Enzymatic activity-changed amino deoxychorismate synthetase mutant T426I and application thereof

A chorismate and synthase technology, applied in the fields of genetic engineering and fermentation engineering, can solve problems such as influence level, and achieve the effect of good industrial application value and prospect.

Active Publication Date: 2020-12-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And since the aminodeoxychorismate synthase (ADC synthase) encoded by pabAB is a key enzyme in the synthetic pathway of p-aminobenzoic acid (PABA), a component of folic acid (Matthews RG. One-carbon metabolism In Escherichia coli and Salmonella Cellular and Molecular Biology Volume 1. Second edition. Edited by: Neidhardt FC, Curtiss R 3rd, Ingraham JL, Lin ECC, LowKB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE. Washington DC, ASM Press; 1996:600-611), speculated amino The difference in the activity of deoxychorismate synthase will directly cause the change of intracellular folic acid content, thereby affecting the level of coenzyme tetrahydrofolate in SHMT, that is to say, aminodeoxychorismate synthase (coding gene pabAB) is related to the degradation of L-serine, Aminodeoxychorismate synthase can promote the degradation of L-serine, and correspondingly, reducing its activity may increase the production of serine
[0003] However, how to improve serine production by carrying out directional transformation of aminodeoxychorismate synthetase has not been reported at present. Therefore, the present invention provides a kind of operable scheme for this problem, which is of great importance for utilizing Corynebacterium glutamicum to produce serine. significance

Method used

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  • Enzymatic activity-changed amino deoxychorismate synthetase mutant T426I and application thereof
  • Enzymatic activity-changed amino deoxychorismate synthetase mutant T426I and application thereof
  • Enzymatic activity-changed amino deoxychorismate synthetase mutant T426I and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Point mutation of aminodeoxychorismate synthetase to T426I

[0037] Site-directed mutagenesis was carried out by PCR method, saturation mutation was performed on the P426 site of ΔSSAAI aminodeoxychorismate synthetase, and the tyrosine at the 426th site was mutated into isoleucine. Using the strain genome as a template, select the 500 bp before and 500 bp after the point mutation as the amplification target, and use primers pabAB-F and pabAB-R to amplify the homology arm gene fragment containing the point mutation. It was ligated with the plasmid pK18mobsacB double-digested by EcoR I and Xba I to construct a reverse mutation plasmid, and electrotransformed Corynebacterium glutamicum; cultured at 30°C for 3 days, and the single colony grown was a recombinant colony. Pick a single colony and inoculate it into a seed liquid medium containing 50 μg / mL Kan, cultivate it at 30°C on a reciprocating shaker at 120 rpm, dilute it to 10% sucrose screening medium, and cu...

Embodiment 2

[0041] Example 2: Point mutation of aminodeoxychorismate synthetase to other amino acids

[0042] Site-directed mutagenesis was carried out by PCR method, saturation mutation was carried out on the P426 site of aminodeoxychorismate synthetase, and the tyrosine at the 426th site was mutated into other 19 kinds of amino acids. Subsequent other steps are the same as in Example 1. The primers used for T426 to carry out other non-T426I mutations are the same as SEQ ID NO.3 and SEQ ID NO.4.

Embodiment 1-2

[0043] Embodiment 1-2 result analysis

[0044] (1) The effect of point mutation on the activity of aminodeoxychorismate synthase

[0045] The obtained series of aminodeoxychorismate synthase mutant strains were tested for enzyme activity, and the effect of point mutations on the aminodeoxychorismate synthase activity was analyzed. The kit was used to measure the aminodeoxychorismate synthesis of the starting strain ΔSSAAI and the mutant strain T426I after 60 hours of cultivation. Enzyme specific enzyme activity.

[0046] The results are shown in Table 2. Taking the unmutated aminodeoxychorismate synthetase starting strain as a control group, the aminodeoxychorismate synthase obtained in Example 1 was point-mutated into T426I and the aminodeoxychorismate synthetase obtained in Example 2. The mutant strains with enzyme point mutations to other amino acids were used as the experimental group, and the specific enzyme activities of amino-deoxychorismate synthetase after cultivatio...

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Abstract

The invention discloses an enzymatic activity-changed amino deoxychorismate synthetase mutant T426I and application thereof, and belongs to the field of genetic engineering and fermentation engineering. According to the enzymatic activity-changed amino deoxychorismate synthetase mutant T426I, a T426 locus of amino deoxychorismate synthetase in parent Corynebacterium glutamicum delta SSAAI (the preservation name of a strain is delta SSAAI, and the preservation number is CGMCC No 15170) is mutated into isoleucine, and the enzymatic activity of the obtained mutant is reduced by 15.2%. A genetically engineered bacterium constructed by the mutant is fermented, and the yield of L-serine is increased by 18.75%. The enzymatic activity-changed amino deoxychorismate synthetase mutant T426I effectively changes the activity of the amino deoxychorismate synthetase through point mutation, creates conditions for constructing the genetically engineered bacterium for efficiently producing the serine, and has good industrial application value and prospects.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and fermentation engineering, and specifically relates to an aminodeoxychorismate synthetase mutant T426I with changed enzyme activity and its application. Background technique [0002] Corynebacterium glutamicum (Corynebacterium glutamicum) is a typical prokaryotic model strain, which has the advantages of clear genetic background and convenient metabolic transformation operation; it is also certified by the FDA as a GRAS (General Regarded AsSafe) strain, and it is the most important strain for the production of amino acids by fermentation. microorganism. In Corynebacterium glutamicum, phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT) and phosphoserine phosphorylase (PSP) are usually involved in the synthesis of L-serine, while serine hydroxymethyltransferase (SHMT) and serine dehydrogenase (SerDH) are then involved in the degradation reaction of L-serine. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N1/21C12P13/06A23L29/00A23K20/189A23K20/142C12R1/15
CPCC12N9/1096C12Y206/01085C12P13/06A23L29/06A23L29/045A23K20/189A23K20/142A23V2002/00A23V2250/0642
Inventor 张晓梅卞金玉武政楠李会史劲松许正宏
Owner JIANGNAN UNIV
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