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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, and can solve problems such as the regulation mechanism hindering the efficient biosynthesis of L-cysteine

Active Publication Date: 2021-09-14
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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] Embodiment 1: Escherichia coli chassis cell 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 hydrogen 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) Different Escherichia coli chassis cells were selected and inserted into the seed medium for activation to prepare calcium-transduced competent cells.

[...

Embodiment 2

[0059] Example 2: Constitutive promoters of different strengths individually 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 fluorescent protein mCherry was used to replace the cysE gene, and the pLH02-1(PcysE-mCherry) plasmid was constructed by one-step ligation (seamless cloning) ( image 3 ), construct the required primers as 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] TGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTCAGCGG...

Embodiment 3

[0081] Example 3: Constitutive promoter combinations of different strengths overexpress genes in the L-cysteine ​​metabolic pathway

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

[0083] (2) Select the single genes 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, construct strain LH2A1M.

[0084] (3) Prepare the strains constructed in steps (1) and (2) to be competent for calcium transduction, transform the pLH03 plasmid, and obtain L-cysteine ​​production strains, which are named LH2A2C1B1M1H-pLH03 and LH2A1M-pLH03 in s...

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Abstract

The invention provides a construction method and application of recombinant escherichia coli for synthesizing L-cysteine. The construction method comprises the following transformation ways: (1) adapting an L-cysteine expression plasmid pLH03 and an escherichia coli chassis cell, wherein the chassis cell is a K-12 series strain; (2) performing individual and / or combined expression on the L-cysteine synthetic pathway gene by using constitutive promoters with different intensities; and (3) knocking out an L-cysteine decomposition pathway gene and a precursor L-serine decomposition pathway gene of the L-cysteine decomposition pathway gene independently and / or in combination. The recombinant strain constructed by the invention is relatively less in gene modification, has high yield of L-cysteine, and has relatively high production and application values.

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 fermenting and producing L-cysteine ​​with the strain. Background technique [0002] L-cysteine ​​(L-cysteine) is an amino acid with an active sulfhydryl group. Due to its physiological functions and chemical properties, it is widely used in industries such as medicine, food, agriculture, and daily chemicals (Adv.Biochem.Eng.Biotechnol .2017, 159, 129-151). [0003] In the field of medicine, L-cysteine ​​has a special sulfhydryl group, which has a significant effect on the treatment of many types of diseases. In organisms, L-cysteine ​​has a detoxification effect on many harmful substances such as formaldehyde. When the body has allergies and inflammations that reduce the activity of thiol enzymes, L-cysteine ​​can maintain the activity of thiol enzy...

Claims

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Application Information

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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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