Recombinant microorganism for producing L-valine, construction method and application thereof

Abstract

Related are a recombinant microorganism for producing L-valine, a construction method and an application thereof. Through enhancing amino acid dehydrogenase activity of L-valine fermentation strain, and/or activating an Entner-Doudoroff (ED) metabolic pathway, a problem in L-valine fermentation process that reducing power is unbalanced is solved, thereby the titer and yield of L-valine produced by Escherichia coli are improved, and L-valine was produced by one-step anaerobic fermentation.

Claims

1. A construction method of a recombinant microorganism for producing L-valine comprising: transferring an amino acid dehydrogenase gene into a microorganism and/or activating an Entner-Doudoroff metabolic pathway in the microorganism.

2. The construction method according to claim 1, wherein the method further comprises one or more of the following modifications (1)-(7) to the recombinant microorganism according to claim 1: (1) knocking out a gene mgsA; (2) knocking out a gene IdhA; (3) knocking out genes pta and/or ackA; (4) knocking out genes tdcD and/or tdcE; (5) knocking out a gene adhE; (6) knocking out genes frd and/or pflB; and (7) enhancing activity of AHAS and/or ilvD; preferably, the above items (7), (2) and (5) are selected for modification; preferably, the above items (7), (2) and (6) are selected for modification; preferably, the above items (7), (1), and (3)-(6) are selected for modification; preferably, the above items (1)-(7) are selected for modification; preferably, the item (6) is achieved by substituting the pflB gene of the microorganism itself with the ilvD gene; preferably, the item (6) is achieved by substituting the frd gene of the microorganism itself with the leuDH gene; and preferably, the item (1) is achieved by substituting the mgsA gene of the microorganism itself with the ilvC gene.

3. The construction method according to claim 1, wherein the microorganism is Escherichia coli; and more preferably, the microorganism is Escherichia coli ATCC 8739.

4. The construction method according to claim 1, wherein at least one regulatory element is used to activate or enhance activity of encoding genes of ilvD, leuDH, ilvBN, zwf, pgl, ilvGM, edd, eda or ilvC; preferably, the regulatory element is selected from an M1-93 artificial regulatory element, an MRS1 artificial regulatory element, a RBS artificial regulatory element or an M1-46 artificial regulatory element; preferably, the M1-93 artificial regulatory element regulates ilvD, leuDH, ilvBN, zwf, pgl and ilvGM genes; the MRS1 artificial regulatory element regulates the edd gene; the RBS artificial regulatory element regulates the gene eda; and the M1-46 artificial regulatory element regulates the ilvC gene.

5. The construction method according to claim 1, wherein one or more copies of the enzyme encoding gene and the regulatory element are integrated into a genome of the microorganism, or a plasmid containing the enzyme encoding gene is transferred into the microorganism; preferably, transfer, mutation, knockout, activation or regulation of the enzyme gene is completed by a method of integrating into the genome of the microorganism; preferably, the transfer, mutation, knockout, activation or regulation of the enzyme gene is completed by a homologous recombination method; and preferably, the transfer, mutation, knockout, activation or regulation of the enzyme gene is completed by a two-step homologous recombination method.

6. A recombinant microorganism obtained by the construction method according to claim 1.

7. The construction method according to claim 1, wherein the construction method further comprises acquiring a recombinant microorganism for highly producing L-valine obtained through metabolic evolution on the basis of the recombinant microorganism obtained by the construction method according to claim 1.

8. A recombinant microorganism, wherein a preservation number thereof is CGMCC 19457.

9. (canceled)

10. A method for producing L-valine, wherein the method comprises: (1) fermenting and culturing the recombinant microorganism according to claim 6; and (2) separating and harvesting L-valine; preferably, the fermentation is carried out under anaerobic conditions.

11. The construction method according to claim 1, wherein the construction method further comprising a step of knocking out a 6-phosphoglucokinase gene pfkA.

12. The construction method according to claim 1, wherein the construction method further comprising a step of transferring an acetohydroxy acid reductoisomerase encoding gene into the microorganism; the acetohydroxy acid reductoisomerase encoding gene is preferably ilvC.

13. The construction method according to claim 1, wherein the amino acid dehydrogenase gene is NADH-dependent.

14. The construction method according to claim 1, wherein the amino acid dehydrogenase gene is a leucine dehydrogenase gene.

15. The construction method according to claim 1, wherein the activation of the Entner-Doudoroff metabolic pathway comprises a step of improving expression intensity of a zwf gene, a pgl gene, an edd gene and an eda gene.

16. The construction method according to claim 2, wherein the AHAS is ilvBN, or ilvGM, or ilvIH; optionally, the activity of the ilvIH is enhanced by releasing feedback inhibition of valine to the ilvH, preferably, the ilvH gene enhanced by mutation.

17. The construction method according to claim 2, wherein the AHAS is ilvBN, or ilvGM, or ilvIH; optionally, the activity of the ilvIH is enhanced by releasing feedback inhibition of valine to the ilvH, preferably, the ilvH gene enhanced by mutation.

18. The construction method according to claim 2, wherein the item (7) is selected for modification.

19. The construction method according to claim 2, wherein the items (7) and (2) are selected for modification.

20. The construction method according to claim 2, wherein the items (7) and (6) are selected for modification.

21. A method for producing L-valine, wherein the method comprises: (1) fermenting and culturing the recombinant microorganism according to claim 8; and (2) separating and harvesting L-valine; preferably, the fermentation is carried out under anaerobic conditions.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Drawings of the description for constituting a part of the present disclosure are used to provide further understanding of the disclosure. Exemplary embodiments of the disclosure and descriptions thereof are used to explain the disclosure, and do not constitute improper limitation to the disclosure. In the drawings:

[0097] FIG. 1: L-valine synthesis pathway.

[0098] FIG. 2: Determination of a standard substance of L-valine by high performance liquid chromatography.

[0099] FIG. 3: Determination of fermentation solution components of strain Sval048 by high performance liquid chromatography.

[0100] FIG. 4: construction of strain Sval049 by metabolic evolution.

[0101] FIG. 5: Determination of a standard substance of L-valine by high performance liquid chromatography.

[0102] FIG. 6: Determination of fermentation solution components of strain Sval049 by high performance liquid chromatography.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0103] The disclosure is further described by the following embodiments, but any embodiments or combinations thereof should not be interpreted as limiting a scope or an embodiment of the disclosure. The scope of the disclosure is defined by appended claims. In combination with the description and common knowledge in the field, those of ordinary skill in the art may clearly understand the scope defined by the claims. Without departing from the spirit and scope of the disclosure, those skilled in the art may make any modifications or changes to technical schemes of the disclosure, and such modifications and changes are also included in the scope of the disclosure.

[0104] Experimental methods used in the following embodiments are conventional methods unless otherwise specified. Materials, reagents and the like used in the following embodiments may be obtained from commercial sources unless otherwise specified.

[0105] Strains and plasmids constructed in this research are shown in Table 1, and primers used are shown in Table 2.

