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CRISPR-Cas9-based ergothioneine biosynthesis method and application

An ergothioneine, gene cluster technology, applied in the directions of microorganism-based methods, biochemical equipment and methods, botanical equipment and methods, etc., to achieve the effect of improving adaptability and fermentation capacity

Active Publication Date: 2022-06-10
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

Most of the existing ergothioneine engineering bacteria are directly as hosts with bacteria such as E. The biosynthesis method with Bacillus and Saccharomyces cerevisiae as the host still has the disadvantages of low yield, long fermentation cycle, and high production cost; while the biosynthesis method with Escherichia coli as the host has the risk of phage infection and endotoxin contamination, resulting in limited application
In addition, traditional gene editing methods have disadvantages such as complicated steps, low operability, and difficulty in integrating into the genome (patent publication number CN109554414A)

Method used

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  • CRISPR-Cas9-based ergothioneine biosynthesis method and application
  • CRISPR-Cas9-based ergothioneine biosynthesis method and application
  • CRISPR-Cas9-based ergothioneine biosynthesis method and application

Examples

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Effect test

preparation example Construction

[0045] (3) Preparation of Competent Corynebacterium glutamicum

[0046] In the following examples, the strain needs to be prepared before electroporation, and the preparation steps for the competence of Corynebacterium glutamicum are as follows:

[0047] (1) Streak the bacterial solution, pick a single colony in LB medium, culture overnight at 30°C, 200r / min;

[0048] (2) The above bacterial solution was inoculated in LB medium at a ratio of 2%, so that the initial OD 600 0.3, 30°C, 200r / min culture to OD 600 0.6-0.9;

[0049] (3) Put the above bacterial solution into a centrifuge tube for 15 minutes on ice, then centrifuge at 4000r / min at 4°C for 10 minutes, and discard the supernatant;

[0050] (4) The above-mentioned bacteria sludge was fully resuspended with pre-cooled 10% glycerol, centrifuged at 4000r / min, 4°C for 10min, discarded the supernatant, and repeated twice;

[0051] (5) The above-mentioned bacteria sludge was resuspended with a small amount of pre-cooled 10...

Embodiment 1

[0061] Construction of CRISPR / Cas9 gene knockout vector:

[0062] The protein A described in this example is a peptidoglycan endonuclease, the nucleotide sequence of its encoding gene mepA is shown in Gene ID: 1020444; the protein B is cgl2736, the nucleotide sequence of its encoding gene As shown in GenBank: 2917628 to 2918758 in BA000036.3; the protein C is an intracellular protease, and the nucleotide sequence of its coding gene clpC is shown in Gene ID: 3345370.

[0063] 1. Design of sgRNA: Design sgRNA using Corynebacterium glutamicum ATCC 13032 as a template. The specific operation method is to input the nucleotide sequence of the target gene on the sgRNA special design website (http: / / crispor.tefor.net / ) , select the genome of Corynebacterium glutamicum, select 20bp-NGG-sp Cas9 as the PAM site for analysis, and select two sgRNA sequences with low off-target rate. In this example, the sgRNA nucleotide sequences sgRNA-A1 and sgRNA-A2 corresponding to protein A are shown ...

Embodiment 2

[0069] Construction of CRISPR-Cas9 gene knock-in vector:

[0070] On the basis of steps 1-2 described in Example 1, primers were designed to amplify the egt12 gene cluster (nucleotide sequence shown in SEQ ID No.15) retained by our company, and the corresponding gene fragments were divided into sgRNA by Golden gate -The sequence of the upstream homology arm-egt12-downstream homology arm was connected to the pCas9 plasmid, and the pCas9-A-egt12, pCas9-B-egt12, pCas9-C-egt12 knock-in vectors were respectively constructed.

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Abstract

The invention discloses an ergothioneine biosynthesis method based on CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9) and application, and belongs to the field of gene engineering. According to the recombinant corynebacterium glutamicum, coding genes of non-essential protein peptidoglycan endonuclease, endoprotease and glutamic acid-cystine ligase are knocked out from the corynebacterium glutamicum, and an egt12 gene cluster is integrated, so that the recombinant corynebacterium glutamicum has better adaptability and better ergothioneine production capacity. The genetically engineered bacterium constructed by the invention is fermented for 56h at 30 DEG C under the condition of a 50L fermentation tank, and the yield of ergothioneine is as high as 5.47 g / L. The industrial production of the ergothioneine is facilitated.

Description

technical field [0001] The invention relates to a CRISPR-Cas9-based ergothioneine biosynthesis method and its application, belonging to the field of genetic engineering. Background technique [0002] Ergothioneine (EGT) was first isolated from Claviceps purpurea, a fungus parasitic on rye, and is a natural thiol-containing small molecule compound derived from histidine; Ergothioneine is a powerful antioxidant with the ability to scavenge reactive oxygen species (ROS) such as hydroxyl radicals, hypochlorous acid, peroxynitrite, and singlet oxygen, as well as regulate cellular hydrogen peroxide, tumor The ability of necrosis factor α or inflammatory response caused by palmitic acid treatment; in addition, ergothioneine can also protect DNA from damage, chelate divalent metal ions, and the compound produced after ergothioneine chelation is stable and will not regenerate Decomposition produces free radicals. Therefore, ergothioneine is expected to be widely used in food, medic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/77C12N15/90C12N15/54C12N15/55C12P17/10C12R1/15
CPCC12N9/13C12N15/77C12N15/902C12N9/22C12P17/10C12Y208/01007Y02A50/30
Inventor 董亮张山甘淼陈永丽张岩峰张艳艳
Owner 深圳中科欣扬生物科技有限公司
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