Construction of recombinant Saccharomyces cerevisiae strains for starch utilisation

[摘要] ENGLISH ABSTRACT:Starch-containing agricultural crops are widely available as feedstocks for the productionof fuel ethanol, potable spirits or beer, single-cell protein (animal feed) and high-fructosecorn syrups (sweeteners). Starch-rich crops, such as maize, rye, barley and wheat, areusually used for the production of whisky. One of the first steps in the production of whiskyis to boil the raw starch at temperatures exceeding 100°C. This gelatinisation step isperformed to disrupt and solubilise the starch granules to make them more accessible forenzymatic hydrolysis. After this cooking process, the starch is liquefied by a-amylase andthen saccharified by glucoamylase and a debranching enzyme.Lipomyces kononenkoae and Saccharomycopsis fibuligera secrete highly effectivea-amylases and glucoamylases, making them two of the most efficient raw-starchdegradingyeasts known. However, L. kononenkoae and S. fibuligera cannot be used inexisting industrial fermentations because of their low ethanol tolerance, slow growth rate,catabolite repression, poorly characterised genetics and lack of GRAS (GenerallyRegarded As Safe) status.This study is divided into two sections. The aim of the first section was to clone a gene(LKA2) encoding a novel starch-degrading enzyme, a second a-amylase (Lka2p) fromL. kononenkoae. LKA2 was cloned into a multicopy plasmid, the yeast episomal plasmid,YEp352, under the control of the phosphoglycerate kinase promoter (PGK1 p) andterminator (PGKh) expression cassette. This recombinant plasmid was designatedpJUL3 and transformed into a laboratory strain of S. cerevisiae, I1278b. Plate and liquidassays revealed that the recombinant yeast secreted active a-amylase into the medium.The optimum pH for Lka2p was pH 3.5 and the optimum temperature 60°C.The aim of the second part of the study was to construct recombinant strains ofS. cerevisiae secreting a-amylase and/or glucoamylase. The individual genes were clonedinto a yeast-integrating plasmid, Ylp5, under the control of the PGK1p-PGK1.,-expressioncassette. Two indigenous yeasts were selected on the basis of their ability to utilise rawstarch, L. kononenkoae and S. fibuligera, as gene donors. Eight constructs containing theL. kononenkoae a-amylase genes, LKA 1 and LKA2, and the S. fibuligera a-amylase(SFA 1) and glucoamylase (SFG1) genes were prepared: four single-cassette plasmidsexpressing the individual coding sequences under the control of the PGK1 p-PGK1.,-expression cassette, resulting in plPLKA 1, pIPLKA2, plPSFA 1 and pIPSFG1, respectively;two double-cassette plasm ids (expressing both LKA 1 and LKA2 under the control of thePGK1p-PGK1 .,-expression cassette, and SFA 1 and SFG1 under their respective nativepromoters and terminators), resulting in pIPLKA1/2 and pIPSFAG, respectively, and twosingle-cassette plasmids expressing SFA 1 and SFG1 with their native promoters andterminators, resulting in pSFA 1 and pSFG1, respectively. The respective constructs weretransformed into a laboratory strain of S. cere visiae , L1278b. By homologousrecombination, each plasmid was integrated into the yeast genome at the ura3 locus.S. cerevisiae L:1278b that had been transformed with plPLKA 1/2, LKA 1 and LKA2 underthe control of the PGK1 rrPGK1,expression cassette resulted in the highest levels ofa-amylase activity when assayed for amylolytic activity in a liquid medium. Thisrecombinant strain resulted in the most efficient starch utilisation in batch fermentations,consuming 80% of starch and producing 6 gIL of ethanol after 156 hours of fermentation.The strain expressing SFG1 under the control of the PGK1rrPGK1,expression cassettegave the highest levels of glucoamylase activity.' These results confirmed thatco-expression of a-amylase and/or glucoamylase synergistically enhance starchdegradation.This study paves the way for the development of efficient starch-degrading strains ofS. cerevisiae for the production of whisky, beer and biofuel ethanol.

[发布机构] Stellenbosch University