TABLE-US-00001 TABLE 1 Strains and plasmids used in the disclosure Related characteristics Sources Strain ATCC Wild type Laboratory preservation 8739 M1-93 ATCC 8739, Lu, et al., Appl Microbiol FRT-Km-FRT::M1-93::lacZ Biotechnol, 2012, 93: 2455- 2462 M1-46 ATCC 8739, Lu, et al., Appl Microbiol FRT-Km-FRT::M1-46::lacZ Biotechnol, 2012, 93: 2455- 2462 M1-37 ATCC 8739, Lu, et al., Appl Microbiol FRT-Km-FRT::M1-37::lacZ Biotechnol, 2012, 93: 2455- 2462 Sval001 ATCC 8739, mgsA::cat-sacB Constructed by the disclosure Sval002 Sval001, ΔmgsA Constructed by the disclosure Sval003 Sval002, ldhA::cat-sacB Constructed by the disclosure Sval004 Sval003, ΔldhA Constructed by the disclosure Sval005 Sval004, ackA-pta::cat-sacB Constructed by the disclosure Sval006 Sval005, Δ ackA-pta Constructed by the disclosure Sval007 Sval006, tdcDE::cat-sacB Constructed by the disclosure Sval008 Sval007, ΔtdcDE Constructed by the disclosure Sval009 Sval008, adhE::cat-sacB Constructed by the disclosure Sval010 Sval009, ΔadhE Constructed by the disclosure Sval011 Sval010, mgsA::cat-sacB Constructed by the disclosure Sval012 Sval011, mgsA::ilvC Constructed by the disclosure Sval013 Sval012, mgsA::cat-sacB::ilvC Constructed by the disclosure Sval014 Sval013, mgsA::M1-46-ilvC Constructed by the disclosure Sval015 Sval014, pflB::cat-sacB Constructed by the disclosure Sval016 Sval015, pflB::ilvD Constructed by the disclosure Sval017 Sval016, pflB::cat-sacB::ilvD Constructed by the disclosure Sval018 Sval017, pflB::RBS4-ilvD Constructed by the disclosure Sval019 Sval018, cat-sacB::ilvB Constructed by the disclosure Sval020 Sval019, M1-93:: ilvB Constructed by the disclosure Sval021 Sval020, cat-sacB::ilvG Constructed by the disclosure Sval022 Sval021, M1-93:: ilvG Constructed by the disclosure Sval023 Sval022, ilvH::cat-sacB Constructed by the disclosure Sval024 Sval023, ilvH:: ilvH* Constructed by the disclosure Sval025 Sval024, frd::cat-sacB Constructed by the disclosure Sval026 Sval025, frd::M1-93-leuDH Constructed by the disclosure Sval041 Sval026, cat-sacB::zwf Constructed by the disclosure Sval042 Sval041, RBS1-zwf Constructed by the disclosure Sval043 Sval042, cat-sacB::pgl Constructed by the disclosure Sval044 Sval043, RBS2-pgl Constructed by the disclosure Sval045 Sval044, edd-eda::cat-sacB Constructed by the disclosure Sval046 Sval045, edd-eda::MRS1-edd- Constructed by the disclosure eda Sval047 Sval046, pfkA::cat-sacB Constructed by the disclosure Sval048 Sval047, ΔpfkA Constructed by the disclosure Sval049 metabolic evolution of of Constructed by the disclosure Sval048 for 100 generations, CGMCC 19457 Plasmid pUC57- Artificial regulatory element Nanjing Genscript M1-93- M1-93 and chemically synthe- Biotechnology Co., Ltd. leuDH sized gene leuDH are linked to a pUC57 vector together pUC57- Artificial regulatory element Nanjing Genscript MRS1- MRS1 and chemically synthe- Biotechnology Co., Ltd. edd-eda sized genes edd and eda are linked to the pUC57 vector together

