D-3-phosphoglycerate-dehydrogenase as well as coding gene and construction method thereof

A technology of phosphoglycerate dehydrogenase and phosphoglycerate, applied in the field of genetic engineering, can solve problems such as C-terminal deletion, incomplete release of L-serine feedback inhibition, and reduction of L-serine sensitivity

Inactive Publication Date: 2012-05-02
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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Problems solved by technology

[0005] Peters-Wendisch et al. (2002, Appl. Microbiol. Biotechnol. 60: 437-441) describe the C-terminal deletion of PGDH in Corynebacterium glutamicum, which achieves the elimination of L-serine feedback inhibition, but at the same time leads to loss of enzyme activity
[0006] Patent applicatio...

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  • D-3-phosphoglycerate-dehydrogenase as well as coding gene and construction method thereof
  • D-3-phosphoglycerate-dehydrogenase as well as coding gene and construction method thereof
  • D-3-phosphoglycerate-dehydrogenase as well as coding gene and construction method thereof

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[0020] Below in conjunction with the examples, the present invention is further described, the following examples are illustrative, not limiting, and the protection scope of the present invention cannot be limited by the following examples.

[0021] A site-directed mutation method of D-3-phosphoglycerate dehydrogenase is as follows:

[0022] 1. Clone the serA gene

[0023] Using the total DNA of Escherichia coli MG1655 strain as a template, the following primers were designed for cloning the expression gene serA of D-3-phosphoglycerate dehydrogenase:

[0024] Upstream primers serA-F: 5'-AGC GAG CTC ATG GCAAAG GTATCG CTG GAG-3' (the underline is the SacI restriction site).

[0025] Downstream primer serA-R: 5'-ACG C GT CGA C TT AGT ACA GCA GAC GGG CGC-3' (the underline is the SalI restriction site).

[0026] Amplification system (50μL):

[0027] wxya 2 O 34.5 μL,

[0028] 10×buffer 5.0μL,

[0029] dNTP (2.5mmol / L) 5.0μL,

[0030] Upstream primers serA-F 2.0 μL,

[...

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Abstract

The invention relates to D-3-phosphoglycerate-dehydrogenase and a coding gene thereof. The amino acid sequence of the D-3-phosphoglycerate-dehydrogenase is shown as a sequence 1; the inhibition constant Ki of L-serine of the D-3-phosphoglycerate-dehydrogenase is greater than 250mM; and the sequence of the coding gene is shown as a sequence 2. The difference between the amino acid sequence of the D-3-phosphoglycerate-dehydrogenase (PGDH) provided by the invention and the amino acid sequence (sequence 3) of the escherichia coli wild type PGDH lies in that amino acid at 344th site is not histidine but lactamine and amino acid at 346th site is not asparagines but lactamine and the PGDH generated by mutation is not inhibited by the L-serine. In addition, the enzyme activity of a PGDH variant disclosed by the invention has no change compared with the wild type PGDH variant.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a D-3-phosphoglycerate dehydrogenase, its coding gene and its construction method. Background technique [0002] PGDH (D-3-phosphoglycerate-dehydrogenase, PGDH), that is, D-3-phosphoglycerate dehydrogenase [EC1.1.1.95], the enzyme is the key enzyme of L-serine synthesis, which converts 3-phosphoglycerate It is phosphohydroxypyruvate, which is the first step in the synthesis of L-serine. [0003] At present, the biological methods to produce serine mainly include precursor method and direct fermentation method. The precursor method mainly uses serine hydroxymethyltransferase (SHMT) to convert glycine into L-serine; the preparation of L-serine by this method has the advantages of high yield and short production cycle, but how to separate and extract L-serine from the enzyme reaction solution Serine is a problem; the direct fermentation method uses sugary carbon sources and engi...

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/70
Inventor 路福平李玉陈谷奎佟新伟刘逸寒
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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