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Recombinant escherichia coli constructed by genetic engineering and method for biosynthesizing 6'-sialyllactose

A technology for recombining Escherichia coli and genetically engineered bacteria, which is applied in the field of industrial microbial metabolic engineering, and can solve problems such as differences in relative reactivity and expensive sugar donors

Active Publication Date: 2021-03-09
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method still has its limitations, such as different steps require different protective group manipulations, there must be certain relative reactivity differences between different steps, and sugar donors activated by monophosphate or diphosphate nucleotides are expensive, etc. , becoming a "bottleneck" restricting its use in mass production

Method used

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  • Recombinant escherichia coli constructed by genetic engineering and method for biosynthesizing 6'-sialyllactose
  • Recombinant escherichia coli constructed by genetic engineering and method for biosynthesizing 6'-sialyllactose
  • Recombinant escherichia coli constructed by genetic engineering and method for biosynthesizing 6'-sialyllactose

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Experimental program
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Embodiment 1

[0072] Gene acquisition

[0073] In this embodiment, the gene of different sources is optimized, including: N-acetyl glucamine isomerase gene derived from E. coli Neuc (Optimized SEQ ID NO.1), acetyl neopropase synthase gene NEUB (Optimized SEQ ID No.2), CMP-Acetyl Neurine Synthetase Gene Neua (Optimize the gene SEQ ID NO.3), from Photobacterium Leiognathi of 2,6st Gene (optimized gene SEQ ID NO.4), derived from the phosphate amine acyllet transferase gene of brewing yeast GNA1 (Optimize the gene SEQ ID No.5), from the compositivity SYNECHOCYSTISTISTISTISTISTISTIS SP. N-acetyl glucosamine differential contrast enzyme gene AGE (Optimize the gene SEQ ID NO. 6).

Embodiment 2

[0075] Preparation of recombinant plasmid

[0076] CMP-acetyl neurine synthetase gene derived from E. coli obtained in Example 1 Neua , Acetyl neurine synthetase gene NEUB , N-acetyl glucosamine isomerase gene Neuc PCR amplification was performed, and the amplified fragment was purified and purified, and the double enzyme digestion was carried out, and the fragment of the enzyme digestion was connected to the plasmid of the plasmid Pacyc184 which was also subjected to bisase. 3 proportion of mixing, adding T4 DNA Ligase to 22 ° C for 5 h at 22 ° C, connection product conversion E. Coli DH5α, screening on chloramphenicol plate, gaining recombinant plasmid PACYC- Neucba .

[0077] The origin obtained in Example 1 Photobacterium Leiognathi of 2,6st The gene was amplified in PCR, and the amplified fragment was purified, and the fragment of the bisase digestion was also passed through a double-cut PCYC- Neucba The plasmid is connected, and the carrier: The segment of the target...

Embodiment 3

[0081] Gene's knockout

[0082] This embodiment uses RED homologous recombination assisted CRISPR-CAS9 methods to further simplify gene editing methods, enabling efficient and rapid and continuous editing of Escherichia coli genotype, rapidly obtaining design induction genetic and physiological metabolic characteristics. Below LDH Gene is an example, and the step of the gene knockout is explained in detail, and the knockout of the remaining gene is the same.

[0083] Find in NCBI E. Coli LDH Nucleotide sequence of genes, design LDH Deletion primers and identification of genes.

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Abstract

The invention discloses recombinant escherichia coli constructed by genes and a method for biosynthesizing 6'-sialyllactose, and belongs to the field of metabolic engineering. The recombinant escherichia coli is obtained by correspondingly modifying 6'-sialyllactose synthesis related genes in escherichia coli through a metabolic engineering method (comprising deletion of competitive pathway genesand expression of key pathway genes). 4-5 g / L of the 6'-sialyllactose can be obtained through carrying out shake-flask fermentation for 24 hours by utilizing the strain. The strain can be used for efficiently synthesizing the 6'-sialyllactose by synergistically utilizing glucose and glycerol carbon sources. The constructed recombinant escherichia coli has a wide application prospect and provides anew thought for generating the 6'-sialyllactose by a biological method.

Description

Technical field [0001] The present invention relates to the technical field of industrial microbial metabolic engineering, and specifically relates to an application of recombinantly transforming Escherichia coli and its industrial fermentation to produce 6'-sialyllactose. Background technique [0002] Breast milk has always been regarded as the best food source for infants and young children. In addition to providing the nutrients required for normal growth of infants and young children, breast milk also contains many prebiotics that are not found in cow's milk. Among them, the important component human milk oligosaccharides (HMOs) is the third largest solid component naturally found in human breast milk after lactose and fat. It is usually composed of 2-10 monosaccharide molecules. A large number of studies have shown that for breastfed infants, human milk oligosaccharides play a very extensive and beneficial biological role, such as promoting the growth of beneficial flor...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P19/44C12R1/19
CPCC12N15/70C12P19/44C12N9/2471C12Y302/01023C12Y203/01018C12Y101/01049C12Y207/01011C12N9/0006C12Y205/01056C12Y207/07043C12N9/1081C12N9/90C12N9/1025C12N9/1085C12N9/1205C12N9/1241C12N2800/22Y02A50/30
Inventor 王磊冯露黄笛刘斌
Owner NANKAI UNIV
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