Synergistic modular promoter and gene optimization to push cellulase secretion by Pichia pastoris beyond existing benchmarks

Although successfully used for heterologous gene expression for more than twenty years, general knowledge about all factors influencing protein expression by Pichia pastoris is still lacking. For high titers of protein clones are optimized individually for each target protein. Optimization efforts in this study were focused on the DNA level, evaluating a set of 48 different individual synthetic genes (TrCBH2) coding for the same protein sequence of a Trichoderma reesei cellulase in combination with three different promoter sequences: PGAP (constitutive) and the synthetic AOX1 promoter variants PDeS (derepressed) and PEn (enhanced, inducible). Expression of active secreted enzyme varied from undetectable to ∼300% of the best known gene, as determined by secreted enzyme activity analyses of supernatants from 96 well plate and bioreactor cultivations. Finally, the best optimized gene and new promoters were combined to engineer highly productive P. pastoris CBH2 expression strains. Although no methanol was used for induction a final titer of more than 18 g/l of secreted protein was produced under controlled conditions in small scale bioreactor cultivations after 60–70 h of growth limiting glycerol feed. This is the highest concentration of secreted enzyme in P. pastoris published so far and single parts of the expression cassette could be independently optimized showing additive effects for improvements in protein production by P. pastoris.