Microorganism for producing pantoic acid, and construction method therefor and application thereof

1 . A method of constructing a recombinant Escherichia coli , comprising modifying a starting Escherichia coli per the following steps of A1-A25 to obtain the recombinant Escherichia coli:

A1. introducing and expressing an alsS gene encoding acetolactate synthase;

A2. replacing the promoter of ilvB gene encoding the large subunit of acetolactate synthase I with M1-93 promoter, wherein the M1-93 promoter is a DNA molecule, wherein the nucleotide sequence of one strand of the DNA molecule is set forth as SEQ ID NO: 3;

A3. replacing the promoter of ilvG gene encoding the large subunit of acetolactate synthase II with the M1-93 promoter;

A4. mutating the ilvH gene encoding a regulatory subunit of acetolactate synthase III to an ilvH mutant gene, wherein the ilvH mutant gene encodes the protein of SEQ ID NO: 5;

A5. introducing and expressing an ilvC gene encoding acetohydroxyl-acid reductoisomerase;

A6. introducing and expressing an ilvD gene encoding dihydroxy-acid dehydratase;

A7. introducing and expressing a panB gene encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase derived from Escherichia coli , which is designated as E-panB gene;

A8. introducing and expressing a panE gene encoding 2-dehydropantothenate-2-reductase;

A9. introducing and expressing a glyA gene encoding glycine hydroxymethyltransferase;

A10. replacing the promoter of gcvT gene encoding aminomethyltransferase with the M1-93 promoter;

A11. replacing the promoter of gcvP gene encoding glycine decarboxylase with the M1-93 promoter;

A12. introducing and expressing a panB gene encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase derived from Corynebacterium glutamicum , which is designated as C-panB gene;

A13. mutating the ilvE gene encoding branched-chain amino acid aminotransferase to an ilvE mutant gene, wherein the ilvE mutant gene encodes the protein of SEQ ID NO: 12;

A14. introducing and expressing a serA gene encoding phosphoglycerate dehydrogenase;

A15. introducing and expressing a serC gene encoding phosphoserine/phosphohydroxythreonine aminotransferase and a serB gene encoding phosphoserine phosphatase;

A16. knocking out the sdaA gene encoding L-serine deaminase I;

A17. knocking out the tdcD gene encoding propionate kinase and the tdcE gene encoding formate acetyltransferase;

A18. knocking out the adhE gene encoding alcohol dehydrogenase;

A19. knocking out the pflB gene encoding pyruvate formate lyase;

A20. knocking out the frd gene encoding fumarate reductase;

A21. knocking out the ldhA gene encoding lactate dehydrogenase;

A22. knocking out the mgsA gene encoding methylglyoxal synthase;

A23. knocking out the pta gene encoding the phosphate acetyltransferase and the ackA gene encoding acetate kinase;

A24. knocking out the ara gene encoding ribokinase and

A25. knocking out the avtA gene encoding valine-pyruvate transaminase.

2 . The method according to claim 1 , characterized in that:

the alsS gene is derived from Bacillus subtilis;

and/or, the ilvC gene is derived from Escherichia coli;

and/or, the ilvD gene is derived from Escherichia coli;

and/or, the panE gene is derived from Escherichia coli;

and/or, the glyA gene is derived from Escherichia coli;

and/or, the serA gene is derived from Corynebacterium glutamicum;

and/or, the serC gene and the serB gene are derived from Escherichia coli.

3 . The method according to claim 2 , characterized in that:

the alsS gene encodes the AlsS protein of SEQ ID NO: 2;

and/or, the C-panB gene encodes the C-panB protein of SEQ ID NO: 10;

and/or, the serA gene encodes the SerA protein of SEQ ID NO: 14;

and/or, the serC gene encodes the SerC protein of SEQ ID NO: 16;

and/or, the serB gene encodes the SerB protein of SEQ ID NO: 17.

4 . The method according to claim 1 , characterized in that:

the sequence of the alsS gene is set forth as SEQ ID NO: 1;

and/or, the sequence of the ilvH mutant gene is set forth as SEQ ID NO: 4;

and/or, the sequence of the C-panB gene is set forth as SEQ ID NO: 9;

and/or, the sequence of the ilvE mutant gene is set forth as SEQ ID NO: 11;

and/or, the sequence of the serA gene is set forth as SEQ ID NO: 13;

and/or, the sequence of the serC gene is from positions 89 to 1177 of SEQ ID NO: 15;

and/or, the sequence of the serB gene is from positions 1199 to 2167 of SEQ ID NO: 15.

