Engineering protein translocation pathway to improve recombinant proteins in Pichia pastoris

Pichia pastoris is one of the most commonly used hosts for producing heterologous proteins, whereas production levels vary depending on the protein of interest and are also regulated by regulatory factors. We conducted RNAseq by expressing the reporter EGFP and observed significant upregulation of certain subunits (Sec61p, Sbh1p, Sss1p, Sec66p and Sec72p) of the Sec complex in the high-expression recombinant GS115 stains. The overexpression of these genes may increase the expression levels of heterogeneous proteins. In this study, the endogenous promoters of the Sec complex subunits Sbh1p, Sss1p, Sec66p and Sec72p were isolated and verified their activity using the Lac -Z reporter gene. Sss1, Sbh1, Sec66 and Sec72 were overexpressed under the control of their own promoters in Pichia pastoris, respectively. The overexpression of Sss1, Sbh1, Sec66 and Sec72 in cells was confirmed by fluorescent microscope and Western blot analysis. The alpha-amylase was employed to evaluate the effect of overexpression of the Sec subunits on the heterologous protein expression. The results demonstrated that the alpha-amylase activity increased by 16%, 58%, 16% and 17% in the strains overexpressing Sss1, Sbh1, Sec66 and Sec72, respectively. Engineering the protein translocation pathway can be an alternative to enhance heterogeneous proteins in Pichia pastoris expression system.