Induction without methanol: novel regulated promoters enable high-level expression in Pichia pastoris

Abstract

BackgroundInducible high-level expression is favoured for recombinant protein production in Pichia pastoris. Therefore, novel regulated promoters are desired, ideally repressing heterologous gene expression during initial growth and enabling it in the production phase. In a typical large scale fed-batch culture repression is desired during the batch phase where cells grow on a surplus of e.g. glycerol, while heterologous gene expression should be active in the feed phase under carbon (e.g. glucose) limitation.ResultsDNA microarray analysis of P. pastoris wild type cells growing in glycerol-based batch and glucose-based fed batch was used for the identification of genes with both, strong repression on glycerol and high-level expression in the feed phase. Six novel glucose-limit inducible promoters were successfully applied to express the intracellular reporter eGFP. The highest expression levels together with strong repression in pre-culture were achieved with the novel promoters PG1 and PG6.Human serum albumin (HSA) was used to characterize the promoters with an industrially relevant secreted protein. A PG1 clone with two gene copies reached about 230% of the biomass specific HSA titer in glucose-based fed batch fermentation compared to a PGAP clone with identical gene copy number, while PG6 only achieved 39%. Two clones each carrying eleven gene copies, expressing HSA under control of PG1 and PG6 respectively were generated by post-transformational vector amplification. They produced about 1.0 and 0.7 g L-1 HSA respectively in equal fed batch processes. The suitability in production processes was also verified with HyHEL antibody Fab fragment for PG1 and with porcine carboxypeptidase B for PG6. Moreover, the molecular function of the gene under the control of PG1 was determined to encode a high-affinity glucose transporter and named GTH1.ConclusionsA set of novel regulated promoters, enabling induction without methanol, was successfully identified by using DNA microarrays and shown to be suitable for high level expression of recombinant proteins in glucose-based protein production processes.

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

“…Both systems have their drawbacks, particularly methanol as a substrate and P GAP as a non-inducible promoter (Kovar et al 2010; Mellitzer et al 2014). Systems are sought in which production can be regulated without using methanol induction (Hartner et al 2008; Prielhofer et al 2013; Stadlmayr et al 2010; Vogl and Glieder 2013; Vogl et al 2016). Such carbon source-dependent promoters are either synthetic variants of the natural P AOX1 promoter, generated by selected (short) sequence deletions/insertions (Hartner et al 2008), or newly identified natural carbon source-regulated promoters (Prielhofer et al 2013; Stadlmayr et al 2010; Vogl et al 2016).…”

“…Systems are sought in which production can be regulated without using methanol induction (Hartner et al 2008; Prielhofer et al 2013; Stadlmayr et al 2010; Vogl and Glieder 2013; Vogl et al 2016). Such carbon source-dependent promoters are either synthetic variants of the natural P AOX1 promoter, generated by selected (short) sequence deletions/insertions (Hartner et al 2008), or newly identified natural carbon source-regulated promoters (Prielhofer et al 2013; Stadlmayr et al 2010; Vogl et al 2016). For example, recombinant protein production can be repressed during batch phase with excess glycerol, and recombinant gene expression can be initiated during substrate-limited fedbatch cultivation with glucose (Prielhofer et al 2013).…”

Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing

“…However, they are still commonly referred to as P. pastoris . In recent years, the genetic toolbox for P. pastoris has been markedly expanded with several newly discovered native promoters, synthetic promoters and other regulatory elements891011. The construction of optimized strains and vectors enabled new applications, e.g.…”

Molecular strategies to increase the levels of heterologous transcripts inKomagataella phaffiifor protein production

“…A novel regulated promoter has recently been identified from a high-affinity glucose transporter gene (GTH1) which is repressed by glycerol. 9 Since P GTH1 is responsive to carbon source depletion it should represent an attractive and cheap alternative to other regulated systems that rely on the addition of an external source of the inducing agent. Recently, 2 inducible promoters from the K. phaffii rhamnose utilization pathway were proposed for the production of food-grade and therapeutically important recombinant proteins.…”

“…18 Promoter libraries have already been constructed using not only random mutagenesis methods but also with specific synthetic core promoter sequences derived from wild-type promoter alignments. 9,19 The P GAP sequence has also been manipulated through error-prone PCR for the construction of a GAP-promoter library with strengths varying from »0,6% to 19,6-fold of that of the wild-type P GAP . 20 …”