TABLE-US-00002 TABLE 2 Primers used in the disclosure Sequence Primer name Sequence number mgsA-cs-up gtaggaaagttaactacggatgtacattatggaactgacgactcgcacttTGTGA 1 CGGAAGATCACTTCGCAG mgsA-cs-down gcgtttgccacctgtgcaatattacttcagacggtccgcgagataacgctTTATTT 2 GTTAACTGTTAATTGTCCT XZ-mgsA-up cagctcatcaaccaggtcaa 3 XZ-mgsA-down aaaagccgtcacgttattgg 4 mgsA-del-down Gcgtttgccacctgtgcaatattacttcagacggtccgcgagataacgctaagtgcg 5 agtcgtcagttcc mgsA-ilvC-up gtaggaaagttaactacggatgtacattatggaactgacgactcgcacttATGGC 6 TAACTACTTCAATACac mgsA-ilvC-down gcgtttgccacctgtgcaatattacttcagacggtccgcgagataacgctTTAACC 7 CGCAACAGCAATACGtttc mgsA-Pcs-up gtaggaaagttaactacggatgtacattatggaactgacgactcgcacttTGTGA 8 CGGAAGATCACTTCGCAG mgsA-Pcs-down agctgtgccagctgctggcgcagattcagtGTATTGAAGTAGTTAGCCA 9 TTTATTTGTTAACTGTTAATTGTCCT mgsA-P46-up gtaggaaagttaactacggatgtacattatggaactgacgactcgcacttTTATCT 10 CTGGCGGTGTTGAC ilvC-P46-down agctgtgccagctgctggcgcagattcagtGTATTGAAGTAGTTAGCCA 11 TAGCTGTTTCCTGGTTTAAACCG ilvC-YZ347-down cgcactacatcagagtgctg 12 IdhA-cs-up ttcaacatcactggagaaagtcttatgaaactcgccgtttatagcacaaaTGTGA 13 CGGAAGATCACTTCGCAG IdhA-cs-down agcggcaagattaaaccagttcgttcgggcaggtttcgcctttttccagaTTATTT 14 GTTAACTGTTAATTGTCCT XZ-IdhA-up GATAACGGAGATCGGGAATG 15 XZ- CTTTGGCTGTCAGTTCACCA 16 IdhA-down IdhA-del-down agcggcaagattaaaccagttcgttcgggcaggtttcgcctttttccagatttgtgctat 17 aaacggcgagt ackA-cs-up aggtacttccatgtcgagtaagttagtactggttctgaactgcggtagttTGTGAC 18 GGAAGATCACTTCGCAG pta-cs-down ggtcggcagaacgctgtaccgctttgtaggtggtgttaccggtgttcagaTTATTT 19 GTTAACTGTTAATTGTCCT XZ-ackA-up cgggacaacgttcaaaacat 20 XZ-pta-down attgcccatcttcttgttgg 21 ackA-del-down ggtcggcagaacgctgtaccgctttgtaggtggtgttaccggtgttcagaaactaccg 22 cagttcagaacca tdcDE-cs-up ccgtgattggtctgctgaccatcctgaacatcgtatacaaactgttttaaTGTGAC 23 GGAAGATCACTTCGCAG tdcDE-cs-down cgcctggggcacgttgcgtttcgataatctttttcatacatcctccggcgTTATTTG 24 TTAACTGTTAATTGTCCT XZ-tdcDE-up TGATGAGCTACCTGGTATGGC 25 XZ-tdcDE-down CGCCGACAGAGTAATAGGTTTTAC 26 tdcDE-del-down cgcctggggcacgttgcgtttcgataatctttttcatacatcctccggcgttaaaacagt 27 ttgtatacgatgttcag adhE-cs-up ATAACTCTAATGTTTAAACTCTTTTAGTAAATCACAGTGAG 28 TGTGAGCGCTGTGACGGAAGATCACTTCGCA adhE-cs-down CCGTTTATGTTGCCAGACAGCGCTACTGATTAAGCGGATT 29 TTTTCGCTTTTTATTTGTTAACTGTTAATTGTCCT adhE-del-down CCGTTTATGTTGCCAGACAGCGCTACTGATTAAGCGGATT 30 TTTTCGCTTTGCGCTCACACTCACTGTGATTTAC XZ-adhE-up CATGCTAATGTAGCCACCAAA 31 XZ-adhE-down TTGCACCACCATCCAGATAA 32 pflB-CS-up aaacgaccaccattaatggttgtcgaagtacgcagtaaataaaaaatccaTGTG 33 ACGGAAGATCACTTCGCAG pflB-CS-down CGGTCCGAACGGCGCGCCAGCACGACGACCGTCTGGG 34 GTGTTACCCGTTTTTATTTGTTAACTGTTAATTGTCCT pflB-ilvD-up aaacgaccaccattaatggttgtcgaagtacgcagtaaataaaaaatccaatgcct 35 aagtaccgttccgc pfIB-ilvD-down CGGTCCGAACGGCGCGCCAGCACGACGACCGTCTGGG 36 GTGTTACCCGTTTttaaccccccagtttcgatttatc XZ-pflB-up600 CTGCGGAGCCGATCTCTTTAC 37 XZ-pflB-down CGAGTAATAACGTCCTGCTGCT 38 pflB-Pcs-up aaacgaccaccattaatggttgtcgaagtacgcagtaaataaaaaatccaTGTG 39 ACGGAAGATCACTTCGCA pflB-Pcs-down CCCGCCATATTACGACCATGAGTGGTGGTGGCGGAACGG 40 TACTTAGGCATTTATTTGTTAACTGTTAATTGTCCT pflB-Pro-up AAACGACCACCATTAATGGTTGTCGAAGTACGCAGTAAAT 41 AAAAAATCCATTATCTCTGGCGGTGTTGAC ilvD-Pro-down cccgccatattacgaccatgagtggtggtggcggaacggtacttaggcatTGCT 42 GACCTCCTGGTTTAAACGTACATG ilvD-YZ496-down caaccagatcgagcttgatg 43 XZ-frd-up TGCAGAAAACCATCGACAAG 44 XZ-frd-down CACCAATCAGCGTGACAACT 45 frd-cs-up GAAGGCGAATGGCTGAGATGAAAAACCTGAAAATTGAGG 46 TGGTGCGCTATTGTGACGGAAGATCACTTCGCA frd-cs-down TCTCAGGCTCCTTACCAGTACAGGGCAACAAACAGGATT 47 ACGATGGTGGCTTATTTGTTAACTGTTAATTGTCCT frd-M93-up GAAGGCGAATGGCTGAGATGAAAAACCTGAAAATTGAGG 48 TGGTGCGCTATTTATCTCTGGCGGTGTTGAC frd-leuDH-down TCTCAGGCTCCTTACCAGTACAGGGCAACAAACAGGATT 49 ACGATGGTGGCTTAACGGCCGTTCAAAATATTTTTTTC ilvB pro-catup ctgacgaaacctcgctccggcggggttttttgttatctgcaattcagtacTGTGAC 50 GGAAGATCACTTCGCA ilvB tctgcgccggtaaagcgcttacgcgtcgatgttgtgcccgaacttgccatTTATTT 51 pro-catdown GTTAACTGTTAATTGTCCT ilvB pro-up ctgacgaaacctcgctccggcggggttttttgttatctgcaattcagtacTTATCTC 52 TGGCGGTGTTGAC ilvB pro-down tctgcgccggtaaagcgcttacgcgtcgatgttgtgcccgaacttgccatAGCTG 53 TTTCCTGGTTTAAAC ilvB pro-YZup gttctgcgcggaacacgtatac 54 ilvB ccgctacaggccatacagac 55 pro-YZdown ilvG tgaactaagaggaagggaacaacattcagaccgaaattgaatttttttcaTGTGA 56 pro-catup CGGAAGATCACTTCGCA ilvG ttcacaccctgtgcccgcaacgcatgtaccacccactgtgcgccattcatTTATTT 57 pro-catdown GTTAACTGTTAATTGTCCT ilvG pro-up tgaactaagaggaagggaacaacattcagaccgaaattgaatttttttcaTTATC 58 TCTGGCGGTGTTGAC ilvG ttcacaccctgtgcccgcaacgcatgtaccacccactgtgcgccattcatAGCTG 59 pro-down TTTCCTGGTTTAAACG ilvG gcataagatatcgctgctgtag 60 pro-YZup ilvG gccagttttgccagtagcac 61 p-YZdown ilvH*-cat-up agaacctgattatgCGCCGGATATTATCAGTCTTACTCGAAAATG 62 AATCATGTGACGGAAGATCACTTCGCA ilvH*-cat-down TTCATCGCCCACGGTCTGGATGGTCATACGCGATAATGTC 63 GGATCGTCGGTTATTTGTTAACTGTTAATTGTCCT ilvH*-mut-up agaacctgattatgCGCCGGATATTATCAGTCTTACTCGAAAATG 64 AATCAGaCGCGTTATtCCGCGTGATTGGC ilvH*-mut-down CACACCAGAGCGAGCAACCTC 65 ilvH*-mutYZ- atgagctggaaagcaaacttagc 66 up Zwf-Pcat-up agttttgccgcactttgcgcgcttttcccgtaatcgcacgggtggataagTGTGAC 67 GGAAGATCACTTCGCA Zwf-PsacB-down qcqccqaaaatqaccaqqtcacaqqcctqqqctqtttqcqttaccqccatTTATT 68 TGTTAACTGTTAATTGTCCT zwf-RBS1-up agttttgccgcactttgcgcgcttttcccgtaatcgcacgggtggataagTTATCTC 69 TGGCGGTGTTGAC zwf-RBS1-down gcgccgaaaatgaccaggtcacaggcctgggctgtttgcgttaccgccatATTG 70 TTTCTCCTGGTTTAAACGTACGTG zwf-YZ442-up cgaatggatcgcgttatcgg 71 zwf-YZ383-down caaattgcgccaaaagtgctg 72 pgl-Pcat-up ttcaqcattcaccqccaaaaqcqactaattttaqctqttacaqtcaqttqTGTGAC 73 GGAAGATCACTTCGCA pgl-PsacB-down acqtqaatttqctqqctctcaqqqctqqcqatataaactqtttqcttcatTTATTTG 74 TTAACTGTTAATTGTCCT pgl-RBS2-up ttcagcattcaccgccaaaagcgactaattttagctgttacagtcagttgTTATCTC 75 TGGCGGTGTTGAC pgl-RBS2-down acgtgaatttgctggctctcagggctggcgatataaactgtttgcttcatACGTTTC 76 CTCCTGGTTTAAACGTACATGCTAACAATAC pgl-YZ308-up gatgaatagcgacgtgatgg 77 pgl-YZ341-down ccatcttccagacgcgttac 78 Edd-cat-up tggtcgttcctggaatgagtttgagtaatatctgcgcttatcctttatggTGTGACG 79 GAAGATCACTTCGCA Eda-sacB-down gcaaaaaaacgctacaaaaatgcccgatcctcgatcgggcattttgacttTTATT 80 TGTTAACTGTTAATTGTCCT Edd-int-up tggtcgttcctggaatgagtttgagtaatatctgcgcttatcctttatggTTATCTCT 81 GGCGGTGTTGAC Eda-int-down gcaaaaaaacgctacaaaaatgcccgatcctcgatcgggcattttgacttTTAG 82 GCAACAGCAGCGCGCTTG Edd-YZ-up gcatctggcggatgcctatg 83 Edd-YZ-down caactgaccagtcagaatgtcac 84 pfkAdel-cat-up GGTATCGACGCGCTGGTGGTTATCGGCGGTGACGGTTC 85 CTACATGGGTGCTGTGACGGAAGATCACTTCGCA pfkAdel-sacB- GTGGCCCAGCACAGTTGCGCGGGTTTCACGACCGGTTT 86 down CTTTCTCGATGATTATTTGTTAACTGTTAATTGTCCT pfkAdel-up ATGATTAAGAAAATCGGTGTG 87 pfkAdel-down GTGGCCCAGCACAGTTGCGCGGGTTTCACGACCGGTTT 88 CTTTCTCGATGAGCACCCATGTAGGAACCGTC pfkAdel-YZ-down GTCGATGATGTCGTGGTGAAC 89

EXAMPLE 1

Knockout of Methylglyoxal Synthase Encoding Gene mgsA in ATCC 8739 Strain

[0106] Started from Escherichia coli ATCC 8739, a two-step homologous recombination method is used to knock out the methylglyoxal synthase encoding gene mgsA, and specific steps are as follows.

[0107] In a first step, a pXZ-CS plasmid DNA is used as a template, 2719 bp of a DNA fragment I is amplified by using primers mgsA-cs-up/mgsA-cs-down, and used for the first step of homologous recombination.

[0108] An amplification system is: Phusion 5× buffer (NewEngland Biolabs) 10 μl, dNTP (10 mM for each dNTP) 1 μl, DNAtemplate 20 ng, primers (10 μM) 2 μl each, Phusion High-Fidelity DNA polymerase (NewEngland Biolabs) (2.5 U/μl) 0.5 μl, distilled water 33.5 μl, and a total volume is 50 μl.

[0109] Amplification conditions are 98° C. pre-denaturation for 2 minutes (1 cycle); 98° C. denaturation for 10 seconds, 56° C. annealing for 10 seconds, 72° C. extension for 2 minutes (30 cycles); and 72° C. extension for 10 minutes (1 cycle).

[0110] The above DNA fragment I is used for the first homologous recombination: firstly, a pKD46 plasmid (purchased from the Coil Genetic Stock Center (CGSC) of Yale University, CGSC#7739) is transformed into Escherichia coli ATCC 8739 by an electrotransformation method, and then the DNA fragment I is electrotransformed to the Escherichia coli ATCC 8739 with the pKD46.

