Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter

Abstract

The methanol-regulated alcohol oxidase promoter (P AOX1 ) of Pichia pastoris (syn.Komagataella spp. ) is one of the strongest promoters for heterologous gene expression.Although increasing the gene dosage is a common strategy to improve recombinant protein productivities, P. pastoris strains harboring more than two copies of a Rhizopus oryzae lipase gene (ROL) have previously shown a decrease in cell growth, lipase production, and substrate consumption, as well as a significant transcriptional downregulation of methanol metabolism. This pointed to a potential titration effect of key transcriptional factors methanol expression regulator 1 (Mxr1) and methanol-induced transcription factor (Mit1) regulating methanol metabolism caused by the insertion of multiple expression vectors. To prove this hypothesis, a set of strains carrying one and four copies of ROL (1C and 4C, respectively) were engineered to coexpress one or two copies of MXR1*, coding for an Mxr1 variant insensitive to repression by 14-3-3 regulatory proteins, or one copy of MIT1. Small-scale cultures revealed that growth, Rol productivity, and methanol consumption were improved in the 4C-MXR1* and 4C-MIT1, strains growing on methanol as a sole carbon source, whereas only a slight increase in productivity was observed for re-engineered 1C strains. We further verified the improved performance of these strains in glycerol-/methanol-limited chemostat cultures. K E Y W O R D S AOX1 promoter, heterologous gene dosage, methanol metabolism, Mit1, Mxr1, Pichia pastoris (Komagataella spp.), recombinant protein production

Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris (Komagataella phaffii) PAOX1-driven recombinant producer strains with different target gene dosage

“…Consequently, the AOX1 gene was less strongly expressed in MCC than it was in SCC owing to competition with three CRL1 copies for the equivalent Mit1 resources. This hypothesis of Mit1 limitation is reinforced by the work of Cámara et al [42] where the overexpression of Mit1 is enough to reverse the transcriptional limitation derived from increasing heterologous gene dosage. Moreover, deregulating the expression of some MUT-related TFs increased protein production driven by the P AOX1 expression system even in absence of methanol [19, 24, 42, 43].…”

Continuous Cultivation as a Tool Toward the Rational Bioprocess Development With Pichia Pastoris Cell Factory

“…- One involves using specific primers for selected target genes either by real-time quantitative PCR (qPCR) (Landes et al, 2016 ) or droplet-digital PCR (ddPCR) analyses (Cámara et al, 2019 ). Both methods provide numerical results and hence quantitative data.…”

“…This information was used to design new clones carrying several copies of MXR1 and methanol-induced transcription factor ( MIT1 ). As revealed by specific analysis with ddPCR, methanol uptake was found to have a positive effect on the transcriptional response; the effect, however, resulted in no increase in product activity (Cámara et al, 2019 ).…”

Development of a New High-Cell Density Fermentation Strategy for Enhanced Production of a Fungus β-Glucosidase in Pichia pastoris

“…Dehydrogenation of NAD + -dependent FLD and FDH are essential for energy generation and protein biosynthesis in P. pastoris (Cámara et al, 2019;Theron et al, 2019). Intracellular FLD and FDH activities are shown in Figures 5B,C.…”