Enhancing the production of Recombinant secretory proteins in Pichia pastoris by manipulation of Transcription terminators

Abstract

The Pichia pastoris (Komagataella phaffii), is a highly successful newlineexpression system for the production of various recombinant heterologous newlineproteins. The focus on high production levels of industrial enzymes and newlineheterologous metabolic pathway genes requires a more sophisticated and newlinetighter control of gene expression at both transcriptional and translational newlinelevels. The components of the entire expression cassette exerts control on net newlineprotein output. Gene expression control is primarily achieved through altering newlinethe promoter driving expression and by changing the copy number of the gene. newlineHowever, not all parts are created equal and not all have been tested adequately newlineenough to ensure function in the expression system. Unlike promoters, very newlinefew studies have examined the influence of terminators on net protein newlineexpression. Transcription terminators (TT) are integral part of the expression newlinecassette that influence 3and#8242;-end processing of mRNA, mRNA stability, and newlinetranslational efficiency, which can modulate protein expression. In Pichia newlinepastoris (P. pastoris), little attention has been paid to the selection of terminator newlineregions in efforts to increase heterologous gene expression. newlineTo examine the potential application of the terminators on enhanced newlineproduction of Candida antarctica lipase B (CalB), we examined the ability of newlinethree different classes of terminator regions 1. Terminator regions of methanol newlineutilization pathway genes of P. pastoris; 2. Terminator regions of wellexpressed newlineand housekeeping genes of P. pastoris; and 3. terminators of other newlineyeast genes like Saccharomyces cerevisiae. newline