[0111] Electrotransformation conditions are as follows: firstly, electrotransformation competent cells of the Escherichia coli ATCC 8739 with the pKD46 plasmid are prepared; 50 μl of the competent cells are placed on ice, and 50 ng of the DNA fragment I is added. The mixture was placed on ice for 2 minutes, and transferred into a 0.2 cm MicroPulser Electroporation Cuvette (Bio-Rad). The electroporation was carried with the MicroPulser (Bio-Rad) electroporation apparatus and the electric voltage was 2.5 kV. After electric shock, 1 ml of LB medium was quickly added into the electroporation cuvette, and transferred into a test tube after pipetting five times. The culture was incubated at 30° C. with shaking at 75 rpm for 2 hours. 200 μl of culture was spread onto a LB plate containing ampicillin (a final concentration is 100 μg/ml) and chloramphenicol (a final concentration is 34 μg/ml). After being cultured overnight at 30° C., colonies were verified with primer set XZ-mgsA-up/XZ-mgsA-down, and a correct colony amplification product is a 3646 bp fragment. A correct single colony was selected, and named as Sval001.

[0112] In a second step, a genomic DNA of wild-type Escherichia coli ATCC 8739 is used as template, and 566 bp of a DNA fragment II is amplified with primer set XZ-mgsA-up/mgsA-del-down. DNA fragment II is used for the second homologous recombination. Amplification conditions and system are the same as those described in the first step. The DNA fragment II is electrotransformed into strain Sval001.

[0113] Electrotransformation conditions are as follows: firstly, electrotransformation competent cells of the Sval001 with the pKD46 plasmid (Dower et al., 1988, Nucleic Acids Res 16:6127-6145) were prepared; 50 μl of the competent cells were placed on ice, and 50 ng of a DNAfragment II is added. The mixture was placed on ice for 2 minutes, and transferred into a 0.2 cm MicroPulser Electroporation Cuvette (Bio-Rad). The electroporation was carried with the MicroPulser (Bio-Rad) electroporation apparatus and the electric voltage was 2.5 kV. After electric shock, 1 ml of LB medium was quickly added into the electroporation cuvette, and transferred into a test tube after pipetting five times. The culture was incubated at 30° C. with shaking at 75 rpm for 4 hours. The culture was then transferred into LB medium containing 10% sucrose but without a sodium chloride (50 ml of a medium is loaded in 250 ml of a flask), and after being cultured for 24 hours, it is streak-cultured on an LB solid medium containing 6% sucrose without a sodium chloride. The correct clone was verified by colony PCR amplification with primer set XZ-mgsA-up/XZ-mgsA-down, and a correct colony amplification product was 1027 bp. A correct single colony is then selected, and named as Sval002 (Table 1).

EXAMPLE 2

Knockout of Lactate Dehydrogenase Encoding Gene IdhA

[0114] Started from Sval002, and a lactate dehydrogenase encoding gene IdhA is knocked out by a two-step homologous recombination method. Specific steps are as follows.

[0115] In a first step, a pXZ-CS plasmid DNA is used as a template, 2719 bp of a DNA fragment I is amplified by using primers IdhA-cs-up/IdhA-cs-down, and used for the first step of the homologous recombination. Amplification system and amplification conditions are the same as those described in Example 1. The DNA fragment I is electrotransformed to the Sval002.

[0116] The DNA fragment I is used for the first homologous recombination: firstly, a pKD46 plasmid is transformed into Escherichia coli Sval002 by an electrotransformation method, and then the DNA fragment I is electrotransformed into the Escherichia coli Sval002 with the pKD46.

[0117] Electrotransformation conditions and steps are the same as the first step method for the mgsA gene knockout described in Example 1. 200 μl of culture solution is spreaded onto a LB plate containing ampicillin (a final concentration is 100 μg/ml) and chloramphenicol (a final concentration is 34 μg/ml). After being cultured overnight at 30° C., colonies were PCR verified with primer set XZ-IdhA-up/XZ-IdhA-down, and a correct PCR product should be 3448 bp. A correct single colony is picked, and named as Sval003.

[0118] In a second step, a DNA of wild-type Escherichia coli ATCC 8739 is used as a template, and 476 bp of a DNA fragment II is amplified with primers XZ-IdhA-up/IdhA-del-down. The DNA fragment II is used for the second homologous recombination. The DNA fragment II is electrotransformed into strain Sval003.

[0119] Electrotransformation conditions and steps are the same as the second step method for the mgsA gene knockout described in Example 1. Colony PCR is used to verify clones, the used primers are XZ-IdhA-up/XZ-IdhA-down, and a correct colony amplification product is 829 bp. A correct single colony is picked, and named as Sval004 (Table 1).

EXAMPLE 3

Knockout of Phosphoacetyl Transferase Encoding Gene pta and Acetate Kinase Encoding Gene ackA

[0120] Started from Sval004, a two-step homologous recombination method is used to knock out a phosphoacetyl transferase encoding gene pta and an acetate kinase encoding gene ackA. Specific steps are as follows.

[0121] In a first step, a pXZ-CS plasmid DNA is used as a template, 2719 bp of a DNA fragment I is amplified by using primers ackA-cs-up/pta-cs-down, and used for the first step of the homologous recombination. Amplification system and amplification conditions are the same as those described in Example 1. The DNA fragment I is electrotransformed to the Sval004.

[0122] The DNA fragment I is used for the first homologous recombination: firstly, a pKD46 plasmid is transformed into Escherichia coli Sval004 by an electrotransformation method, and then the DNA fragment I is electrotransformed into the Escherichia coli Sval004 with the pKD46.

[0123] Electrotransformation conditions and steps are the same as the first step method for the mgsA gene knockout described in Example 1. 200 μl of bacterial solution is spreaded onto an LB plate containing ampicillin (a final concentration is 100 μg/ml) and chloramphenicol (a final concentration is 34 μg/ml). After being cultured overnight at 30° C., colonies were PCR verified using primers XZ-ackA-up/XZ-pta-down, and a correct PCR product should be 3351 bp. A correct single colony is picked, and named as Sval005.

[0124] In a second step, a DNA of wild-type Escherichia coli ATCC 8739 is used as a template, and 371 bp of a DNA fragment II is amplified with primers XZ-ackA-up/ackA-del-down. The DNA fragment II is used for the second homologous recombination. The DNA fragment II is electrotransformed into strain Sval005.

[0125] Electrotransformation conditions and steps are the same as the second step method for the mgsA gene knockout described in Example 1. Colony PCR is used to verify clones using primers XZ-ackA-up/XZ-pta-down, and a correct colony amplification product is 732 bp. A correct single colony is picked, and named as Sval006 (Table 1).

EXAMPLE 4

Knockout of Propionate Kinase Encoding Gene tdcD and Formate Acetyltransferase Encoding Gene tdcE

[0126] Started from Sval006, a two-step homologous recombination method is used to knock out the propionate kinase encoding gene tdcD and the formate acetyltransferase encoding gene tdcE. Specific steps are as follows.

[0127] In a first step, a pXZ-CS plasmid DNA is used as a template, 2719 bp of a DNA fragment I is amplified by using primers tdcDE-cs-up/tdcDE-cs-down, and used for the first step of the homologous recombination. Amplification system and amplification conditions are the same as those described in Example 1. The DNA fragment I is electrotransformed to the Sval006.

[0128] The DNA fragment I is used for the first homologous recombination: firstly, a pKD46 plasmid is transformed into Escherichia coli Sval006 by an electrotransformation method, and then the DNA fragment I is electrotransformed into the Escherichia coli Sval006 with the pKD46.

[0129] Electrotransformation conditions and steps are the same as the first step method for the mgsA gene knockout described in Example 1. 200 μl of culture solution is spreaded onto a LB plate containing ampicillin (a final concentration is 100 μg/ml) and chloramphenicol (a final concentration is 34 μg/ml). After being cultured overnight at 30° C., colonies were PCR verified using primers XZ-tdcDE-up/XZ-tdcDE-down, and a correct PCR product should be 4380 bp. A correct single colony is picked, and named as Sval007.

[0130] In a second step, a DNA of wild-type Escherichia coli ATCC 8739 is used as a template, and 895 bp of a DNA fragment II is amplified with primers XZ-tdcDE-up/tdcDE-del-down. The DNAfragment II is used for the second homologous recombination. The DNA fragment II is electrotransformed into strain Sval007.

[0131] Electrotransformation conditions and steps are the same as the second step method for the mgsA gene knockout described in Example 1. Colonies were PCR verified using primers XZ-tdcDE-up/XZ-tdcDE-down, and a correct colony amplification product is 1761 bp. A correct single colony is picked, and named as Sval008 (Table 1).

