Variable production windows for porcine trypsinogen employing synthetic inducible promoter variants in Pichia pastoris

Abstract

Natural tools for recombinant protein production show technological limitations. Available natural promoters for gene expression in Pichia pastoris are either constitutive, weak or require the use of undesirable substances or procedures for induction. Here we show the application of deletion variants based on the well known methanol inducible AOX1 promoter and small synthetic promoters, where cis-acting elements were fused to core promoter fragments. They enable differently regulated target protein expression and at the same time to replace methanol induction by a glucose or glycerol feeding strategy. Trypsinogen, the precursor of the serine protease trypsin, was expressed using these different promoters. Depending on the applied promoter the production window (i.e. the time of increasing product concentration) changed significantly. In fedbatch processes trypsinogen yields before induction with methanol were up to 10 times higher if variants of the AOX1 promoter were applied. In addition, the starting point of autoproteolytic product degradation can be predetermined by the promoter choice.

Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris: functional characterisation of a novel promoter

“…Several promoters for methanol-free production of recombinant protein by P. pastoris have recently been developed, and the general principle of regulation by repression/derepression with glucose or glycerol as the sole substrate has been investigated (Capone et al 2015; Hartner et al 2008; Mellitzer et al 2014; Prielhofer et al 2013; Ruth et al 2010; Vogl et al 2016). However, little is known beyond the dynamic conditions for switching on and off in order to control these modified promoters using appropriate process strategies.…”

“…Regulation of synthetic P AOX1 variants is based on principles of repression/derepression, using glucose or glycerol as the only substrate (Capone et al 2015; Mellitzer et al 2014; Ruth et al 2010). At high concentrations of glycerol or glucose, all P AOX1 variants were repressed but were derepressed by lower substrate availability (also described as being substrate limited) during fedbatch culture (Capone et al 2015; Mellitzer et al 2014; Ruth et al 2010).…”

“…At high concentrations of glycerol or glucose, all P AOX1 variants were repressed but were derepressed by lower substrate availability (also described as being substrate limited) during fedbatch culture (Capone et al 2015; Mellitzer et al 2014; Ruth et al 2010). For these new P AOX1 variants, little is known about their functionalities and possible control by process strategies, i.e.…”

Yeast synthetic biology for the production of recombinant therapeutic proteins

“…Based on an in silico analysis for putative conserved eukaryotic transcription factor binding sites within the P AOX1 , the transcriptional activity of this promoter was rationally optimized by mutagenesis (Hartner et al, 2008;Xuan et al, 2009). In addition, it was shown that short semisynthetic variants of P AOX1 constructed by fusing natural core promoter fragments with cis-acting elements had greater transcriptional activity than the full-length wildtype promoter (Ruth et al, 2010).…”

Perspectives on Synthetic Promoters for Biocatalysis and Biotransformation

“…[5] In contrast, placing the same promoter in control of porcine trypsinogen, an enzyme used for in vitro processing of biopharmaceuticals, caused lower yields, but improved product quality due to a delay, or even prevention, of autoproteolytic product degradation. [27] In addition, employing a novel short synthetic promoter generated by fusing an identified cis-acting sequence to a core promoter enabled the production of significant amounts of trypsinogen, even without induction with methanol. Another library variant with increased promoter strength successfully increased the expression of secreted industrial enzymes [28] and improved P. pastoris whole cell conversions that required dehydrogenases.…”