Co-expression of an Erwinia chrysanthemi pectate lyase-encoding gene (pelE) and an E. carotovora polygalacturonase-encoding gene (peh1) in Saccharomyces cerevisiae
A pectate lyase (PL)-encoding gene (pelE from Erwinia chrysanthemi and a polygalacturonase (PG)-encoding gene (peh1) from E. carotovora were each inserted between a novel yeast expression-secretion cassette and a yeast gene terminator, and cloned separately into a yeast-centromeric shuttle vector (YCp50), generating recombinant plasmids pAMS12 and pAMS13. Transcription initiation signals present in the expression-secretion cassette were derived from the yeast alcohol dehydrogenase gene promoter (ADC1(P)), whereas the transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5(T)). Secretion of PL and PG was directed by the signal sequence of the yeast mating pheromone α-factor (MFα1(S)). A pectinase cassette comprising ADC1(P)-MFα1(S)-pelE-TRP5(T) and ADC1(P)-MFα1(S)-peh1-TRP5(T) was subcloned into YCp50, generating plasmid pAMS14. Subsequently, the dominant selectable Geneticin G418-resistance (Gt(R)) marker, APH1, inserted between the yeast uridine diphosphoglucose 4-epimerase gene promoter (GAL10(P)) and yeast orotidine-5'-phosphate carboxylase gene terminator (URA3(T)), was cloned into pAMS14, resulting in plasmid pAMS15. Plasmids pAMS12, pAMS13 and pAMS14 were transformed into a laboratory strain of Saccharomyces cerevisiae, whereas pAMS15 was stably introduced into two commercial wine yeast strains. DNA-DNA and DNA-RNA hybridization analyses revealed the presence of these plasmids, and the pelE and peh1 transcripts in the yeast transformants, respectively. A polypectate agarose assay indicated the extracellular production of biologically active PL and PG by the S. cerevisiae transformants and confirmed that co-expression of the pelE and peh1 genes synergistically enhanced pectate degradation.