EXAMPLE 5

Knockout of Alcohol Dehydrogenase Gene adhE

[0132] Started from Sval008, a two-step homologous recombination method is used to knock out the alcohol dehydrogenase gene adhE. Specific steps are as follows.

[0133] In a first step, a pXZ-CS plasmid DNA is used as a template, 2719 bp of a DNA fragment I is amplified by using primers adhE-cs-up/adhE-cs-down, and used for the first step of the homologous recombination. Amplification system and amplification conditions are the same as those described in Example 1.

[0134] The DNA fragment I is used for the first homologous recombination: firstly, a pKD46 plasmid is transformed into Escherichia coli Sval008 by an electrotransformation method, and then the DNA fragment I is electrotransformed into the Escherichia coli Sval008 with the pKD46.

[0135] Electrotransformation conditions and steps are the same as the first step method for the mgsA gene knockout described in Example 1. 200 μl of bacterial solution is spreaded onto a LB plate containing ampicillin (a final concentration is 100 μg/ml) and chloramphenicol (a final concentration is 34 μg/ml). After being cultured overnight at 30° C., colonies were PCR verified using primers XZ-adhE-up/XZ-adhE-down, and a correct PCR product should be 3167 bp. A correct single colony is picked, and named as Sval009.

[0136] In a second step, a DNA of wild-type Escherichia coli ATCC 8739 is used as a template, and 271 bp of a DNA fragment II is amplified with primers XZ-adhE-up/adhE-del-down. The DNA fragment II is used for the second homologous recombination. The DNA fragment II is electrotransformed into strain Sval009.

[0137] Electrotransformation conditions and steps are the same as the second step method for the mgsA gene knockout described in Example 1. Colonies were PCR verified using primers XZ-adhE-up/XZ-adhE-down, and a correct colony amplification product is 548 bp. A correct single colony is picked, and named as Sval010 (Table 1).

EXAMPLE 6

Integration of Acetohydroxy Acid Reductoisomerase Encoding Gene ilvC in Methylglyoxal Synthase Encoding Gene mgsA Site

[0138] Started from Sval010, an acetohydroxy acid reductoisomerase encoding gene ilvC from Escherichia coli is integrated into the methylglyoxal synthase encoding gene mgsA site through a two-step homologous recombination method. Specific steps are as follows.

[0139] In a first step, a cat-sacB fragment is integrated into the mgsA site of strain Sval010. PCR, integration, and verification of the cat-sacB fragment are exactly the same as the first step of the mgsA gene knockout in Example 1, and an obtained clone is named as Sval011.

[0140] In a second step, a DNA of wild-type Escherichia coli ATCC 8739 is used as a template, 1576 bp of a DNA fragment II is amplified by using primers mgsA-ilvC-up/mgsA-ilvC-down. The DNA fragment II is used for the second homologous recombination. The DNA fragment II is electrotransformed into strain Sval011.

[0141] Electrotransformation conditions and steps are the same as the second step method for the mgsA gene knockout described in Example 1. Colonies were PCR verified using primers XZ-mgsA-up/XZ-mgsA-down and sequenced, and a correct colony amplification product is 2503 bp. A correct single colony is picked, and named as Sval012 (Table 1).

EXAMPLE 7

Regulation of Acetohydroxy Acid Reductoisomerase Encoding Gene ilvC

[0142] Started from Sval012, and an artificial regulatory element is used to regulate expression of the acetohydroxy acid reductoisomerase encoding gene ilvC integrated in a methylglyoxal synthase encoding gene mgsA site. Specific steps are as follows.

[0143] In a first step, a pXZ-CS plasmid DNA is used as a template, 2719 bp of a DNA fragment I is amplified by using primers mgsA-Pcs-up/mgsA-Pcs-down, and used for the first step of the homologous recombination. Amplification system and amplification conditions are the same as those described in Example 1. The DNAfragment I is electrotransformed into the Sval012.

[0144] The DNA fragment I is used for the first homologous recombination: firstly, a pKD46 plasmid (purchased from the Coil Genetic Stock Center (CGSC) of Yale University, CGSC#7739) is transformed into Escherichia coli Sval012 by an electrotransformation method, and then the DNA fragment I is electrotransformed into the Escherichia coli Sval012 with the pKD46.

[0145] Electrotransformation conditions and steps are the same as the first step method for the mgsA gene knockout described in Example 1.200 μl of bacterial solution is spreaded onto a LB plate containing ampicillin (a final concentration is 100 μg/ml) and chloramphenicol (a final concentration is 34 μg/ml). After being cultured overnight at 30° C., colonies were PCR verified using primers XZ-mgsA-up/ilvC-YZ347-down, and a correct PCR product should be 3482 bp. A correct single colony is picked, and named as Sval013.

[0146] In a second step, a genomic DNA of M1-46 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93:2455-2462) is used as a template, and 188 bp of a DNA fragment II is amplified by using primers mgsA-P46-up/ilvC-P46-down. The DNA fragment II is used for the second homologous recombination. The DNA fragment II is electrotransformed into strain Sval013.

[0147] Electrotransformation conditions and steps are the same as the second step method for the mgsA gene knockout described in Example 1. Colonies were PCR verified using primers XZ-mgsA-up/ilvC-YZ347-down and sequenced, and a correct colony amplification product is 951 bp. A correct single colony is picked, and named as Sval014 (Table 1).

EXAMPLE 8

Integration of Dihydroxy Acid Dehydratase Encoding Gene ilvD

[0148] Started from Sval014, a dihydroxy acid dehydratase encoding gene ilvD from Escherichia coli is integrated into the pyruvate formate lyase encoding gene pflB site and replaces the pflB gene through a two-step homologous recombination method, namely the pflB gene is knocked out while the ilvD is integrated. Specific steps are as follows.

[0149] In a first step, a pXZ-CS plasmid DNA is used as a template, 2719 bp of a DNA fragment I is amplified by using primers pflB-CS-up/pflB-CS-down, and used for the first step of the homologous recombination. Amplification system and amplification conditions are the same as those described in Example 1. The DNA fragment I is electrotransformed into the Sval014.

[0150] The DNA fragment I is used for the first homologous recombination: firstly, a pKD46 plasmid is transformed into Escherichia coli Sval014 by an electrotransformation method, and then the DNA fragment I is electrotransformed into the Escherichia coli Sval014 with the pKD46.

[0151] Electrotransformation conditions and steps are the same as the first step method for the mgsA gene knockout described in Example 1. 200 μl of bacterial solution is spreaded onto a LB plate containing ampicillin (a final concentration is 100 μg/ml) and chloramphenicol (a final concentration is 34 μg/ml). After being cultured overnight at 30° C., colonies were PCR verified using primers XZ-pflB-up600/XZ-pflB-down, and a correct PCR product should be 3675 bp. A correct single colony is picked, and named as Sval015.

[0152] In a second step, a genomic DNA of Escherichia coli MG1655 (from ATCC, No. 700926) is used as a template, and 1951 bp of a DNA fragment II is amplified by using primers pflB-ilvD-up/pflB-ilvD-down. The DNAfragment II is used for the second homologous recombination. The DNA fragment II is electrotransformed into strain Sval015.

[0153] Electrotransformation conditions and steps are the same as the second step method for the mgsA gene knockout described in Example 1. Colonies were PCR verified using primers XZ-pflB-up600/XZ-pflB-down and sequenced, and a correct colony amplification product is 2907 bp. A correct single colony is picked, and named as Sval016 (Table 1).

EXAMPLE 9

Regulation of Dihydroxy Acid Dehydratase Encoding Gene ilvD

[0154] Started from Sval016, and an artificial regulatory element is used to regulate expression of the dihydroxy acid dehydratase encoding gene ilvD integrated in the pyruvate formate lyase encoding gene pflB site. Specific steps are as follows.

[0155] In a first step, a pXZ-CS plasmid DNA is used as a template, 2719 bp of a DNA fragment I is amplified by using primers pflB-Pcs-up/pflB-Pcs-down, and used for the first step of the homologous recombination. Amplification system and amplification conditions are the same as those described in Example 1. The DNA fragment I is electrotransformed into the Sval016.

