Improving ethanol yields in sugarcane molasses fermentation by engineering the high osmolarity glycerol pathway while maintaining osmotolerance in Saccharomyces cerevisiae.

The ever-increasing demand of energy has made it imperative to increase the production of renewable fuels like ethanol. Many studies have reported increase in ethanol production by reducing fermentation by-products like glycerol. Deletion of structural genes like gpd1and gpd2 leads to an increase in ethanol by reducing glycerol; however, it makes the yeast osmosensitive that is not desirable for industrial strains. In this study, genes in the HOG pathway which regulates glycerol synthesis in Saccharomyces cerevisiae were targeted for improving ethanol yields in fermentation of sugarcane molasses. Deletion strains of ssk1, hot1, and smp1 were tested and they did not show osmosensitivity. Δssk1 and Δsmp1 recombinant strains showed consistent improved ethanol yields. As a result, a double-deletion strain, Δssk1Δsmp1, was also constructed, which showed a synergistic effect leading to 6% increase in ethanol yield and 35% decrease in glycerol yield. It was also observed that there was a significant decrease in acetic acid yields of all the recombinant strains. Overall, the study demonstrates an industrially viable technique of engineering the HOG pathway resulting in decrease of glycerol and no loss of osmotolerance. These S. cerevisiae strains showed a significant increase in ethanol yields.

Heterologous production of (-)-geosmin in Saccharomyces cerevisiae.

(-)-Geosmin has high demand in perfumes and cosmetic products for its earthy congenial aroma. The current production of (-)-geosmin is either by distillation of sun-baked soil or by inefficient chemical synthesis because of the presence of multiple chiral centers. Fermentation processes are not viable as the titers of the Streptomyces sp. based processes are low. This work presents an alternative route by the heterologous synthesis of (-)-geosmin in Saccharomyces cerevisiae. The enzyme involved is the bifunctional geosmin synthase that catalyzes the conversion of farnesyl diphosphate to germacradienol and germacradienol to geosmin. This study evaluated the activity of many orthologs of geosmin synthase when expressed heterologously in S. cerevisiae. When the well-characterized CAB41566 from Streptomyces coelicolor origin was tested, germacradienol and germacrene D were detected but no geosmin. Bioinformatic analysis based on high/low identities to N-terminal and C-terminal domains of CAB41566 was carried out to identify different orthologs of geosmin synthase proteins from different bacterial and fungal origins. ADO68918 of Stigmatella aurantiaca origin showed the best activity among the tested orthologs, not only in terms of geosmin production but also an order of magnitude higher total abundance of the products of geosmin synthase as compared to CAB41566. This study successfully demonstrated the production of (-)-geosmin in S. cerevisiae and offers an alternative, sustainable and environment-friendly approach to producing (-)-geosmin.