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Bacterial strain of producing pyruvic acid and construction method of bacterial strain

A construction method and technology of pyruvate bacteria, which can be applied to bacteria, introduction of foreign genetic material using vectors, recombinant DNA technology, etc., can solve problems such as unoptimized conditions

Active Publication Date: 2014-06-25
HEFEI BAIMAI BIOTECH CO LTD
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Problems solved by technology

At present, a variety of key enzyme genes involved have been cloned and studied, but the conditions for using these enzymes for biotransformation have not been optimized

Method used

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  • Bacterial strain of producing pyruvic acid and construction method of bacterial strain
  • Bacterial strain of producing pyruvic acid and construction method of bacterial strain
  • Bacterial strain of producing pyruvic acid and construction method of bacterial strain

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Embodiment Construction

[0014] The following examples are further illustrations of the present invention, and do not constitute a limitation to the essence of the present invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the materials and reagents used can be purchased from commercial sources.

[0015] Synthesis and Cloning of Manipulated 1D-Amino Acid Oxidase Gene

[0016] The synthesis and cloning of D-amino acid oxidase (DAAO) are divided into the following operations:

[0017] Step 1: Whole gene synthesis of D-amino acid oxidase (DAAO):

[0018] D-amino acid oxidase genes from Cyprinus carpio, Rhodotorula gracilis and Trigonopsis variabilis were synthesized by total gene synthesis. Gene synthesis was completed by Jinweizhi Biotechnology (Beijing) Co., Ltd. The synthetic gene was cloned in the pUC57 plasmid by EcoRV. The three genes were named CcDAAO, RgDAAO and TvDAAO respectively.

[0019] The...

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Abstract

The invention discloses a bacterial strain of producing pyruvic acid and a construction method of the bacterial strain. The construction method comprises the following steps: cloning a D-amino acid oxidase gene DAAO (D Amino Acid Oxidase) into the pUC57 plasmids through EcoRI or BamHI to obtain a plasmid pUC57-DAAO; carrying out gene amplification by taking plasmids pUC57-DAAO as a template; cloning a product obtained by gene DAAO amplification to a plasmid pET28A or pET21a; selecting and culturing positive clone plasmids connected to a DNA segment containing DAAO from the plasmids; converting the constructed positive clone plasmids to an expression host bacterium BL21(DE3); and selecting and culturing to obtain a first bacterial strain which can convert D-alanine to pyruvic acid, wherein the fragment size of the DAAO gene is 1124bp, and the gene contains cleavage sites of endonuclease NdeI and EcoRI. The bacterial strain constructed by the construction method can directly convert D-alanine into pyruvic acid through a biotransfer approach.

Description

technical field [0001] The invention relates to the field of pyruvate production, specifically, the invention provides a strain for producing pyruvate and a construction method of the strain. Background technique [0002] Pyruvate is an α-keto acid that plays an important role in biochemical metabolic pathways. As an important organic acid, pyruvic acid is not only an important intermediate in the human body's tricarboxylic acid cycle and amino acid synthesis, but also has a wide range of uses in food, chemical and pharmaceutical industries and scientific research. In the manufacturing industry, it is often used as a food additive, weight control additive, health food and antioxidant. At the same time, because pyruvic acid has both active ketone groups and active carboxyl groups, it is often used as an important raw material in the production of chemical, pharmaceutical and agricultural chemicals, such as it can be used for the synthesis of L-tyrosine , N-acetyl-D-mannosam...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21
Inventor 张学礼张冬竹
Owner HEFEI BAIMAI BIOTECH CO LTD
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