Construction and application of strains of over-expression Tm-AHAS-CSU

Abstract

The invention discloses a construction method and application of strains of an over-expression Tm-AHAS-CSU. The construction method of the recombination strains includes the steps that according to the gene sequence of the Tm-AHAS-CSU, upstream and downstream primers are designed, the designed upstream primer carries NdeI restriction enzyme cutting sites, the designed downstream primer carries BamHI restriction enzyme cutting sites, and then with thermotoga maritime genome DNA being a template, a target gene fragment is obtained through a PCR amplification technology; the target gene fragment is connected to an expression carrier pET28a to obtain recombined plasmids pET28a-TmAHAS-CSU, and the recombined plasmids are transformed into competent cells through a thermal shock method to construct the recombination strains of over-expression Tm-AHAS-CSU proteins. On the basis, the function of the recombination strains and the application of the recombination strains in the aspects of selectively synthesizing R-PAC and other analogues in a three-dimensional and catalyzed mode are 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...

AI Technical Summary

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; ②E.coliAHAS 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.coliAHAS in the catalytic synthesis of R-PAC and its analogues

Images