Efficient method for in-vitro evolution of serine hydroxymethyltransferase

Abstract

The invention discloses an efficient method for in-vitro evolution of serine hydroxymethyltransferase. The method comprises the following steps: performing PCR amplification on serine hydroxymethyltransferase genes of multiple resources, performing in-vitro low-energy ion injection on the serine hydroxymethyltransferase genes of multiple resources so as to establish a carrier, transforming a recipient bacterium, screening high-activity serine hydroxymethyltransferase strains and sequencing. The method has the beneficial effects that a low-energy ion radiation technique is adopted, low-energy ions directly act on the serine hydroxymethyltransferase genes of different resources so as to cause in-vitro radiation damage and modification on the serine hydroxymethyltransferase genes, the serine hydroxymethyltransferase genes are further connected with the carrier, the recipient bacterium is transformed, after in-vivo remediation and mutation, a mutant gene library is expressed and screened so as to obtain a serine hydroxymethyltransferase gene, and the in-vitro evolution is accomplished. The method can also be applied to in-vitro evolution of other enzyme molecules.

Description

Technical field [0001] The invention relates to a method for in vitro evolution of high-efficiency serine hydroxymethyltransferase, which belongs to the technical field of directed evolution of microbial enzyme genes and molecular modification technology. Background technique [0002] In vitro evolution refers to artificially creating special evolutionary conditions, simulating the natural evolutionary mechanism, randomly mutating genes in vitro, and finally obtaining certain properties expected in advance through certain screening or selection methods from one or more artificial mutant enzyme libraries The dominant mutant enzyme. In vitro evolution is a process of polymer evolution completed in a test tube, which provides a powerful means for changing the substrate specificity and catalytic activity of enzymes. It does not need to understand the relationship between the spatial structure and function of the protein. It only needs to construct a mutation library through multiple...

AI Technical Summary

Problems solved by technology

[0004] Although there are many strategies for building a library, there are also obvious shortcomings and deficiencies. Although error-prone PCR is simple and easy to implement, it is a non-preferred amino acid substitution, with a low mutation rate and only point mutations; the method of mutagenic strains is often the whole organism or Plasmids are mutated, and can only be point mutations; the advantage of DNA recombination is that it requires the use of several parental genes, can create chimeras, merge useful mutations and discard harmful mutations, but requires sequence homology, which is often difficult to meet

The method of staggered extension is similar to DNA rearrangement, without fragment purification, which is simpler, but still requires sequence homology, and PCR operation must be specifically optimized

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