[0156] The DNA fragment I is used for the first homologous recombination: firstly, a pKD46 plasmid is transformed into Escherichia coli Sval016 by an electrotransformation method, and then the DNA fragment I is electrotransformed into the Escherichia coli Sval016 with the pKD46.

[0157] Electrotransformation conditions and steps are the same as the first step method for the mgsA gene knockout described in Example 1.200 μl of bacterial solution is spreaded onto a LB plate containing ampicillin (a final concentration is 100 μg/ml) and chloramphenicol (a final concentration is 34 μg/ml). After being cultured overnight at 30° C., colonies were PCR verified using primers XZ-pflB-up600/ilvD-YZ496-down, and a correct PCR product should be 3756 bp. A correct single colony is picked, and named as Sval017.

[0158] In a second step, a genomic DNA of M1-93 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93:2455-2462) is used as a template, and 189 bp of a DNA fragment II is amplified by using primers pflB-Pro-up/ilvD-Pro-down. The DNA fragment II is used for the second homologous recombination. The DNA fragment II is electrotransformed into strain Sval017.

[0159] Electrotransformation conditions and steps are the same as the second step method for the mgsA gene knockout described in Example 1. Colonies were PCR verified using primers XZ-pflB-up600/ilvD-YZ496-down and sequenced, and a correct colony amplification product is 1226 bp. A correct single colony is picked, and named as Sval018 (Table 1).

EXAMPLE 10

Regulation of Acetolactate Synthase Gene ilvBN

[0160] An artificial regulatory element M1-93 is used to regulate expression of an acetolactate synthase gene ilvBN through a two-step homologous recombination method. Specific steps are as follows.

[0161] In a first step, a pXZ-CS plasmid DNA is used as a template, 2719 bp of a DNA fragment I is amplified by using primers ilvB pro-catup/ilvB pro-catdown, and used for the first step of the homologous recombination. Amplification system and amplification conditions are the same as those described in Example 1.

[0162] The DNA fragment I is used for the first homologous recombination: firstly, a pKD46 plasmid is transformed into Escherichia coli Sval018 by an electrotransformation method, and then the DNA fragment I is electrotransformed into the Escherichia coli Sval018 with the pKD46.

[0163] Electrotransformation conditions and steps are the same as the first step method for the mgsA gene knockout described in Example 1. 200 μl of bacterial solution is spreaded onto a LB plate containing ampicillin (a final concentration is 100 μg/ml) and chloramphenicol (a final concentration is 34 μg/ml). After being cultured overnight at 30° C., colonies were PCR verified using primers ilvB pro-YZup/ilvB pro-YZdown, and a correct PCR product should be 2996 bp. A correct single colony is picked, and named as Sval019.

[0164] In a second step, a genomic DNA of M1-93 is used as a template, and 188 bp of a DNA fragment II is amplified by using primers ilvB pro-up/ilvB pro-down. The DNA fragment II is used for the second homologous recombination. The DNA fragment II is electrotransformed into strain Sval019.

[0165] Electrotransformation conditions and steps are the same as the second step method for the mgsA gene knockout described in Example 1. Colonies were PCR verified using primers ilvB pro-YZup/ilvB pro-YZdown, and a correct colony amplification product is 465 bp. A correct single colony is picked, and named as Sval020.

EXAMPLE 11

Regulation of Acetolactate Synthase Gene ilvGM

[0166] An artificial regulatory element M1-93 is used to regulate expression of the acetolactate synthase gene ilvGM through a two-step homologous recombination method. Specific steps are as follows.

[0167] In a first step, a pXZ-CS plasmid DNA is used as a template, 2719 bp of a DNA fragment I is amplified by using primers ilvG pro-catup/ilvG pro-catdown, and used for the first step of the homologous recombination. Amplification system and amplification conditions are the same as those described in Example 1.

[0168] The DNA fragment I is used for the first homologous recombination: firstly, a pKD46 plasmid is transformed into Escherichia coli Sval020 by an electrotransformation method, and then the DNA fragment I is electrotransformed into the Escherichia coli Sval020 with the pKD46.

[0169] Electrotransformation conditions and steps are the same as the first step method for the mgsA gene knockout described in Example 1. 200 μl of bacterial solution is spreaded onto a LB plate containing ampicillin (a final concentration is 100 μg/ml) and chloramphenicol (a final concentration is 34 μg/ml). After being cultured overnight at 30° C., colonies were PCR verified using primers ilvG pro-YZup/ilvG p-YZdown, and a correct PCR product should be 2993 bp. A correct single colony is picked, and named as Sval0121.

[0170] In a second step, a genomic DNA of M1-93 is used as a template, and 188 bp of a DNA fragment II is amplified by using primers ilvG pro-up/ilvG pro-down. The DNAfragment II is used for the second homologous recombination. The DNA fragment II is electrotransformed into strain Sval021.

[0171] Electrotransformation conditions and steps are the same as the second step method for the mgsA gene knockout described in Example 1. Colonies were PCR verified using primers ilvG pro-YZup/ilvG p-YZ down and sequenced, and a correct colony amplification product is 462 bp. A correct single colony is picked, and named as Sval022.

EXAMPLE 12

Mutation of Acetolactate Synthase Gene ilvH

[0172] A mutation is transferred into the ilvH gene so as to release feedback inhibition of L-valine through a two-step homologous recombination method. Specific steps are as follows.

[0173] In a first step, a pXZ-CS plasmid DNA is used as a template, 2719 bp of a DNA fragment I is amplified by using primers ilvH*-cat-up/ilvH*-cat-down, and used for the first step of the homologous recombination. Amplification system and amplification conditions are the same as those described in Example 1.

[0174] The DNA fragment I is used for the first homologous recombination: firstly, a pKD46 plasmid is transformed into Escherichia coli Sval022 by an electrotransformation method, and then the DNA fragment I is electrotransformed into the Escherichia coli Sval022 with the pKD46.

[0175] Electrotransformation conditions and steps are the same as the first step method for the mgsA gene knockout described in Example 1. 200 μl of bacterial solution is spreaded onto a LB plate containing ampicillin (a final concentration is 100 μg/ml) and chloramphenicol (a final concentration is 34 μg/ml). After being cultured overnight at 30° C., colonies were PCR verified using primers ilvH*-mutYZ-up/ilvH*-mut-down, and a correct PCR product should be 3165 bp. A correct single colony is picked, and named as Sval023.

[0176] In a second step, a DNA of wild-type Escherichia coli ATCC 8739 is used as a template, and 467 bp of a DNA fragment II is amplified by using primers ilvH*-mut-up/ilvH*-mut-down. The DNA fragment II is used for the second homologous recombination. The DNAfragment II is electrotransformed into strain Sval023.

[0177] Electrotransformation conditions and steps are the same as the second step method for the mgsA gene knockout described in Example 1. Colonies were PCR verified using primers ilvH*-mutYZ-up/ilvH*-mut-down, and a correct colony amplification product is 619 bp. A correct single colony is picked, and named as Sval024.

EXAMPLE 13

Fermentation and Production of L-Valine Using Recombinant Strain Sval024

[0178] A seed culture medium is formed by the following components (a solvent is water):

[0179] Glucose 20 g/L, corn syrup dry powder 10 g/L, KH.sub.2PO.sub.4 8.8 g/L, (NH.sub.4).sub.2SO.sub.4 2.5 g/L, and MgSO.sub.4.7H.sub.2O 2 g/L.

[0180] The fermentation culture medium is most the same as the seed culture medium, and a difference is only that the glucose concentration is 50 g/L.

[0181] Anaerobic fermentation of Sval024 includes the following steps:

[0182] (1) Seed culture: a fresh clone on an LB plate is inoculated into a test tube containing 4 ml of the seed culture medium, and shake-cultured overnight at 37° C. and 250 rpm. Then, a culture is transferred to 250 ml of a triangular flask containing 30 ml of the seed culture medium according to an inoculum size of 2% (V/V), and seed culture solution is obtained by shake culture at 37° C. and 250 rpm for 12 hours, and used for fermentation medium inoculation.

[0183] (2) Fermentation culture: a volume of the fermentation culture medium in 500 ml of an fermenter is 250 ml, and the seed culture solution is inoculated into the fermentation culture medium according to an inoculum size of final concentration OD550=0.1, and fermented at 37° C. and 150 rpm for 4 days, to obtain fermentation solution. The neutralizer is 5M ammonia, the pH was is controlled at 7.0. No air was sparged during the fermentation.

