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Genetically engineered bacterium for high-yield production of ergothioneine

An ergothioneine and genome technology, applied in the field of genetic engineering, can solve the problems of high ergothioneine price, low production capacity, hindering the development and promotion of the ergothioneine market, and achieve the effect of expanding market supply

Pending Publication Date: 2022-08-05
深圳中科欣扬生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, the main production method of ergothioneine is still plant extraction, and its low production capacity leads to high prices of ergothioneine, which seriously hinders the market development and promotion of ergothioneine

Method used

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  • Genetically engineered bacterium for high-yield production of ergothioneine
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  • Genetically engineered bacterium for high-yield production of ergothioneine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Construction of recombinant Escherichia coli containing ergothioneine synthesis gene cluster

[0042] (1) The ergothioneine synthesis genes RsEGT1 (genebank: RCH97401.1) and RsEGT2 (genebank: RCI05990.1) derived from Rhizopus stolonifer were selected, and the nucleotide sequences were obtained after codon optimization for Escherichia coli The gene fragments shown in SEQ ID NO. 1 and SEQ ID NO. 2 were subjected to DNA synthesis to obtain candidate genes.

[0043] (2) Design relevant primers (Table 1), take the RsEGT1 gene fragment whose nucleotide sequence is as shown in SEQ ID NO.1 in step (1) as a template, and use primers F1 and R1 to amplify the fragment RsEGT1 by PCR; The RsEGT2 gene fragment whose nucleotide sequence is shown in SEQ ID NO. 2 is used as a template, and the fragment RsEGT2 is obtained by PCR amplification using primers F2 and R2. Using pBAD / HisA vector (Beijing Jiangchen Biotechnology) as the template, the linear pBAD vector was amplified...

Embodiment 2

[0053] Example 2: Optimization of ergothioneine synthesis pathway

[0054] (1) Two ergothioneine synthases, the gene RrEgtB (NCBI Reference Sequence: WP_038682659.1) and the gene RrEgtC (NCBI Reference Sequence: WP_003419806.1) derived from Rubrobacter radiotolerans, were selected to target E. After optimization, gene fragments whose nucleotide sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4 are obtained, and DNA synthesis is performed to obtain candidate genes.

[0055] (2) Design relevant primers (Table 1), take the RrEgtB gene fragment whose nucleotide sequence is shown in SEQ ID NO.3 as a template, and use primers F4 and R4 to amplify the fragment RrEgtB by PCR; The RrEgtC gene fragment shown in SEQ ID NO. 4 was used as a template, and the fragment RrEgtC was obtained by PCR amplification using primers F5 and R5. Using R3 and pBAD-F as primers and pBAD-RsEGT1-RsEGT2 plasmid as template, PCR was performed to obtain the vector fragment. Fragment RrEgtB, fragment RrEgt...

Embodiment 3

[0057] Example 3: Construction of Chassis Cells

[0058] Chromosomal editing of Escherichia coli BW25113 using CRISPR / CAS9 gene editing technology to enhance the supply of three precursor amino acids and adenosylmethionine (SAM). CRISPR-Cas9 System".

[0059] (1) CRISPR / CAS9 gene editing technology:

[0060] (1) Preparation of DNA fragments: use CRISPR / CAS9 gene editing technology for gene knockout, design primer pairs to amplify the upstream and downstream homology arms of the target gene with 100-500bp each, and fuse the upstream and downstream fragments by fusion PCR to obtain DNA fragments , recovered by DNA gel recovery or PCR product purification kit. Gene insertion using CRISPR / CAS9 gene editing technology amplifies the target gene while amplifying the upstream and downstream homology arms of the insertion site, and fuses the square fragments of fusion PCR to obtain DNA fragments.

[0061] (2) Design of sgRNA: Use the CRISPR-ERA website (crispr-era.stanford.edu) to d...

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Abstract

The invention discloses a genetically engineered bacterium for high yield of ergothioneine, and belongs to the field of genetic engineering. The ergothioneine synthesis yield of the recombinant bacterium BWEGT-12 provided by the invention reaches the highest within 72 hours, and the yield is 110 mg / L. According to the method, ergothioneine synthesis gene clusters with different sources are screened firstly, ergothioneine synthesis genes from rhizopus nigricans and radiation-resistant red bacillus are screened, and the yield of 65mg / L and 77mg / L of ergothioneine is achieved preliminarily. Furthermore, chassis cells are improved, enhanced expression or knockout is carried out on related genes in metabolic pathways of histidine, cysteine and methionine, and the yield of 81mg / L of ergothioneine is achieved. In order to achieve higher yield, genes in the whole ways of methionine circulation and folic acid circulation are adjusted in a targeted manner, and finally the yield of 110mg / L of ergothioneine is achieved. According to the invention, efficient synthesis of ergothioneine is realized in escherichia coli, and the market supply of ergothioneine can be expanded.

Description

technical field [0001] The invention relates to a genetically engineered bacterium with high ergothioneine production, belonging to the field of genetic engineering. Background technique [0002] Ergothioneine (ergothioneine) is a sulfur-containing amino acid derivative, it is a unique cell physiological protection agent, widely exists in human blood, semen, liver and kidney and other tissues, with strong anti-oxidation, anti-aging , Anti-radiation activity, with the maintenance of DNA synthesis and normal cell growth and metabolism and a variety of physiological functions, is regarded as a functional active substance necessary to maintain human health. However, the human body cannot synthesize ergothioneine itself, and it needs to be obtained from other foods that synthesize ergothioneine, such as mushrooms, and accumulate in most cells and tissues of the human body through a new type of cation transporter (OCTN1). With the increase of age, the concentration of ergothionei...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/67C12N15/31C12P17/10C12R1/19
CPCC07K14/37C07K14/195C12N15/70C12P17/10C12N2800/22Y02A50/30
Inventor 张山丁利平焦银山
Owner 深圳中科欣扬生物科技有限公司
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