A single-cell factory for efficiently synthesizing α-aminobutyric acid and its construction and application

Abstract

The invention discloses a single-cell factory for efficiently synthesizing α-aminobutyric acid and its construction and application, belonging to the technical field of microorganisms. In the present invention, L-threonine deaminase, L-amino acid dehydrogenase, and the dehydrogenase gene that provides the cofactor NADH cycle are expressed in series to construct a recombinant E. coli single-cell factory for efficient synthesis of α-aminobutyric acid, through Optimized rbs sequence controls the expression of L-threonine deaminase, solves the problem of rapid accumulation of the intermediate product ketobutyric acid and inhibits transformation, and controls the expression of dehydrogenase that provides the cofactor NADH cycle through promoter and rbs sequence optimization amount, enhancing the rate of NADH regeneration ultimately increases production. The use of single-cell factories for whole-cell transformation can reduce material entry and exit barriers, speed up the transformation rate, and promote the intracellular circulation of cofactors without exogenous additions, and the cost is low. Within 20 hours, the single-cell factory yield of recombinant E. coli in a 5L fermenter was 204g / L, and the space-time yield was 10.2g / L·h, providing a practical and effective strategy for its industrial production.

Description

technical field [0001] The invention relates to a single-cell factory for efficiently synthesizing α-aminobutyric acid and its construction and application, belonging to the technical field of microorganisms. Background technique [0002] Unnatural α-amino acids are a large class of amino acids that are different from the 22 natural α-amino acids that can be synthesized by organisms themselves. They have important biological activities and physiological effects, and are widely used in compounds such as polypeptides, chiral drugs, and alkaloids. Synthesis. α-aminobutyric acid is an unnatural amino acid that inhibits the transmission of human nerve information. It can enhance the activity of glucose phosphatase and promote the metabolism of brain cells. α-aminobutyric acid is also an important chemical raw material and pharmaceutical intermediate, which has been widely used in the synthesis of drugs, such as the synthesis of anti-tuberculosis drug ethambutol hydrochloride and...

AI Technical Summary

Problems solved by technology

During the enzyme conversion process, it was found that the amount of L-threonine deaminase needs to be precisely controlled, otherwise it will cause the accumulation of the intermediate product ketobutyric acid, thereby inhibiting the conversion of ketobutyric acid to α-aminobutyric acid, causing the enzyme conversion Interruption of production of alpha-aminobutyric acid

At the same time, using the enzyme transformation system to produce α-aminobutyric acid requires cell disruption of the three enzyme-producing recombinant bacteria, which is cumbersome and costly. At the same time, the stability of the transformation is affected by the inactivation of the enzyme during the transformation process. In addition, due to the loss of cofactors, it is necessary to continuously add exogenous sources, which further increases the production cost of α-aminobutyric acid

Images