Recombinant escherichia coli capable of high-yielding L-methionine and application thereof

A technology for recombining Escherichia coli and methionine, which is applied in the direction of recombinant DNA technology, bacteria, and microbial-based methods, and can solve problems such as the lack of in-depth and detailed research on the fermentation and production of methionine

Active Publication Date: 2018-12-21
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Based on the above reasons, there has not been in-depth and detaile

Method used

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  • Recombinant escherichia coli capable of high-yielding L-methionine and application thereof
  • Recombinant escherichia coli capable of high-yielding L-methionine and application thereof
  • Recombinant escherichia coli capable of high-yielding L-methionine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Global search—L-methionine synthesis gene in situ promoter replacement in the genome

[0047] (1) E.coli W3110ΔmetJΔmetI / pTrc99A / metA* / yjeH (Jiangfeng Huang et al.2016.Metabolic engineering of Escherichia coli for microbial production of L-methionine.Biotechnology and Bioengineering.114:843-851) was used as the starting strain, and the global Search strategy, using CRISPR-Cas9-mediated gene editing technology (Yu Jiang et al.2015MultigeneEditing in the Escherichia coli Genome via the CRISPR-Cas9System.AppliedEnvironmental Microbiology.81:2506-2514), using the trc promoter derived from pTrc99A ( The nucleotide sequence is shown in SEQ ID No.1), the original promoters of all genes involved in L-methionine biosynthesis are replaced in the genome, the genes involved and the corresponding pathways are shown in Table 1, identified in Key genes affecting L-methionine in the starting strain.

[0048] Table 1 Genes and corresponding pathways involved in gene editing ...

Embodiment 2

[0063] Example 2: Global Search—L-Methionine Synthetic Gene in Genome Shake Flask Fermentation Test of Strains with In Situ Promoter Replacement

[0064]A series of strains of different genotypes constructed in Example 1 (as shown in Table 4) were inoculated into 10 ml of LB medium containing 50 mg / l of Amp, cultivated at 37° C. and 200 rpm as a preculture, wherein E.coli W3110ΔmetJΔmetI / pTrc99A / metA* / yjeH was a negative control. After 8-12 hours, inoculate 1ml of the preculture into a 500ml shake flask containing 20ml of MS medium containing 50mg / L Amp. Then culture and ferment to OD at 30°C and 150rpm 600 =0.8-1.0, add IPTG with a final concentration of 0.1mM, continue to cultivate for 48h, take 1ml of fermentation broth to measure OD after fermentation 600 Take 1ml of fermentation broth, centrifuge at room temperature at 12000rpm for 3min, dilute the fermentation supernatant 100 times, and analyze the amino acid titer with a fully automatic amino acid analyzer (SYKAM S-4...

Embodiment 3

[0067] Example 3: Global Search—Down-regulation of the expression of genes not involved in the synthesis of L-methionine and methionine in the central metabolic pathway

[0068] In order to down-regulate the expression of genes not involved in the biosynthesis of L-methionine and methionine in the central metabolic pathway in the genome, E.coli W3110ΔmetJΔmetI / pTrc99A / metA* / yjeH was used as the starting bacterium to implement CRISPR-dCas9-mediated The sequence-specific gene expression interference technology (CRISPRi) (Qi LS.et al.Repurposed CRISPR as an RNA-Guided Platform for Sequence-Specific Control of GeneExpression.Cell.2013.152(5):1173-1183), this technology can make dCas9 The protein binds to a specific position in the gene coding sequence and reduces gene expression by inhibiting the movement of RNA polymerase. In order to realize this process, the following primers (Table 6) were used to construct pTarget plasmid, pdCas plasmid.

[0069] Use CRISPRi technology to kn...

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Abstract

The invention discloses a recombinant escherichia coli capable of high-yielding L-methionine and an application thereof. The recombinant escherichia coli is constructed according to a method as follows: adopting a CRISPR-Cas9 gene editing technology for respectively replacing L-methionine synthase gene promoters with trc promoters in escherichia coli genomes, introducing a host strain genome and screening, thereby acquiring the recombinant escherichia coli capable of high-yielding L-methionine. The host strain is E.coli W3110 Delta metJ Delta metI/pTrc99A/metA*/yjeH which is constructed by knocking out metJ and metI in escherichia coli E.coli W3110 and then introducing a pTrc99A/metA*/yjeH plasmid. The maximal titer of L-methionine can reach up to 2.8g/L and DAP of by-products is obviouslyreduced to above 60% (reduced from 1g/L to below 0.4g/L).

Description

(1) Technical field [0001] The present invention relates to related technologies in the fields of molecular biology, synthetic biology and metabolic engineering. Specifically, it involves the use of molecular biology techniques to construct recombinant expression plasmids to up-regulate the expression of key enzymes, and to transform the regulatory system and metabolic pathways of parental strains. , proposed a global search, based on the functional modular metabolic network and the flux allocation optimization strategy between modules, systematically analyzed the limiting factors of L-methionine biosynthesis, and systematically reprogrammed the metabolic pathways of the parental strains to remove the restrictions layer by layer Bottleneck, finally use the obtained engineered bacteria to enhance the production of L-methionine. (2) Background technology [0002] For animals, methionine (also known as methionine) is a sulfur-containing essential amino acid that plays an import...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/113C12P13/12C12R1/19
CPCC12N15/113C12P13/12
Inventor 柳志强郑裕国黄建峰张博牛坤沈臻旸毛巧利
Owner ZHEJIANG UNIV OF TECH
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