[0184] Analytical method: an Agilent (Agilent-1260) high performance liquid chromatograph is used to determine components in the fermentation solution after fermentation for 4 days. The concentrations of glucose and organic acid in the fermentation solution are determined by using an Aminex HPX-87H organic acid analytical column of Biorad Company. A Sielc amino acid analysis column primesep 100 250×4.6 mm is used for amino acid determination.

[0185] It is discovered from results that: strain Sval024 could produce 1.3 g/L of L-valine (L-valine peak corresponding to a position in FIG. 2 appears) with a yield of 0.31 mol/mol after 4 days fermentation under anaerobic conditions.

EXAMPLE 14

Cloning and Integration of Leucine Dehydrogenase Encoding Gene leuDH

[0186] Referring to the reported (Ohshima, T. et. al, Properties of crystalline leucine dehydrogenase from Bacillus sphaericus. The Journal of biological chemistry 253, 5719-5725 (1978)) sequence of a leuDH from Lysinibacillus sphaericus IFO 3525, a leuDH gene was codon optimized and chemically synthesized (an optimized sequence is as shown in a sequence number 90). During the synthesis, an M1-93 artificial regulatory element is added before the leuDH gene to initiate expression of the leuDH gene, and inserted into a pUC57 vector to construct a plasmid pUC57-M1-93-leuDH (gene synthesis and vector construction are completed by Nanjing Genscript Biotechnology Co., Ltd.). The M1-93 artificial regulatory element and the leuDH gene are integrated into the fumarate reductase encoding gene frd site in strain Sval024 through a two-step homologous recombination method and substitute the frd gene, namely the frd gene is knocked out while the leuDH is integrated. Specific steps are as follows.

[0187] In a first step, a pXZ-CS plasmid DNA is used as a template, 2719 bp of a DNA fragment I is amplified by using primers frd-cs-up/frd-cs-down, and used for the first step of the homologous recombination. Amplification system and amplification conditions are the same as those described in Example 1.

[0188] The DNA fragment I is used for the first homologous recombination: firstly, a pKD46 plasmid is transformed into Escherichia coli Sval024 by an electrotransformation method, and then the DNA fragment I is electrotransformed into the Escherichia coli Sval024 with the pKD46.

[0189] Electrotransformation conditions and steps are the same as the first step method for the mgsA gene knockout described in Example 1. 200 μl of bacterial solution is spreaded onto a LB plate containing ampicillin (a final concentration is 100 μg/ml) and chloramphenicol (a final concentration is 34 μg/ml). After being cultured overnight at 30° C., colonies were PCR verified using primers XZ-frd-up/XZ-frd-down, and a correct PCR product should be 3493 bp. A correct single colony is picked, and named as Sval025.

[0190] In a second step, a pUC57-M1-93-leuDH plasmid DNA is used as a template, and 1283 bp of a DNA fragment II is amplified by using primers frd-M93-up/frd-leuDH-down. The DNA fragment II is used for the second homologous recombination. The DNA fragment II is electrotransformed into strain Sval025.

[0191] Electrotransformation conditions and steps are the same as the second step method for the mgsA gene knockout described in Example 1. Colonies were PCR verified using primers XZ-frd-up/XZ-frd-down, and a correct colony amplification product is 2057 bp. A correct single colony is picked, and named as Sval026.

EXAMPLE 15

Fermentation and Production of L-Valine Using Recombinant Strain Sval026

[0192] Components and preparation of seed culture medium and fermentation culture medium are the same as those described in Example 13.

[0193] The fermentation is performed in 500 mL of a fermentation vessel, and a fermentation process and an analysis process are the same as the fermentation process and the analysis process of the Sval024 described in Example 13.

[0194] It is discovered from results that: the strain Sval026 could produce 1.8 g/L of L-valine (L-valine peak corresponding to a position in FIG. 2 appears) 0.56 mol/mol after 4 days fermentation under anaerobic conditions.

EXAMPLE 16

Regulation of 6-phosphate Glucose Dehydrogenase Gene Encoding zwf

[0195] Started from Sval026, an artificial regulatory element is used to regulate expression of zwf gene through a method of two-step homologous recombination, and recombinant Escherichia coli Sval041 are obtained. It specifically includes the following steps.

[0196] In a first step, a pXZ-CS plasmid DNA is used as a template, 2719 bp of a DNA fragment I is amplified by using primers Zwf-Pcat-up/Zwf-PsacB-down, and used for the first step of the homologous recombination.

[0197] The DNA fragment I is used for the first homologous recombination: firstly, a pKD46 plasmid is transformed into Escherichia coli Sval026 by an electrotransformation method, and then the DNA fragment I is electrotransformed into the Escherichia coli Sval026 with the pKD46.

[0198] Electrotransformation conditions and steps are the same as the first step method for the mgsA gene knockout described in Example 1. 200 μl of bacterial solution is spreaded onto a LB plate containing ampicillin (a final concentration is 100 μg/ml) and chloramphenicol (a final concentration is 34 μg/ml). After being cultured overnight at 30° C., colonies were PCR verified using primers zwf-YZ442-up/zwf-YZ383-down, and a correct colony amplification product is 3339 bp. A correct single colony is picked, and named as Sval041.

[0199] In a second step, a genomic DNA of M1-93 is used as a template, and 189 bp of a DNA fragment II is amplified by using primers zwf-RBS1-up/zwf-RBS1-down, and used for the second homologous recombination. Amplification conditions and system are the same as those described in (1). The DNA fragment II is electrotransformed into strain Sval041.

[0200] Electrotransformation conditions and steps are the same as the second step method for the mgsA gene knockout described in Example 1. Colonies were PCR verified using primers zwf-YZ442-up/zwf-YZ383-down, and a correct colony amplification product is 809 bp. A correct single colony is picked, and named as Sval042 (Table 1).

EXAMPLE 17

Regulation of Lactonase Encoding Gene pgl

[0201] Started from Sval033, an artificial regulatory element is used to regulate expression of a pgl gene through a method of two-step homologous recombination, and recombinant Escherichia coli Sval042 are acquired. It specifically includes the following steps.

[0202] In a first step, a pXZ-CS plasmid DNA is used as a template, 2719 bp of a DNA fragment I is amplified by using primers pgl-Pcat-up/pgl-PsacB-down, and used for the first step of the homologous recombination.

[0203] The DNA fragment I is used for the first homologous recombination: firstly, a pKD46 plasmid is transformed into Escherichia coli Sval033 by an electrotransformation method, and then the DNA fragment I is electrotransformed into the Escherichia coli Sval042 with the pKD46.

[0204] Electrotransformation conditions and steps are the same as the first step method for the mgsA gene knockout described in Example 1. 200 μl of bacterial culture is spreaded onto a LB plate containing ampicillin (a final concentration is 100 μg/ml) and chloramphenicol (a final concentration is 34 μg/ml). After being cultured overnight at 30° C., colonies were PCR verified using primers pgl-YZ308-up/pgl-YZ341-down, and a correct PCR product should be 3248 bp. A correct single colony is picked, and named as Sval043.

[0205] In a second step, a genomic DNA of M1-93 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93:2455-2462) is used as a template, and 189 bp of a DNA fragment II is amplified by using primers pgl-RBS2-up/pgl-RBS2-down, and used for the second homologous recombination. Amplification conditions and system are the same as those described in (1). The DNA fragment II is electrotransformed into strain Sval043.

[0206] Electrotransformation conditions and steps are the same as the second step method for the mgsA gene knockout described in Example 1. Colonies were PCR verified using primers pgl-YZ308-up/pgl-YZ341-down, and a correct colony amplification product is 718 bp. A correct single colony is picked, and named as Sval044 (Table 1).

EXAMPLE 18

Cloning and Integration of edd and eda Genes from Zymomonas mobilis ZM4 Strain

[0207] According to sequences of edd and eda genes derived from Zymomonas mobilis ZM4 reported by a literature (The genome sequence of the ethanologenic bacterium Zymomonas mobilis ZM4, Nat. Biotechnol., 2005, 23(1):63-68), the edd and eda genes derived from the Zymomonas mobilis ZM4 are synthesized by whole genes, herein an artificial regulatory element MRS1 is added before the edd gene, and the edd and eda genes are connected by an artificial RBS regulatory element (CAGGAAACAGCT). A whole-gene-synthesized MRS1-edd-RBS-eda fragment is linked to a pUC57 vector through EcoRV. The whole gene synthesis of the sequence is completed by Nanjing Genscript Biotechnology Co., Ltd., and the pUC57 vector is also from Nanjing Genscript Biotechnology Co., Ltd. The MRS1-edd-RBS-eda is integrated into an edd-eda site of the Escherichia coli itself through a two-step homologous recombination method and replaces the edd-eda gene of the Escherichia coli itself. Specific steps are as follows.

