unpublished work).Brewing strains containing four DEXI, DEX2 and STA3 genes have been constructed and found to ferment worts to a much lower gravity than normal brewing strains (Russell & Stewart, 1986), but do not have any debranching activity. Faster fermentations have been obtained using diastatic strains which were selected for catabolite derepression using 2-deoxyglucose resistance. Progress is still being made in the genetic analysis of brewing and distilling yeasts. Recently improved sporulation rates have been obtained from brewing strains using improved sporulation media (Bilinski et al., 1986) and the genetics of aneuploid and tetraploid distilling strains have been analysed by the hybridization of restriction fragments with selected gene probes (Keiding, 1985).In deciding a strategy for cloning amylolytic genes into yeasts to be used for fermenting distilled beverages it is necessary to consider: (i) the enzyme activity required; (ii) the most appropriate donor organism, bearing in mind consumer acceptability; (iii) selection of an appropriate gene banking technique; (iv) selection of a procedure for introducing the amylase gene into the distilling strain which does not introduce other undesirable characteristics; and (v) selection of an appropriate expression system providing the level of expression required. These requirements are probably more difficult to satisfy when the product is a potable spirit than when it is industrial or fuel alcohol.
The methylotrophic yeast Hansenula polymorpha, a host organism for the production of heterologous proteins, has been applied to produce the alpha-galactosidase from the plant Cyamopsis tetragonoloba (guar). The yeast/Escherichia coli shuttle expression vector used is based on the origin of replication of the endogenous 2 microns plasmid of Saccharomyces cerevisiae and the LEU2 gene of S. cerevisiae for selection in H. polymorpha. In the expression vector, the alpha-galactosidase is controlled by the methanol-regulated promoter from the methanol oxidase gene, MOX, of H. polymorpha. The signal sequence of SUC2 (invertase) from the yeast S. cerevisiae, was used to ensure secretion of the alpha-galactosidase enzyme. After transformation and stabilization, the expression vector was stably integrated in the genome. The active alpha-galactosidase enzyme was efficiently secreted (greater than 85%) and after methanol induction, the expression level was 42 mg/l. Amino-terminal sequencing of the purified alpha-galactosidase enzyme synthesized by H. polymorpha showed that the S. cerevisiae invertase signal sequence was correctly processed by H. polymorpha. The secreted alpha-galactosidase was glycosylated and had a sugar content of 9.5%. The specific activity of the alpha-galactosidase produced by H. polymorpha was 38 U mg-1 compared to 100 U mg-1 for the guar alpha-galactosidase. Deglycosylation of the H. polymorpha alpha-galactosidase restored the specific activity completely.
A strain of the methylotrophic yeast Hansenula polymorpha, A16, has been developed that expresses the guar alpha-galactosidase gene to 22.4 mg/g dry cell weight in chemostat cultures at a dilution rate of 0.1 h(-1). This corresponds to more than 13.1% of soluble cell protein, of which 56-62% is secreted into the medium. The alpha-galactosidase gene was flanked by the promoter and terminator sequences of the H.polymorpha mox gene, which can direct expression of the mox gene itself more than 30% of total cell protein under methanol growth. The expression cassette (pUR3510) based on the Saccharomyces cerevisiae plasmid, YEp13, was integrated into the genome. Such transformants were stable in chemostat cultures and exhibited 100% stability for both alpha-galactosidase+ and leu+ phenotypes. Chemostat cultures produced higher levels of alpha-galactosidase with higher specific productivities expressed as mg alpha-galactosidase g(-1) h(-1) compared to batch cultures.
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