Novel method for co-transformation of pichia pastoris through multiple genes

Abstract

The invention discloses a novel method for co-transformation of pichia pastoris through multiple genes. By adopting the novel method, the fact that multiple exogenous genes required to be expressed are connected to the same pichia pastoris expression vector is realized, and in the expression vector, each gene is positioned in an independent expression frame; the fact that multiple genes are efficiently, simultaneously and stably integrated into a pichia pastoris genome is achieved by adopting a built coexpression vector to transform the pichia pastoris. The invention provides the efficient novel method provided for achieving that multiple genes are simultaneously and independently expressed in the same pichia pastoris cell.

Description

technical field [0001] The present invention relates to a new method for multi-gene co-transformation of Pichia pastoris, in particular to a method for co-transforming yeast with two or more genes and obtaining multi-gene integration into the same Pichia genome. The source genes have the ability to express independently under the control of the same and independent gene expression regulatory elements. Background technique [0002] The methanolic yeast expression system is a very widely used yeast expression system. The exogenous gene expression system using Pichia pastoris (P. pastoris, Pichia pastoris) as the host has developed the fastest in recent years and is also the most widely used. The reason is that in addition to the characteristics of general yeast, the system also has The following advantages: ① It has a unique strong alcohol oxidase gene AOX1 promoter, and the expression of foreign proteins can be strictly regulated by methanol; ② As a eukaryotic expression sys...

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Problems solved by technology

However, there are obvious deficiencies in the above three methods: firstly, the different genes are connected by PCR, fused and then constructed into an expression vector.

However, the expression level of the product obtained by this method is often very low

The main reason is that two or more genes are connected in series in the same reading frame, coupled with the IRES sequence, the gene fragments are larger than the gene fragments expressed by fusion, and when only the common expression regulatory elements are regulated, especially It is under the control of the only promoter, which will affect the transcription and translation of the gene, so it is difficult to obtain high-level secretory expression of Pichia transformants; finally, construct the expression vectors of foreign genes respectively, and transform one of them to complete Red yeast, obtain yeast transformants, and then retransform another gene into the yeast that has been transformed into one gene, so as to realize the simultaneous integration of two genes into the yeast genome, it is often difficult to screen for yeast transformation with simultaneous insertion of two genes Because when the second gene is transformed, the first gene is likely to be removed from the chromosome by recombination; if the probability of yeast transformants co-expressing 3 or more genes is lower, it is difficult to screen all Yeast transformants with integrated genes

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