Method for screening recombinant pichia pastoris strain for high expression of FAD-GDH

Abstract

The invention provides a method for screening a recombinant pichia pastoris strain for high expression of FAD-dependent glucose dehydrogenase (FAD-GDH). The method comprises the following steps: concentration gradient screening is carried out by using Zeocin, G418 and G418 sequentially, so as to increase a copy number of an FAD-GDH gene in recombinant bacteria. Firstly, codon preference optimized FAD-GDH gene segments obtained at an earlier stage are linked with expression vectors pPICZ alpha A and pMD to construct recombinant expression vectors pPICZ alpha A-GDH and pMD-GDH; then, the pPICZ alpha A-GDH is electrically transferred into pichia pastoris X33 to construct a recombinant, and screening is carried out by using Zeocin and test tube fermentation to obtain a pichia pastoris recombinant strain X33-GDH-2, and the pichia pastoris recombinant strain is made into a competent state; and finally, the pMD-GDH is electrically transferred into X33-GDH-2 competent cells, and screening is carried out by utilizing G418 concentration gradient plate and test tube fermentation. The result shows that the enzyme activity of a transformant X33-GDH-2-1 on a 100 [mu] g / ml G418 flat plate after the pMD-GDH is transferred is 94% higher than that of a transformant X33-GDH-2; the enzyme activity of the strain X33-GDH-2-17 on a G418 gradient plate with a concentration of 2000 [mu] g / ml is highest, and is 97% higher than that of X33-GDH-2-1. The result shows that the expression quantity of the FAD-GDH is favorably improved by sequentially using different selection markers and concentration gradient strategies.

Description

technical field [0001] The invention relates to the technical field of yeast strain screening, and more specifically relates to a method for screening recombinant Pichia pastoris strains highly expressing FAD-GDH. Background technique [0002] FAD-dependent glucose dehydrogenase (FAD-dependent glucose dehydrogenase, referred to as FAD-GDH, EC 1.1.99.10), and glucose oxidase (glucose oxidase, referred to as GOD), pyranose dehydrogenase, choline dehydrogenase and Methanol oxidase belongs to the GMC oxidoreductase (glucose-methanol-choline-oxidoreductase) family. It uses FAD as a prosthetic group to catalyze β-D-glucose to generate D-glucono-δ-lactone in the presence of NAD(P)+, etc., and D-glucono-δ-lactone will spontaneously form glucose acid. [0003] Among the currently used enzymes for blood glucose detection, glucose oxidase (GOD) can use oxygen as an electron acceptor, and the change of oxygen partial pressure in the sample will cause errors in the test results, while ...

AI Technical Summary

Problems solved by technology

Although feasible, only less than 5% of the high-concentration antibiotic-resistant clones obtained by this method have multi-copy recombinant strains, and only about 1-2% of the recombinants contain more than 10 copies of foreign genes; it can be seen that using The efficiency of this method to screen and obtain high-copy recombinant bacteria is still low

This method is simple and practical, but the copy number of the foreign gene in the recombinant strain constructed by it is often limited by the number of selection markers available in Pichia pastoris

Images