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.