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dna molecule, expression vector, recombinant bacteria and its construction method, and preparation method of hydroxyproline

A technology of expression vector and construction method, which is applied in the field of amino acid fermentation, can solve the problems of low dissolved oxygen and low yield, and achieve the effects of increasing fermentation yield, short fermentation cycle, and increasing yield

Active Publication Date: 2021-07-06
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are few research reports on the production of hydroxyproline by microbial transformation. According to the degeneracy of codons and the codon preference of E. coli, the researchers optimized the proline 4-hydroxylase gene (DSp4h) in the large intestine. Bacillus expression; Knock out the putA gene to block the degradation pathway of proline to glutamic acid; Introduce the vgb gene to solve the problem of low dissolved oxygen in the fermentation process of the strain and further increase the fermentation yield of hydroxyproline
However, at present, the yield of hydroxyproline produced by fermentation with glucose as substrate is still low (25g / L, 96h), and there is a big gap with the realization of industrialized production. Therefore, it is urgent to select and breed engineering strains that efficiently synthesize hydroxyproline , to realize the production of hydroxyproline by microbial direct fermentation

Method used

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  • dna molecule, expression vector, recombinant bacteria and its construction method, and preparation method of hydroxyproline
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  • dna molecule, expression vector, recombinant bacteria and its construction method, and preparation method of hydroxyproline

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Embodiment 1

[0043] The construction of embodiment 1 chassis bacterium CG415

[0044] According to the metabolic regulation network of L-proline, 5-phosphoglutamate kinase (encoded by proB), the first enzyme on the L-proline synthesis pathway, is regulated by feedback inhibition of the final product L-proline.

[0045] According to the method described in the patent (CN101084312A), the glycine at the 149th position of point mutation ProB is aspartic acid in this embodiment, and the wild-type strain ATCC13032 is used as the starting strain to construct the chassis bacterium CG415 (WT -proB G446A ).

[0046] The specific construction steps of the strain are as follows:

[0047] Chromosomal proB gene site-directed mutation adopts a two-step replacement method. First, the proB gene is knocked out, and then the site-directed mutation proB gene is inserted into the chromosome.

[0048] 1) Knockout of the proB gene: First, design primers based on the proB gene of Corynebacterium glutamicum ATC...

Embodiment 2

[0071] The construction of embodiment 2 hydroxyproline recombinant bacteria Hyp-1

[0072] The original sequence of DSp4h was derived from Dactylosporangium sp. (GenBank ID: D78338.1), optimized according to the codon usage frequency of Corynebacterium glutamicum and the GC percentage content of its highly expressed genes, the optimized DSp4h gene See SEQ ID NO:2 for the sequence.

[0073] Primers were designed according to the optimized DSp4h gene sequence, and the conserved RBS sequence (AAAGGAGGA) of the highly expressed gene of Corynebacterium glutamicum was added before the start codon ATG. Using the synthesized DSp4h gene as a template and using WZ2286 and WZ2287 as primers to PCR amplify the connected RBS and DSp4h genes, the 883bp PCR product was obtained as RBS and DSp4h fragments (SEQ ID NO: 35).

[0074] Then, the purified PCR product was ligated with pXMJ19 (purchased from BiovectorScience Lab, Inc, Cat. No. SMD1168H) digested with BamHI and EcoRI in one step usin...

Embodiment 3

[0081] The construction of embodiment 3 hydroxyproline recombinant bacteria Hyp-2

[0082] Primers were designed according to the sucCD gene of Corynebacterium glutamicum ATCC13032 in Genbank (Genbank ID: 1020511 and 1020512) and its upstream and downstream sequences. Genomic DNA of Corynebacterium glutamicum ATCC13032 was used as template, and the upstream homology arm of sucCD gene was amplified by PCR with primers WZ2206 and WZ2207; the downstream homology arm of sucCD gene was amplified with primers WZ2208 and WZ2209. Using the purified PCR product as a template and using WZ2206 and WZ2209 as primers, the overlap extension PCR technique was used to amplify to obtain a 1086 bp fragment containing the upstream and downstream homology arms of the gene sucCD to be knocked out (SEQ ID NO: 21).

[0083] After the purified and recovered PCR product was digested with EcoRI and XbaI, it was ligated with the homologous recombination vector pK18mobsacB (purchased from American Type M...

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Abstract

The invention relates to a DNA molecule, an expression vector, a recombinant bacterium and a construction method thereof, and a preparation method of hydroxyproline. The nucleotide sequence of the DNA molecule RBS1 is shown in SEQ ID NO.1. The RBS1 provided by the invention can improve the expression of proline hydroxylase coding gene.

Description

technical field [0001] The invention generally relates to the field of amino acid fermentation, and specifically relates to a DNA molecule, an expression vector, a recombinant bacterium and a construction method thereof, and a preparation method of hydroxyproline. Background technique [0002] Trans-4-hydroxy-L-proline, also known as hydroxyproline (trans-4-hydroxy-L-proline, Hyp) is an imino acid, which is the product obtained by hydroxylation of L-proline . Hydroxyproline is found in abundant amounts in collagen, but also in elastin, tooth enamel, complement C1, and extensin. As a rare imino acid, hydroxyproline is widely used in pharmaceutical, chemical, food and beauty industries. In the field of medicine, hydroxyproline can be used as the synthetic precursor of chemical drugs such as anti-inflammatory drugs and carbapenems, and its derivative N-acetyl-trans-4-hydroxyproline is a drug for treating inflammation with no toxic side effects The active ingredient of oxacei...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N15/53C12N15/63C12N1/21C12N1/15C12P13/24
Inventor 温廷益张宇张芸商秀玲刘树文
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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