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Construction method and application of recombinant Escherichia coli for synthesizing l-cysteine

A technology for recombining Escherichia coli and cysteine, which is applied in the field of L-cysteine ​​fermentation and production, can solve the problems of the regulation mechanism hindering the efficient biosynthesis of L-cysteine, and achieve good production and application value, genetic modification little effect

Active Publication Date: 2022-07-26
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the toxicity of L-cysteine ​​to cells and complex regulatory mechanisms hinder the efficient biosynthesis of L-cysteine, making L-cysteine ​​fermentation production one of the main challenges facing the amino acid fermentation industry

Method used

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  • Construction method and application of recombinant Escherichia coli for synthesizing l-cysteine
  • Construction method and application of recombinant Escherichia coli for synthesizing l-cysteine
  • Construction method and application of recombinant Escherichia coli for synthesizing l-cysteine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Escherichia coli chassis cells and expression plasmid pLH03 ( figure 1 ) fit comparison

[0036] In this example:

[0037] The composition of the seed medium: 10g / L peptone, 5g / L yeast powder, 10g / L sodium chloride; solid medium plus agar 15g / L.

[0038] Fermentation medium composition: 10g / L glucose, 12g / L dipotassium phosphate, 3g / L potassium dihydrogen phosphate, 0.1g / L sodium chloride, 5g / L ammonium thiosulfate, 0.3g / L magnesium sulfate, 0.015 g / L calcium chloride 0.002g / L ferrous sulfate, 1g / L sodium citrate, 5mg / L vitamin B1, 1mL / L trace elements (0.15g / L sodium molybdate, 2.5g / L boric acid, 0.7g / L L cobalt chloride, 0.25g / L copper sulfate, 1.6g / L manganese chloride, 0.3g / L zinc sulfate), 50mg / L apramycin sulfate.

[0039] The specific implementation process is as follows:

[0040] (1) Select different Escherichia coli chassis cells to be activated in seed medium to prepare calcium transcompetent cells.

[0041] (2) Expression plasmid pLH03 (derive...

Embodiment 2

[0059] Example 2: Constitutive promoters of different strengths alone overexpress genes in the L-cysteine ​​metabolic pathway

[0060] (1) with pLH02(PcysE-cysE) (derived from pACYC184 plasmid, Biotechnol.J.2018, No.e1700695) ( figure 2 ) as the starting plasmid, the cysE gene was replaced with fluorescent protein mCherry, and the pLH02-1 (PcysE-mCherry) plasmid was constructed by one-step ligation (seamless cloning) ( image 3 ), and the primers required for construction are shown in Table 2.

[0061] Table 2 Plasmid Construction Primer List (SEQ ID NO.3-SEQ ID NO.6)

[0062]

[0063] (2) pLH02-1 (PcysE-mCherry) constructed in step (1) ( image 3 ) as the starting plasmid, by randomly mutating the lacO binding site of the inducible promoter Ptrc (derived from the commonly used high-copy plasmid pTrc99a), two constitutive promoters Ptrc1 and Ptrc2 with different strengths were obtained (see

[0064] SEQ ID NO. 1:

[0065] TGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTCAGC...

Embodiment 3

[0081] Example 3: Combination of different strengths of constitutive promoters to overexpress genes in the L-cysteine ​​metabolic pathway

[0082](1) Select the single gene with improved yield and significant difference (p<0.05) in Example 2 for combination, namely LH2A-pLH03, LH2C-pLH03, LH1B-pLH03, LH1M-pLH03, LH1H-pLH03 , and combined and overexpressed these single-gene operations with better results to construct strain LH2A2C1B1M1H, and the primers required for the construction are the same as Table 5.

[0083] (2) Select the single gene with improved yield and extremely significant difference (p<0.01) in Example 2 for combination, that is, LH2A-pLH03, LH1M-pLH03, and combine these better single genes. Expression, construction of strain LH2A1M.

[0084] (3) The strains constructed in step (1) and step (2) were prepared for calcium transduction, transformed into pLH03 plasmid to obtain L-cysteine ​​producing strains, which were named LH2A2C1B1M1H-pLH03 and LH2A1M-pLH03 in ...

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Abstract

The present invention provides a construction method and application of recombinant Escherichia coli for synthesizing L-cysteine, and the construction method includes the following transformation approaches: (1) adapting L-cysteine ​​expression plasmid pLH03 to Escherichia coli chassis cells, the Chassis cells are K-12 series strains; (2) L-cysteine ​​synthesis pathway genes are expressed individually and / or in combination using constitutive promoters of different strengths; (3) L-Cysteine ​​synthesis pathway genes are individually and / or in combination knocked out Cysteine ​​catabolism pathway gene and its precursor L-serine catabolism pathway gene. The recombinant strain constructed by the invention has less genetic modification, high L-cysteine ​​production and good production and application value.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to the construction of a strain for biosynthesizing L-cysteine ​​by recombinant Escherichia coli and a method for using the strain for L-cysteine ​​fermentation production. Background technique [0002] L-cysteine ​​(L-cysteine) is an amino acid with active sulfhydryl group. Due to its physiological functions and chemical properties, it is widely used in medicine, food, agriculture, daily chemicals and other industries (Adv.Biochem.Eng.Biotechnol .2017, 159, 129-151). [0003] In the field of medicine, L-cysteine ​​has a special sulfhydryl group, which plays a significant role in the treatment of many types of diseases. In vivo, L-cysteine ​​has detoxification effect on many harmful substances such as formaldehyde. When allergies and inflammations in the body reduce the activity of thiolase, L-cysteine ​​can maintain the activity of thiolase, and has anti-allerg...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N1/21C12P13/12C12R1/19
CPCC12N15/70C12P13/12
Inventor 李志敏刘晗
Owner EAST CHINA UNIV OF SCI & TECH
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