Cloning of PpKAR2 and PpPDI1 genes promoters from yeast Pichia pastoris, an evaluation of their activity and efficacy for the heterologous genes expression

Abstract

Background. Yeast Pichia pastoris is successfully used in biotechnology, with their help synthesized various compounds. Promoters are a key factor in the productivity of an expression system, since they determine the expression level of a heterologous gene.
 The aim of our work was to study the promoter regions of the PpKAR2 and PpPDI1 genes and to evaluate their use for effective expression of heterologous genes.
 Materials and Methods. To evaluate the activity of promoters, we used a reporter system based on the structural gene of acid phosphatase of yeast Saccharomyces cerevisiae – PHO5. To determine the effect of overproduction of native and heterologous protein on the activity of the promoters under study, we used the producer strains of P. pastoris protein disulfide isomerase and maize delta-zein. To evaluate the effectiveness of the use of the promoters under study for the expression of heterologous genes, we have expressed under their control a gene encoding human interferon-alpha16.
 Results. The promoters of the yeast genes – PpKAR2 and PpPDI1 were cloned. Their activity was compared with the promoter of the PpAOX1 gene in the native strains, as well as in strains with overproduction of native and heterologous proteins. Under the control of these promoters, the gene encoding human interferon-alpha 16 is expressed.
 Conclusion. The promoters studied were weaker than the promoter of the AOX1 gene, but increase their activity in response to the production of heterologous proteins and can be used to express hete­rologous genes.