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Method for improving yield of S-adenosylmethionine through gene expression regulation

A technology of adenosylmethionine and adenosylmethionine decarboxylase, which is applied in the field of genetic engineering and can solve the problems of nutritional deficiencies of recombinant bacteria and increased fermentation costs

Inactive Publication Date: 2014-09-10
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
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Problems solved by technology

In view of this, He et al. (He J, Deng J, Zheng Y, et al. A synergistic effect on the production of S-adenosyl-L-methionine in Pichia pastoris by knocking in of S-adenosyl-L-methionine synthase and knocking out of cystathionine -beta synthase.J Biotechnol.2006,126:519-527) deleted the CBS synthesis gene of the Pichia pastoris strain overexpressing SAM synthase by gene knockout method, and as a result, the accumulation of SAM increased by more than two times, and the effect Quite obvious; however, the SAM catabolism pathway β-cystathionine (CBS) is completely blocked, the recombinant bacteria become auxotrophic, and the medium needs to be supplemented with glutathione (GSH)
Therefore, the cost of fermentation is also greatly increased

Method used

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  • Method for improving yield of S-adenosylmethionine through gene expression regulation
  • Method for improving yield of S-adenosylmethionine through gene expression regulation
  • Method for improving yield of S-adenosylmethionine through gene expression regulation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Embodiment 1, the construction of recombinant bacteria

[0106] 1. Construction of pDsmvA and pDsmvP plasmids

[0107] Such as image 3 As shown, using pCmG as the starting plasmid, first replace the homology arm and connect the vgb gene, which are regulated by PADH2 (SEQ ID NO: 3) and PAOX (SEQ ID NO: 2) respectively, wherein PADH2 is a low-lying gene from Pichia stipitis An oxygen-inducible promoter, PAOX is a methanol-inducible promoter in Pichia pastoris, and two plasmids pDsmvA and pDsmvP were constructed. The specific construction process is as follows:

[0108] Using spe2 5'F and spe2 5'R as primers and using Pichia pastoris genomic DNA as a template to amplify Spe2 5' (SEQ ID NO: 4), insert it into the XhoI and SpeI sites of pCmG, replacing the original CBS5; using spe2 3'F and spe2 3'R as primers and using Pichia pastoris genomic DNA as a template to amplify Spe2 3' (SEQ ID NO: 5), insert it into the XhoI and SpeI sites of pCmG, replacing Original CBSORF; A...

Embodiment 2

[0118] Embodiment 2, research on the characteristics of recombinant bacteria

[0119] 1. Shake flask fermentation under normal conditions

[0120] Under normal conditions (250mL shake flask liquid volume is 50mL, rotating speed 220rpm), without taking artificial oxygen limitation measures, culture DS16 / DsvA, DS16 / DsvP and G / Dspe respectively, with DS16 as a control, investigate the effect of vgb expression on the bacteria Effects on growth and survival. The result is as Figure 9 As shown in A, the maximum specific growth rate of DS16 is after induction for 12 hours (0.018h -1 ), the maximum specific growth rate of G / Dspe in 24 hours (0.016h -1 ), and the two strains (DS16 / DsvA, DS16 / DsvP) introduced into the vgb gene, their maximum specific growth rate was 12 hours after induction, and DS16 / DsvA was 0.021h -1 , DS16 / DsvP is 0.023h -1 . It can be seen that after the introduction of the vgb gene, according to the numerical calculation in the figure, the results show that ...

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Abstract

The invention relates to a method for improving the yield of S-adenosylmethionine through gene expression regulation. Coding genes of Vitreoscilla hemoglobin (VHb) are transformed into an S-adenosylmethionine production strain, and after S-adenosylmethionine decarboxylase spe2 genes are removed, the yield of the S-adenosylmethionine of the S-adenosylmethionine production strain can be obviously improved.

Description

technical field [0001] The invention relates to the field of genetic engineering, more specifically, the invention relates to a method for increasing the production of S-adenosylmethionine by regulating gene expression. Background technique [0002] S-adenosyl-methionine (S-adenosyl-L-methionine, SAM) was discovered by Cantoni et al. in 1952, as an important methyl donor in all organisms, and referring to a variety of metabolic reactions, SAM plays an important role in depression , liver disease and arthritis treatment fields have good clinical application value. Therefore, more and more researchers use metabolic engineering to change the metabolic pathway of cells to obtain high-production SAM strains. [0003] In organisms, with L-Met and ATP as precursors, SAM can be synthesized by SAM synthase ( figure 1 ). The reaction process is that the adenosine part of ATP collides with the sulfur atom part of L-Met, so that the adenosine structure combines with methionine to gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/40C12N1/19C12R1/84
Inventor 钱江潮陆俊杰乔雪锋秦秀林储炬庄英萍张嗣良
Owner EAST CHINA UNIV OF SCI & TECH
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