Resumo

Due to the large volumes involved, sugarcane fermentation into fuel ethanol is one of the most important industrial processes in Brazil. Saccharomyces cerevisiae yeasts ferment sucrose through extracellular hydrolysis of the sugar, mediated by the periplasmatic invertase encoded by SUC genes, releasing glucose plus fructose that are transported and fermented by the cells. The rapid hydrolysis of high sucrose concentrations produces deleterious effects to the cells due to the osmotic shock produced by high glucose plus fructose concentrations, while these sugars may also allow the growth of contaminant bacterial and non-Saccharomyces yeast in the vats, decreasing fermentation yields. Alternatively, sucrose can be directly transported into the cells by an active H⁺-symporter mediated by AGT1 permease, and hydrolyzed by intracellular invertases or maltases, allowing increased ethanol production. In this work, we modified the expression of the SUC2 and AGT1 genes in a Brazilian fuel-ethanol industrial strain by genomic engineering, in order to improve its fermentation performance. The analysis of the genome of strain CAT-1 revealed a diploid yeast with only SUC2 in the sub-telomeric region of chromosome IX, and a AGT1 gene with a defective promoter in the telomere of chromosome VII. Thus, we first over-expressed the intracellular form of invertase (iSUC2), replacing the signal sequence of one copy of the SUC2 gene by a strong promoter (P_PGK) linked to a recyclable dominant marker (loxP-KanMX-loxP) to allow selection of the transformants. Trough a self-cloning strategy we disrupted the second copy of the SUC2 gene by replacing it with the AGT1 gene under the control of a constitutive promoter (P_GPD::AGT1). The resulting strain lacked extracellular invertase activity, and over-expressed the intracellular invertase and the AGT1 permease, allowing efficient growth on sucrose. The modified strain is able to ferment rapidly and efficiently high sucrose concentration (<200 g L-1), with no glucose or fructose been released into the medium. We also observed a significant decrease in glycerol production, which is normally synthesized in response to the osmotic stress generated by high sugar concentrations. We have now successfully removed the marker gene (KanMX) from the modified strain, allowing the use of this yeast by the industry without any restrictions.