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Recombinant escherichia coli for synthesizing 3-fucosyllactose and construction method thereof

A technology for recombining Escherichia coli and fucosyllactose, applied in the field of genetic engineering, can solve the problems of limited chemical properties of protective groups, expensive guanosine diphosphate fucose, unsuitable for large-scale food production, etc. Potential for industrial application, significant actual effect, good application prospect

Pending Publication Date: 2021-04-16
量子高科(广东)生物有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the industrial production of 3-fucosyllactose is not easy
In the early days, 3-fucosyllactose was obtained by chromatographic separation from donated breast milk, which could not be produced on a large scale due to limited material sources
3-Fucosyllactose can be directly chemically synthesized, however, limited by the chemical properties of the protecting group, it cannot be scaled up, and chemical synthesis requires the use of toxic reagents, which is not suitable for large-scale food production
Fucosyltransferase can catalyze the synthesis of 3-fucosyllactose in vitro, but the donor substrate guanosine diphosphate fucose is very expensive
Therefore, the enzymatic synthesis of 3-fucosyllactose is not suitable for industrial production with an annual output of hundreds of tons

Method used

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  • Recombinant escherichia coli for synthesizing 3-fucosyllactose and construction method thereof
  • Recombinant escherichia coli for synthesizing 3-fucosyllactose and construction method thereof
  • Recombinant escherichia coli for synthesizing 3-fucosyllactose and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Gene acquisition:

[0043] In the present embodiment, the gene manB (gene accession number GI: 946574) derived from Escherichia coli MG1655, the gene manC (gene accession number GI: 946580), the gene gmd (gene accession number GI: 946562), the gene fcl (gene accession number No. GI: ​​946563), gene lacY (gene accession number GI: 949083), and gene cafF (gene accession number GI: 187425317) derived from Akkermansia muciniphila ATCCBAA-835.

[0044] In this example, the E. coli genes manB, manC, gmd, fcl and lacY were successfully obtained by extracting the genomic DNA of E. coli MG1655; Gene cafF of Akkermansia mucinica ATCC BAA-835. The successful acquisition of the gene lays the foundation for the construction of the recombinant plasmid.

Embodiment 2

[0046] Preparation of recombinant plasmids

[0047] Use designed primers (upstream primer sequence as shown in SEQ ID NO.15, downstream primer sequence as shown in SEQ ID NO.16) the gene manC and gene manB derived from Escherichia coli MG1655 obtained in Example 1 are carried out PCR amplification , the amplified fragments were gel-cut and purified, and double-digested with NcoI and HindIII restriction endonucleases, and the digested fragments were ligated with the plasmid pCOLADuet-1, which was also double-digested with NcoI and HindIII, and Vector: The target fragments were mixed at a molar ratio of 1:3, added with T4 DNA ligase, and ligated for 5 hours at 22°C. The ligated product was transformed into E. coli DH5α competent cells, and screened on a kanamycin plate , to obtain the recombinant plasmid pCOLA-CB. Use designed primers (upstream primer sequence as shown in SEQ ID NO.17, downstream primer sequence as shown in SEQ ID NO.18) the gene gmd and gene fcl derived from E...

Embodiment 3

[0051] gene editing

[0052] In this example, CRISPR gene editing technology was used to knock out the wcaJ and lacZ genes and to knock in the T7lac promoter. The steps of gene editing are described in detail below.

[0053] 1) The test showed that the original strain of Escherichia coli BL21 (DE3) was in normal growth state. The detection of target gene wcaJ and lacZ and T7lac promoter knock-in positions and their upstream and downstream sequences showed that the size of PCR amplified bands was in line with expectations, and the sequencing results were consistent with NCBI Sequence is consistent;

[0054] 2) According to the insertion position of the target gene sequence and the surrounding sequence characteristics, design and prepare gRNA, clone the gRNA and Donor sequence into the gene editing vector Donor plasmid, and verify by sequencing to ensure that the gRNA and Donor sequence in the constructed vector are consistent with the target sequence consistent;

[0055] 3) ...

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Abstract

The invention provides recombinant Escherichia coli for synthesizing 3-fucosyllactose and a construction method of the recombinant Escherichia coli, and the recombinant Escherichia coli is prepared by knocking out a beta- galactosidase gene in the genome of Escherichia coli, synthesizing a key enzyme gene of Colanic acid from guanosine diphosphate fucose, enhancing expression of lactose permease genes and key enzyme genes of a guanylate diphosphate fucose de novo synthesis route and expressing exogenous fucosyltransferase genes. According to the scheme, lactose hydrolysis can be reduced, and the rate of transferring exogenous lactose into cells is increased, so that the concentration of lactose in the cells is effectively increased; meanwhile, the consumption of the guanosine diphosphate fucose is reduced, and the concentration of the guanosine diphosphate fucose in cells is effectively improved; and through codon optimization, the expression of an exogenous fucosyltransferase gene is improved, and the synthesis of 3-fucosyllactose is effectively promoted.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant Escherichia coli for synthesizing 3-fucosyllactose and a construction method thereof. Background technique [0002] Breast milk is a complex mixture containing water, fat, protein, minerals, vitamins, polysaccharides, monosaccharides, disaccharides (lactose), and oligosaccharides. Lactose is the main solid component of breast milk (~6.8% of total solids), and is widely present in animal milk. However, the structurally more complex breast milk oligosaccharides (-1% of total solids) are unique to humans. At present, there are about 200 human milk oligosaccharides whose structures have been confirmed, with degrees of polymerization ranging from 3 to 32. The structural features of human milk oligosaccharides include lactose-N-acetylglucosamine backbone, linear β-(1,3 / 4)-glycosidic bonds, branched β-(1,6)-glycosidic end and L-fucose or sialic acid at the n...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/113C12N15/56C12N15/60C12N15/54C12N15/61C12N15/53C12N15/52C12N15/70C12P19/00C12R1/19
CPCY02A50/30
Inventor 郝占西曾宪维杨新球魏远安许本宏阮鸿波吴嘉仪吴少辉霍金洪
Owner 量子高科(广东)生物有限公司
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