A kind of mutant of serine hydroxymethyltransferase and use thereof

A serine hydroxymethyl and transferase technology, applied in the biological field, can solve problems such as difficulty in confirming the impact of secondary mutations and hindering the improvement of strains

Active Publication Date: 2022-07-22
SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, a number of L-serine production strains have been gradually cultivated at home and abroad through the combination of traditional mutation breeding and metabolic engineering. However, the construction process of the above-mentioned engineering bacteria involves multiple rounds of mutagenesis, and it is difficult to confirm the mutagenesis process. The impact of secondary mutations on strains, which in turn hinders further improvement of strains

Method used

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  • A kind of mutant of serine hydroxymethyltransferase and use thereof
  • A kind of mutant of serine hydroxymethyltransferase and use thereof
  • A kind of mutant of serine hydroxymethyltransferase and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Construction of serine hydroxymethyltransferase site-directed mutant engineering bacteria

[0046] Construction of BL21(DE3) / pT 7-7-glyA: The genome of the wild-type strain E.coli W3110 (ATCC 23275) was used as the template, and the primers glyA-F / R (see Table 1 for the sequence) were used to synthesize E.coli E.coli. The serine hydroxymethyltransferase expression gene glyA in W3110 (ATCC 23275) (the glyA gene sequence is shown in the sequence 2682276-2683529 in Genbank accession number U00096.2) was amplified by PCR, verified by electrophoresis, and purified by electrophoresis gel recovery. The glyA fragment. The purified glyA fragment and pT 7-7 plasmid (Addgene, MA, USA) were double digested with NdeI and HindIII, respectively, and the double digested product of the glyA fragment and the pT 7-7 plasmid were subjected to T4 digestion. Ligase overnight at 4°C. The ligation product was transformed into Escherichia coli BL21 (DE3), spread on the LB solid plate containi...

Embodiment 2

[0059] Induced expression of serine hydroxymethyltransferase parent engineering bacteria and serine hydroxymethyltransferase site-directed mutant engineering bacteria:

[0060] The serine hydroxymethyltransferase parent engineering bacteria and the serine hydroxymethyltransferase mutant engineering bacteria in Example 1 were respectively inoculated into the LB liquid medium containing the final concentration of 100 μg / mL ampicillin, and cultured at 37°C for 8 hours, inoculated into fresh TB liquid medium containing a final concentration of 100 μg / mL ampicillin with an inoculum volume of 1% by volume, and cultured at 37°C at 200 rpm to OD=0.5 to induce induction by adding 0.1 mM IPTG, and continued. Incubate for 16h. The fermentation broth was centrifuged at 4°C and 10000rpm for 10min, the supernatant was discarded, the cells were washed with phosphate buffer and then centrifuged at 10000r / min for 10min, the supernatant was discarded, and the cells obtained after washing twice ...

Embodiment 3

[0062] Serine hydroxymethyltransferase enzyme activity assay:

[0063] Take the serine hydroxymethyltransferase parent engineering bacteria and the serine hydroxymethyltransferase mutant engineering bacteria wet thalline prepared by the method of Example 2 as a catalyst, press 1mg wet thalli / 1mL substrate solution (50mmol / L DL- Phenylserine, 30 μmol / L pyridoxal phosphate) to prepare a reaction solution, the reaction solution was added with 0.03 g / L cetyltrimethylammonium bromide (CTAB) in a certain proportion to treat the bacteria, and the reaction solution was shaken at 30 °C for 1 h. Centrifuge at 5000r / min for 10min, take the supernatant, measure the absorbance value at A279, and determine the benzaldehyde concentration in the reaction solution according to the A279-benzaldehyde concentration regression equation. In 1 L of substrate solution at 30°C, U is 1 g of wet cells to produce 1 mol of benzaldehyde for 1 h. Using serine hydroxymethyltransferase enzyme activity unit a...

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Abstract

The present invention relates to the field of biotechnology, in particular to a mutant of serine hydroxymethyltransferase and use thereof. The present invention provides a mutant of serine hydroxymethyltransferase. The amino acid sequence of the mutant of serine hydroxymethyltransferase is relative to the amino acid sequence of wild-type serine hydroxymethyltransferase. The amino acid residue at position 229, or the amino acid residue at position 229, is replaced by other amino acid residues, and the amino acid sequence of the wild-type serine hydroxymethyltransferase includes the sequence shown in SEQ ID NO.24. The mutant of serine hydroxymethyltransferase and related genetic engineering bacteria provided by the invention can effectively improve the ability of Escherichia coli to produce L-serine.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a mutant of serine hydroxymethyltransferase and use thereof. Background technique [0002] L-serine (L-serine, L-ser) is an aliphatic polar amino acid with molecular formula C 3 H 7 NO 3 , has important physiological role and application value. In organisms, L-serine, as an important intermediate metabolite, is the main source of one-carbon units in the process of cell proliferation, and is involved in the synthesis of glycine and cysteine, and between sulfhydryl (-SH) and hydroxyl (-OH) transformation, and the synthesis of purine, thymine and choline. At the same time, studies have shown that L-serine also plays an important role in the formation and development of the central nervous system, and plays a very important role in regulating the body. Using L-serine as a raw material can synthesize more than 20 kinds of anti-cancer, anti-AIDS, Drugs with different effects s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N1/21C12P13/06C12R1/19
CPCC12N9/1014C12Y201/02001C12N9/0006C12N9/1096C12N9/1217C12Y101/01095C12Y206/01052C12Y207/02003C12P13/06
Inventor 赵志军王晨阳史吉平陈枭嘉李沁雨李立马新新
Owner SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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