5 . The method according to claim 1 , characterized in that:

A1 is achieved by introducing an alsS gene expression cassette into the recipient Escherichia coli , wherein the alsS gene expression cassette contains a promoter and the alsS gene driven by the promoter;

and/or, A5 is achieved by introducing an ilvC gene expression cassette into the recipient Escherichia coli , wherein the ilvC gene expression cassette contains a promoter and the ilvC gene driven by the promoter;

and/or, A6 is achieved by introducing an ilvD gene expression cassette into the recipient Escherichia coli , wherein the ilvD gene expression cassette contains a promoter and the ilvD gene driven by the promoter;

and/or, A7 is achieved by introducing an E-panB gene expression cassette into the recipient Escherichia coli , wherein the E-panB gene expression cassette contains a promoter and the E-panB gene driven by the promoter;

and/or, A8 is achieved by introducing a panE gene expression cassette into the recipient Escherichia coli , wherein the panE gene expression cassette contains a promoter and the panE gene driven by the promoter;

and/or, A9 is achieved by introducing a glyA gene expression cassette into the recipient Escherichia coli , wherein the glyA gene expression cassette contains a promoter and the glyA gene driven by the promoter;

and/or, A12 is achieved by introducing a C-panB gene expression cassette into the recipient Escherichia coli , wherein the C-panB gene expression cassette contains a promoter and the C-panB gene driven by the promoter;

and/or, A14 is achieved by introducing a serA gene expression cassette into the recipient Escherichia coli , wherein the serA gene expression cassette contains a promoter and the serA gene driven by the promoter;

and/or, A15 is achieved by introducing a serCB gene expression cassette into the recipient Escherichia coli , wherein the serCB gene expression cassette contains a promoter and the serC gene and the serB gene driven by the promoter.

6 . The method according to claim 5 , characterized in that:

the promoter in A1, A7, A12, A14 or A15 is the M1-93 promoter;

the promoter in A5 or A9 is M1-46 promoter, wherein the M1-46 promoter is any selected from the group consisting of the following DNA molecules:

1) A DNA molecule, wherein the nucleotide sequence of one strand of the DNA molecule is set forth as SEQ ID NO: 6;

2) A DNA molecule having at least 80% identity with the DNA molecule in 1) and having a promoter function;

the promoter in A6 is RBSL1 promoter, wherein the RBSL1 promoter is any selected from the group consisting of the following DNA molecules:

a1) a DNA molecule, wherein the nucleotide sequence of one strand of the DNA molecule is set forth as SEQ ID NO: 7;

a2) a DNA molecule having at least 80% identity with the DNA molecule in a1) and having a promoter function;

the promoter in A8 is RBSL2 promoter, wherein the RBSL2 promoter is any selected from the group consisting of the following DNA molecules:

c1) a DNA molecule, wherein the nucleotide sequence of one strand of the DNA molecule is set forth as SEQ ID NO: 8;

c2) a DNA molecule having at least 80% identity with the DNA molecule in c1) and having a promoter function;

and/or, the starting Escherichia coli is Escherichia coli ATCC 8739.

7 . A recombinant Escherichia coli obtained using the method according to claim 1 .

8 . The recombinant Escherichia coli according to claim 7 , characterized in that: the recombinant Escherichia coli is the strain deposited in the China General Microbiological Culture Collection Center with the accession number of CGMCC No. 21699.

9 . A method of producing pantoic acid, comprising: culturing the recombinant Escherichia coli according to claim 7 to obtain fermentation products; and obtaining pantoic acid from the fermentation products.

10 . A method of producing pantoic acid, comprising: culturing the recombinant Escherichia coli according to claim 8 to obtain fermentation products; and obtaining pantoic acid from the fermentation products.

11 . The method of claim 10 , further comprising converting the pantoic acid obtained from the fermentation products to calcium pantothenate.

12 . The method of claim 9 , further comprising converting the pantoic acid obtained from the fermentation products to calcium pantothenate.