Construction and application of a bacterial strain overexpressing the catalytic subunit of thermotoga maritima acetolactate synthase

Abstract

The invention discloses a method for constructing a strain that overexpresses the catalytic subunit of Thermotoga maritima acetolactate synthase and its use. The construction method of the recombinant strain includes the following steps: design upstream and downstream primers based on the gene sequence of TmAHAS-CSU. The designed upstream primer has an NdeI restriction site, and the downstream primer has a BamHI restriction site. Then, Thermotoga maritima is used. Genomic DNA is used as a template, and the target gene fragment is obtained through PCR amplification technology; the target fragment is connected to the expression vector pET28a to obtain the recombinant plasmid pET28a-TmAHAS-CSU, and the recombinant plasmid is transformed into competent cells by heat shock method. Construct a recombinant strain overexpressing Tm‑AHAS‑CSU protein. On this basis, the function of the recombinant strain and its use in the stereoselective catalytic synthesis of R-PAC and its analogs were verified.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for constructing bacterial strains for catalytically synthesizing (R)-phenylacetylcarbinol (R-PAC) and analogues thereof. Background technique [0002] (R)-Phenylacetylmethanol (R-PAC) is a precursor substance for the synthesis of α / β-adrenaline in the pharmaceutical industry, and is also used to synthesize L-ephedrine, pseudoephedrine and benzene Propanolamine (Norephedrin) and other drugs, and it is also a chiral intermediate commonly used in the organic synthesis process, which can be used for the synthesis of chiral drugs. R-PAC is currently prepared by a biotransformation method, mainly using yeast cells to catalyze the condensation reaction between pyruvate and benzaldehyde, but due to the low reaction rate and the low conversion rate of the substrate into the target compound, this method is not an effective method. Very efficient R-PAC synthesis rou...

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Problems solved by technology

However, this reaction also has disadvantages: ① As mentioned above, AHAS is usually composed of two large subunits and two small subunits. When preparing the enzyme, these two subunits must be prepared at the same time and then remodeled in vitro to obtain it. This makes the enzyme preparation process complicated; ② E.coli AHAS is usually less stable, although its large subunit also shows weaker catalytic activity when it exists alone, but the stability is worse

These two shortcomings largely limit the application of E.coli AHAS in the catalytic synthesis of R-PAC and its analogues

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