Starch fermentation characteristics of Saccharomyces cerevisiae strains transformed with amylase genes from Lipomyces kononenkoae and Saccharomycopsis fibuligera

Three strains of Saccharomyces cerevisiae transformed with different combinations of foreign yeast amylase genes were evaluated in aerobic and anoxic bioreactor cultures in respect of their growth characteristics and ability to hydrolyse and ferment starch to ethanol. The cloned genes were the Lipomyces kononenkoae LKA1 and LKA2 alpha-amylase genes, both with the S. cerevisiae PGK1 promoter and terminator, and the Saccharomycopsis fibuligera SFG1 glucoamylase gene with its natural promoter and terminator. Preliminary evaluation on starch agar plates failed to reflect the relative performance of these recombinant strains on starch in submerged cultures. Although LKA2 is described in literature as a gene encoding alpha-amylase, we found negligible alpha-amylase activity using the Phadebas assay. Anoxic cultivation on 55 g starch per litre resulted in the production of 21 g ethanol per litre at a yield of 0.4 g ethanol per gram starch within 120 h by a strain transformed with a double gene casette containing LKA1 plus SFG1. A strain expressing LKA1 plus LKA2 fermented starch slowly, producing low amylase activities that appeared only late in the fermentation. With all strains plasmid loss was negligible. The rate of starch hydrolysis was the rate-limiting step in the fermentation of starch. The data presented here illustrate the need to evaluate and characterise genetic transformants thoroughly, since genetic engineering procedures sometimes yield unexpected outcomes. (C) 2004 Elsevier Inc. All rights reserved.