Construction Of Amylolytic Industrial Strains Of Saccharomyces Cerevisiae

A 1560bp EcoRI/Kpnl gene fragment, 2950bp BamHl/SalI gene fragment and 291 bp HindIII/Pstl sequence were prepared by PCR with Saccharomycopsis fibuligera genomic DNA, Saccharomyces cerevisiae genomic DNA and Pichia methanolica expression vector pMETaB as the template. They were designated AMY gene ILV2 gene and MFalS, respectively, and inserted into vector YEp352 or M13", generating the recombinant plasmid pLA6, pLV2 and Ml3a. The restriction endonuclease digestion analysis showed the cloned AMY gene and ILV2 gene has similar sequence with the reported sequence of GenBank. Only a few differences exist. There is a Sacl site in the cloned AMY gene at about 100bp from the starting codon, while a BglI site in the reported AMY gene at 72bp and the cloned ILV2 gene has one Xbal site less than the reported sequence.The AMY gene, together with phosphoglycerate kinasel promoter and ethanol dehydrogenasel terminator from S. cerevisiae was cloned into a yeast expression vector (YEp352), generating the recombinant plasmid pLA8. The recombinant plasmid pLA8 was introduced into laboratory S. cerevisiae with LiAC method and the transformants were screened on the YNB+His+Leu+Trp medium. The transformant became URA auxotrophy and can grow on the medium containing starch as the sole carbon source. This result indicated that the cloned AMY can express bioactive amylase. The recombinant plasmid pLA8 was introduced into an industrialstrain of S. cerevisiae Sc-11 and the transformants were screened on the SC medium in which starch is the sole carbon source. The average amylase activities of the transformants in cellular extract and culture supernatants were 0.3U/ml and l.lU/ml respectively, while no amylase activity was detected in that of the host strain. The transformants were able to hydrolyze 38% starch. This verified that the amylase secretion signal sequence of S. fibuligera was not able to efficiently guide the secretion of amylase in S. cerevisiae, part of amylase kept in the cell. The genetic stability of transformant was 71% under non-selective condition.As the expression level of amylase in the transformant-pLA8 was low and only part of it was secreted into the culture medium, the yeast a-factor secretion signal sequence was inserted before the A MY starting codon and the recombinant plasmid containing a-factor secretion signal sequence was constructed. The recombinant plasmid designated pLA8ot was introduced into the industrial strain of S. cerevisiae Sc-11 and transformants Sc-11-pLA8a were screened on the YNBS medium. The activity of the amylase enzyme was 6.3U/ml in the culture supernatant and the transformant, with 80% genetic stability, was able to hydrolyze 83% starch under optimal fermentation condition. The wort fermentation experiments under simulating brewing conditions were carried out and the residue sugar, ethanol and flavor compounds were examined by chromatographic analyses. The fermentation characteristics of the engineered strain were similar to that of the control strain and only minor changes in the concentration of flavor compounds could be observed.As YEp-type plasmids are not stably maintained in the yeast cells under nonselective conditions, the transformant might lose the amylase activity during successive fermentation, and the recombinant industrial strains of S. cerevisiae, manipulated with shuttle vectors, contain bacterial antibiotic-resistance markers and yeast drug-resistance markers and are generally not suitable for application in food industry. To overcome the genetic instability and construct an amylolytic brewer's yeast suitable for commercial use, integrating cassette was constructed. During primary wort fermentation, acetolactate is formed by brewer's yeast and leaks out of the yeast cells into the fermented wort and slowly decarboxylates to diacetylnon-enzymatically. The diacetyl is a well-known component of yeast-derived off-flavor in beer and its removal is time-consuming and adds production cost. The integrating cassette was devised to achieve double-crossover integration by homolo...