Cloning of alpha-amylase gene from Schwanniomyces occidentalis and expression in Saccharomyces cerevisiae.

The cloning of alpha-amylase gene of S. occidentalis and the construction of starch digestible strain of yeast, S. cerevisiae AS. 2. 1364 with ethanol-tolerance and without auxotrophic markers used in fermentation industry were studied. The yeast/E. coli shuttle plasmid YCEp1 partial library of S. occidentalis DNA was constructed and alpha-amylase gene was screened in S.cerevisiae by amylolytic activity. Several transformants with amylolysis were obtained and one of the fusion plasmids had an about 5.0 kb inserted DNA fragment, containing the upstream and downstream sequences of alpha-amylase gene fromS. occidentalis. It was further confirmed by PCR and sequence determination that this 5.0 kb DNA fragment contains the whole coding sequence of alpha-amylase. The amylolytic test showed that when this transformant was incubated on plate of YPDS medium containing 1% glucose and 1% starch at 30 degrees Celsius for 48 h starch degradation zones could be visualized by staining with iodine vapour. alpha-amylase activity of the culture filtratate is 740-780 mU/mL and PAGE shows that the yeast harboring fusion plasmids efficiently secreted alpha-amylase into the medium, and the amount of the recombinant alpha-amylase is more than 12% of the total proteins in the culture filtrate. These results showed that alpha-amylase gene can be highly expressed and efficiently secreted inS. cerevisiae AS. 2.1364, and the promotor and the terminator of alpha-amylase gene fromS. occidentalis work well inS. cerevisiae AS. 2.1364.