Gene of coded 6-phosphogluconate dehydrogenase and application thereof

A technology of glucose phosphate and acid dehydrogenase, which is applied in the field of genetic engineering and can solve the problems of low yield and the like

Active Publication Date: 2016-05-25
GUANGXI UNIV
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Problems solved by technology

[0007] Aiming at the problems of feedback inhibition regulation and relatively low yield of L-serine produced by the fermentation of methylobacterium sp.MB200, the present invention uses plasmid transposon insertion technology to construct a mutant library of methylobacterium sp.MB200, and screens out A mutant with D-serine resistance, cloned the 6-phosphogluconate dehydrogenase (6-PGDH) gene with L-serine inhibitory effect, and constructed a pgdh gene-deficient L-serine high-yielding strain, relieved Inhibitory effect of 6-PGDH on the production of L-serine during the metabolic process of methylotrophic bacteria, thereby increasing the production of L-serine

Method used

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  • Gene of coded 6-phosphogluconate dehydrogenase and application thereof
  • Gene of coded 6-phosphogluconate dehydrogenase and application thereof
  • Gene of coded 6-phosphogluconate dehydrogenase and application thereof

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Embodiment 1

[0033] 1.1 Cloning and analysis of the D-serine tolerance mutant gene of methylotrophic bacteria Methylobacteriumsp.MB200

[0034] 1.1.1 The mutant library of methylobacterium sp.MB200 was constructed by plasmid transposon insertion technology, and the mutant D-2 with D-serine resistance was screened out.

[0035] 1.1.2 Extract the total DNA from the mutant D-2, connect it to the cloning vector pMD-18T after enzyme digestion, and introduce it into the competent E. Plasmids were further sequenced after digestion and verification, and Blast sequence alignment and analysis were performed in NCBI. As a result, the obtained DNA fragment was 1875bp, and the sequence had the highest similarity with the 6-phosphogluconate dehydrogenase gene of MethylobacteriumpopuliBJ001, with a consistency of 89%. See Table 1 for details.

[0036] Mutant DNA fragments cloned in table 1

[0037]

[0038] 1.2 Bioinformatics analysis of inserted DNA fragments in mutant strain D-2

[0039] The DNA ...

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Abstract

The invention provides a gene of coded 6-phosphogluconate dehydrogenase and application thereof. The nucleotide sequence is shown in SEQ ID NO:1. The 6-phosphogluconate dehydrogenase gene pgdhD2 is derived from Methylobacterium sp. MB200 and has the L-serine inhibiting effect. The Methylobacterium sp. MB200 is used as an original strain, pgdh gene deletion mutation DMB is built through a homologous double-crossover method, an L-serine high-yield mutant strain with the L-serine producing capacity greatly higher than that of original strain Methylobacterium sp. MB200 is obtained, and the tolerated D-serine concentration of the L-serine high-yield mutant strain is greatly higher than that of the original strain. The pgdh gene deletion mutation stain has the good application prospect in production of L-serine through a fermentation method.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a gene encoding 6-phosphogluconate dehydrogenase and its application. Background technique [0002] L-serine has important physiological functions and application value, and is widely used in the fields of medicine, food and chemical industry. At present, the production methods of L-serine mainly include chemical synthesis, proteolysis, enzymatic transformation and microbial fermentation. The production of L-serine by microbial fermentation is a green and environmentally friendly production method, but due to the feedback inhibition regulation, it is difficult for microorganisms to accumulate L-serine in large quantities, so the yield of L-serine produced by microbial fermentation is relatively low. application of this method. [0003] The method for removing the feedback inhibition regulation of L-serine and improving the yield of L-serine produced by fermentation ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/04C12N15/70
CPCC12N9/0006
Inventor 申佩弘武波李献蒋承建
Owner GUANGXI UNIV
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