One-step enzymatic hydrolysis of starch using a recombinant strain of Saccharomyces cerevisiae producing α-amylase, glucoamylase and pullulanase

A recombinant strain of Saccharomyces cerevisiae was constructed that contained the genes encoding a bacterial α-amylase (AMY1), a yeast glucoamylase (STA2) and a bacterial pullulanase (pulA). The Bacillus amyloliquefaciens α-amylase and S. cerevisiae var. diastaticus glucoamylase genes were expressed in S. cerevisiae using their native promoters and the encoded enzymes secreted under direction of their native leader sequences. In contrast, the Klebsiella pneumoniae pullulanase gene was placed under the control of the yeast alcohol dehydrogenase gene promoter (ADC1_P) and secreted using the yeast mating pheromone α-factor secretion signal (MFα1_S). Transcription termination of the pullulanase gene was affected by the yeast tryptophan synthase gene terminator (TRP5T), whereas termination of the glucoamylase and α-amylase genes was directed by their native terminators. Pullulanase (PUL1) produced by recombinant yeasts containing ADC1_PMFα1_SpulA TRP5_T (designated PUL1) was further characterized and compared to its bacterial counterpart (PulA). The different genes were introduced into S. cerevisiae in different combinations and the various amylolytic Saccharomyces transformants compared to Schwanniomyces occidentalis. Introduction of PUL1 into a S. cerevisiae strain containing both STA2 and AMY1, resulted in 99% assimilation of starch