-LDH Gene through Fed-Batch Fermentation

[0221] The nucleotide sequence encoding the protein D-LDH (SEQ ID No. 78) was integrated into Bacillus lichenformis, thereby obtaining recombinant Bacillus lichenformis. The recombinant Bacillus lichenformis was incubated in a seed culture medium B for fermentation (components: 100 g L.sup.1glucose, 10 g L.sup.1 yeast extract, 5 g L.sup.1 peptone, 50 g L.sup.1 CaCO.sub.3) at 50 C. without agitation overnight, making an OD.sub.620 nm value reach 2.0 to 8.0.

[0222] The fermentation seed was inoculated at 10% (v/v) into a fermentation medium B (components: 40 g L.sup.1 peanut meal, 100 g L.sup.1 glucose, 0.3 g L.sup.1 neutral protease) for fermentation culture. Fermentation conditions: a 5-L fermenter was used, 25% (w/v) Ca(OH).sub.2 was used to control a pH at 7.0, a speed of agitation was 80 rpm, a fermentation temperature was 50 C. An initial glucose concentration was 100 g L.sup.1, in the process of fermentation, when the glucose concentration was 0.0 g/L to 40.0 g/L, to the fermenter, glucose was added to maintain the glucose concentration in the range of 0 to 100 g/L, a fermentation period was 60 hours.

[0223] The fermentation broth was boiled at 100 C. for 10 minutes, then 2 mL was taken and placed in a 100 mL volumetric flask, and 2 mL of 2M sulfuric acid was used for acid-hydrolysis for 10 minutes, the volume was then brought to 100 mL, finally, the above solution was centrifuged at 8,000 rpm for 10 minutes, and after being filtered through a 0.22-micron syringe membrane filter for aqueous filtration, subjected to high-performance liquid chromatography for detecting D-lactic acid.

[0224] According to the results of high-performance liquid chromatography, in the fermentation broth resulting from fermentation for 60 hours, a D-lactic acid titer was 48.05 g L.sup.1, a D-lactic acid yield was 56.00%, a D-lactic acid production rate was 0.80 g L.sup.1 h.sup.1.

[0225] The results of D-lactic acid production by the recombinant microorganisms through fed-batch fermentation in Examples 9 to 11 are summarized in Table 6 below.

TABLE-US-00010 TABLE 6 D-Lactic Acid Production by Recombinant Microorganisms through Fed-Batch Fermentation Example Example 9 Example 10 Example 11 Recombinant Recombinant Recombinant Recombinant Microorganism Geobacillus Bacillus Bacillus thermoglucosidasius licheniformis with licheniformis with with D-LDH.sup.P101Q Gene D-LDH.sup.P101Q Gene D-LDH Gene Carbon Source glucose glucose glucose Culture Temperature 60 50 50 ( C.) Fermentation Time 48 60 60 (hours) Titer of D-lactic Acid 153.07 145.91 48.05 (g L.sup.1) Yield of D-lactic Acid 93.04 86.00 56.00 (%) Rate of D-Lactic Acid 3.189 2.43 0.80 Production (g L.sup.1 h.sup.1)

[0226] As can be seen from the results of Table 6, when the recombinant Geobacillus thermoglucosidasius with the D-LDH.sup.P101Q-coding sequence or the recombinant Bacillus licheniformis with the D-LDH.sup.P101Q-coding sequence according to the present invention is used to produce D-lactic acid via fed-batch fermentation, a resulting D-lactic acid titer is up to 145 g L.sup.1 or above, a D-lactic acid yield is up to 86% or above, and a D-lactic acid production rate is up to 3.189 g L.sup.1 h.sup.1.

[0227] In contrast, when Bacillus lichenformis with the unmutated D-LDH gene is used in Example 11 to produce D-lactic acid via fed-batch fermentation, under the same fermentation conditions as Example 10 using the recombinant Bacillus lichenformis with the mutant gene D-LDHP.sup.101Q, a D-lactic acid titer is only 48.05 g L.sup.1, much lower than a D-lactic acid titer of 145.91 g L.sup.1 in Example 10. At the same time, a D-lactic acid yield in Example 11 is only 56%, a D-lactic acid production rate is only 0.80 g L.sup.1 h.sup.1, both significantly lower than corresponding values in Example 10. This indicates that the D-lactate dehydrogenase mutants according to the present invention significantly improve the titer, yield and production rate in D-lactic acid production through fed-batch fermentation, reflecting significantly improved catalytic performance.

[0228] Preferred specific embodiments of the present application are described in detail above. It should be understood that those of ordinary skill in the art can make various modifications and changes based on the concept of the present application without exerting any creative effort. Accordingly, all technical solutions that can be obtained by those skilled in the art through logical analysis, inference or limited experimentation in accordance with the concept of the present invention on the basis of the prior art should fall within the scope as defined by the appended claims.