[0208] In a first step, a pXZ-CS plasmid (Tan, et al., Appl Environ Microbiol, 2013, 79:4838-4844) DNA is used as a template, 2719 bp of a DNA fragment I is amplified by using primers edd-cat-up/eda-sacB-down, and used for the first step of the homologous recombination.

[0209] The DNA fragment I is used for the first homologous recombination: firstly, a pKD46 plasmid is transformed into Escherichia coli Sval044 by an electrotransformation method, and then the DNA fragment I is electrotransformed into the Escherichia coli Sval044 with the pKD46.

[0210] Electrotransformation conditions and steps are the same as the first step method for the mgsA gene knockout described in Example 1. 200 μl of bacterial solution is spreaded onto a LB plate containing ampicillin (a final concentration is 100 μg/ml) and chloramphenicol (a final concentration is 34 μg/ml). After being cultured overnight at 30° C., colonies were PCR verified using primers edd-YZ-up/edd-YZ-down, and a correct PCR product should be 3123 bp. A correct single colony is picked, and named as Sval045.

[0211] In a second step, a plasmid DNA of pUC57-MRS1-edd-eda is used as a template, and 2651 bp of a DNA fragment II is amplified by using primers Edd-int-up/Eda-int-down, and used for the second homologous recombination. Amplification conditions and system are the same as those described in (1). The DNA fragment II is electrotransformed into strain Sval045.

[0212] Electrotransformation conditions and steps are the same as the second step method for the mgsA gene knockout described in Example 1. Colonies were PCR verified using primers edd-YZ-up/edd-YZ-down, and a correct colony amplification product is 3055 bp. A correct single colony is picked, and named as Sval046 (Table 1).

EXAMPLE 19

Knockout of 6-phosphoglucokinase Encoding Gene pfkA

[0213] In an strain Sval037, a key enzyme 6-phosphoglucokinase gene pfkA in a glycolytic pathway is knocked out to achieve that a recombinant strain uses an ED pathway to ferment and generate L-valine. The pfkA gene is knocked out through a method of two-step homologous recombination, and specific steps are as follows.

[0214] In a first step, a pXZ-CS plasmid (Tan, et al., Appl Environ Microbiol, 2013, 79:4838-4844) DNA is used as a template, 2719 bp of a DNA fragment I is amplified by using primers pfkAdel-cat-up/pfkAdel-sacB-down, and used for the first step of the homologous recombination.

[0215] The DNA fragment I is used for the first homologous recombination: firstly, a pKD46 plasmid (Datsenko and Wanner 2000, Proc Natl Acad Sci USA 97:6640-6645; and the plasmid is purchased from Coil Genetic Stock Center (CGSC) of Yale University, CGSC#7739) is transformed into Escherichia coli Sval046 by an electrotransformation method, and then the DNA fragment I is electrotransformed into the Escherichia coli Sval046 with the pKD46.

[0216] Electrotransformation conditions and steps are the same as the first step method for the mgsA gene knockout described in Example 1. 200 μl of bacterial solution is spreaded onto a LB plate containing ampicillin (a final concentration is 100 μg/ml) and chloramphenicol (a final concentration is 34 μg/ml). After being cultured overnight at 30° C., colonies were PCR verified using primers pfkAdel-up/pfkAdel-YZ-down, and a correct PCR product should be 3145 bp. A correct single colony is picked, and named as Sval047.

[0217] In a second step, a genomic DNA of wild-type ATCC8739 is used as a template, and 379 bp of a DNA fragment II is amplified by using primers pfkAdel-up/pfkAdel-down, and used for the second homologous recombination. Amplification conditions and system are the same as those described in (1). The DNA fragment II is electrotransformed into strain Sval047.

[0218] Electrotransformation conditions and steps are the same as the second step method for the mgsA gene knockout described in Example 1. Colonies were PCR verified using primers pfkAdel-up/pfkAdel-YZ-down, and a correct colony amplification product is 526 bp. A correct single colony is picked, and named as Sval048 (Table 1).

EXAMPLE 20

Production of L-Valine Using Recombinant Strain Sval048

[0219] Components and preparation of seed culture medium and fermentation culture medium are the same as those described in Example 13.

[0220] The fermentation is performed in 500 mL of a fermentation vessel, a fermentation process and an analysis process are the same as the fermentation process and the analysis process of the Sval024 described in Example 13.

[0221] It is discovered from results that: the strain Sval048 could produce 1.9 g/L of L-valine with a yield of 0.82 mol/mol after fermentation for 4 days under anaerobic conditions. FIG. 2 is a spectrum of L-valine standard substance, and FIG. 3 is a spectrum of Sval048 fermentation solution.

EXAMPLE 21

Construction of Recombinant Strain Sval049

[0222] Started from Sval048, cell growth and L-valine production capacity are synchronously improved through metabolic evolution.

[0223] Metabolic evolution was carried out in 500 ml fermentation vessel with 250 ml fermentation culture medium. The fermentative pH was controlled at 7.0 by using 5 Mammonia as neutralizer. Components and preparation of the fermentation culture medium used for the metabolic evolution are the same as those of the fermentation culture medium described in Example 18. Every 24 hours, fermentation solution is transferred into a new fermentation vessel, and the initial OD550 is 0.1. After 100 generations of the evolution, a strain Sval049 is obtained (FIG. 4). The strain Sval049 is preserved in China General Microbiological Culture Collection Center (CGMCC) with a preservation number CGMCC 19457.

EXAMPLE 22

Fermentation of Strain Sval049 to Produce L-Valine in 500 mL Fermentation Vessel

[0224] Components and preparation of a seed culture medium are the same as those described in Example 13.

[0225] The fermentation is performed in 500 mL of a fermentation vessel, and a fermentation culture medium is 250 ml. The fermentation culture medium is basically the same as the seed culture medium. A difference is that a glucose concentration is 100 g/L, and a neutralizer used is 5M ammonia, so that fermentative pH is controlled in 7.0.

[0226] It is discovered from results that: after fermented in 500 mL of the fermentation vessel for 48 hours, strain Sval049 produced 47 g/L with a yield of 0.91 mol/mol, and impurities such as a heteroacid are not generated.

EXAMPLE 23

Production of L-Valine by Fermentation of Recombinant Strain Sval049 in 5 L Fermentation Vessel

[0227] Components, preparation and analytical method of a seed culture medium are the same as those described in Example 13. A fermentation culture medium is basically the same as the seed culture medium, and a difference is that a glucose concentration is 140 g/L.

[0228] The fermentation is performed in 5 L of a fermentation vessel (Shanghai Baoxing, BIOTECH-5BG) under anaerobic conditions, including the following steps:

[0229] (1) Seed culture: the seed culture medium in 500 ml of a triangular flask is 150 ml, and it is sterilized at 115° C. for 15 min. After cooling, recombinant Escherichia coli Sval041 are inoculated into the seed culture medium according to an inoculum size of 1% (V/V), and cultured at 37° C. and 100 rpm for 12 hours to obtain seed solution for inoculation of the fermentation culture medium.

[0230] (2) Fermentation culture: a volume of the fermentation culture medium in 5 L is 3 L, and it is sterilized at 115° C. for 25 min. The seed solution is inoculated into the fermentation culture medium according to an inoculum size of final concentration OD550=0.2, and cultured under anaerobic conditions at 37° C. for 48 hours, and a stirring speed is 200 rpm, fermentation solution is obtained. The fermentation solution is all of substances in the fermentation vessel. No air was sparged during the fermentation.

[0231] It is discovered from results that: after fermented in 5 L of the fermentation vessel for 48 hours, Sval049 produced 87 g/L L-valine with a yield of 0.92 mol/mol, and impurities such as a heteroacid are not generated. FIG. 5 is a spectrum of L-valine standard substance, and FIG. 6 is a spectrum of Sval049 fermentation solution.