Characterization of novel promoters for heterologous protein production in pichia pastoris

Abstract

Heterologous expression of the gene of interest (GOI) follows newlinetranscription, translation, folding and possible posttranslational modifications newlineby the select host. The initial transcription of the GOI is often a critical step in newlineheterologous protein production in both prokaryotes and eukaryotes. newlineTherefore, substantial, moderate and controllable promoters are a crucial tool newlinefor efficient heterologous protein production. Intracellular metabolic flux is newlineregulated by a series of distinct and interwoven regulatory control levels newlineoccurring at the transcriptional, translational, and protein levels. Control newlinetranscript production at the promoter level is one of the fundamental access newlinepoints to alter this metabolic flux. Therefore, metabolic engineering newlineapplications have long relied on the characterization and design of useful and newlinediverse promoters. newlinePichia pastoris (syn. Komagataella phaffii) is a popular host system newlinefor heterologous protein production. However, as compared to other yeast newlinespecies like Saccharomyces cerevisiae, the number of useable promoter newlinesequences is relatively fewer and also limited to promoters like PAOX1 and newlinePGAP having a strong activity. For metabolic engineering applications, such as newlinepathway engineering, the co-expression of metabolic pathway genes, newlinesecretion helpers, or the individual expression of multiple proteins, e.g. the newlineheavy (HC) and light chain (LC) of antibodies, it might be of interest to use newlinepromoters with varying strength. Therefore, it is an advantage to have a newlineselection of different promoter sequences suitable for recombinant expression newlineof a heterologous or homologous gene, varying from strong promoter activity newlineto weak or reduced promoter activity newline