Construction method and application of microorganism capable of realizing high production of lacto-N-neotetraose

Patent metadata

Field	Value
Publication number	US-12098402-B2
Application number	US-202318485543-A
Country	US
Kind code	B2
Filing date	Oct 12, 2023
Priority date	Aug 6, 2021
Publication date	Sep 24, 2024
Grant date	Sep 24, 2024

Abstract

Disclosed are a construction method and application of a microorganism capable of realizing high production of lacto-N-neotetraose, belonging to the field of microbial genetic engineering. Coding genes of ß-1,3-acetyl glucosamine transferase, ß-1,4-galactosyl transferase and/or UDP-glucose 4 epimerase are over-expressed on the basis of a strain which is previously constructed by the team and is subjected to related-gene knockout, thus enabling the strain to have a synthesis capability of producing the lacto-N-neotetraose. The present disclosure accurately regulates the carbon flux of a metabolic pathway and relieves the metabolic stress by screening the high-efficiency ß-1,4-galactosyl transferase gene and regulating the expression of IgtA, Aa-β-1,4-GalT and galE in a lacto-N-neotetraose synthesis pathway in a combined manner. In a shake flask experiment, the lacto-N-neotetraose production capacity of Escherichia coli is 0.91 g/L. The lacto-N-neotetraose yield in a 3 L fermentation tank reaches 12.14 g/L. Therefore, the microorganism has an industrial application prospect.

First claim

What is claimed is:

1. A recombinant Escherichia coli , wherein ß-1,4-galactosyl transferase derived from Aggregatibacter actinomycetemcomitans NUM4039, ß-1,3-acetyl glucosamine transferase derived from Neisseria meningitidis , and UDP-glucose 4 epimerase derived from E. coli are expressed, and a gene encoding UDP-N-acetyl glucosamine-2-epimerase, a gene encoding glucosamine-6 phosphate deaminase, and a gene encoding ß-galactosidase are knocked out; and the amino acid sequence of the ß-1,4-galactosyl transferase is as set forth in SEQ ID NO:4, and wherein the sequence of gene IgtA encoding the ß-1,3-acetyl glucosamine transferase of N. meningitidis is as set forth in SEQ ID NO:1.

2. The recombinant E. coli according to claim 1 , wherein the gene IgtA encoding the ß-1,3-acetyl glucosamine transferase is expressed by using a pACYCDuet-1, pCDFDuet-1, pRSFDuet-1, pCOLADuet-1 or pETDuet-1 vector, and a gene encoding the UDP-glucose 4 epimerase and a gene encoding the ß-1,4-galactosyl transferase are co-expressed by using a pACYCDuet-1, pCDFDuet-1, pRSFDuet-1, pCOLADuet-1 or pETDuet-1 vector.

3. The recombinant E. coli according to claim 2 , wherein the gene IgtA encoding the ß-1,3-acetyl glucosamine transferase is expressed by using the pRSFDuet-1 vector, and gene galE encoding the UDP-glucose 4 epimerase and gene Aa-β-1,4-GalT encoding the ß-1,4-galactosyl transferase are simultaneously expressed by using the pRSFDuet-1 vector.

4. The recombinant E. coli according to claim 2 , wherein the gene IgtA encoding the ß-1,3-acetyl glucosamine transferase is expressed by using the pETDuet-1 vector, and gene galE encoding the UDP-glucose 4 epimerase and gene Aa-β-1,4-GalT encoding the ß-1,4-galactosyl transferase are simultaneously expressed by using the pRSFDuet-1 vector.

5. The recombinant E. coli according to claim 2 , wherein the gene IgtA encoding the ß-1,3-acetyl glucosamine transferase is expressed by using the pCDFDuet-1 vector, and gene galE encoding the UDP-glucose 4 epimerase and gene Aa-β-1,4-GalT encoding the ß-1,4-galactosyl transferase are expressed by using the pETDuet-1 vector.

6. The recombinant E. coli according to claim 2 , wherein the gene IgtA encoding the ß-1,3-acetyl glucosamine transferase is expressed by using the pACYCDuet-1 vector, and gene galE encoding the UDP-glucose 4 epimerase and gene Aa-β-1,4-GalT encoding the ß-1,4-galactosyl transferase are simultaneously expressed by using the pCOLADuet-1 vector.

7. The recombinant E. coli according to claim 1 , wherein the nucleotide sequence of gene Aa-β-1,4-GalT encoding the ß-1,4-galactosyl transferase is as set forth in SEQ ID NO:2.

8. The recombinant E. coli according to claim 7 , wherein gene galE encoding the UDP-glucose 4 epimerase is derived from E. coli K-12, and the nucleotide sequence of the gene galE is as set forth in SEQ ID NO:3.

9. The recombinant E. coli according to claim 8 , wherein the amino acid sequence of the UDP-N-acetyl glucosamine-2-epimerase is set forth in SEQ ID NO:5, wherein the amino acid sequence of the glucosamine-6 phosphate deaminase is set forth in SEQ ID NO:6, wherein the amino acid sequence of the ß-galactosidase is set forth in SEQ ID NO:7.

10. The recombinant E. coli according to claim 9 , wherein the E. coli is E. coli BL21 (DE3).

Assignees

• Univ Jiangnan

Inventors

• Mu Wanmeng
• Zhu Yingying
• Luo Guocong
• Zhang Wenli

Classifications

• C12Y204/01146

  Beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,3-N-acetylglucosaminyltransferase (2.4.1.146) · CPC title

• C12Y204/01038

  Beta-N-acetylglucosaminylglycopeptide beta-1,4-galactosyltransferase (2.4.1.38) · CPC title

• C12N2500/35

  Polyols, e.g. glycerin, inositol · CPC title

• C12N2500/34

  Sugars · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title