ABSTRACT
This is the first report of an insect esterase efficiently expressed in the methylotrophic yeast Pichia pastoris (so far insect esterases have been produced only in the baculovirus system). Having isolated a Tribolium castaneum carboxylesterase cDNA (TCE), we were initially unable to express it in Escherichia coli or P. pastoris despite significant transcription levels. As codon usage bias is different in T. castaneum and P. pastoris, we assumed this was a possible explanation for the translational barrier observed in yeast. Accordingly, we designed and constructed by recursive PCR a synthetic TCE gene (synTCE) optimized for heterologous expression in P. pastoris, i.e., a gene in which certain TCE codons are replaced with synonymous codons 'preferred' in P. pastoris. When the altered gene was placed under the control of either the P. pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter or the inducible alcohol oxidase (AOX1) promoter and introduced on an expression vector into P. pastoris, its product was produced intracellularly. We also successfully explored the possibility of obtaining a secreted product: P. pastoris cells expressing an in-frame fusion of synTCE with the alpha-factor secretion signal under the control of the GAP promoter were found to secrete the recombinant esterase into the external medium (to a concentration of 7 mg/L). In addition to this demonstration of TCE production in yeast, our results suggest that the GAP promoter could advantageously replace the AOX1 promoter as a driver of synTCE expression. TCE specific activity was approximately 5 U/mg when p-nitrophenyl acetate was used as substrate.