Genome shuffling is a powerful strategy for rapid engineering of microbial strains for desirable industrial phenotypes. Here we improved the thermotolerance and ethanol tolerance of an industrial yeast strain SM-3 by genome shuffling while simultaneously enhancing the ethanol productivity. The starting population was generated by protoplast ultraviolet irradiation and then subjected for the recursive protoplast fusion. The positive colonies from the library, created by fusing the inactivated protoplasts were screened for growth at 35, 40, 45, 50 and 55°C on YPD-agar plates containing different concentrations of ethanol. Characterization of all mutants and wild-type strain in the shake-flask indicated the compatibility of three phenotypes of thermotolerance, ethanol tolerance and ethanol yields enhancement. After three rounds of genome shuffling, the best performing strain, F34, which could grow on plate cultures up to 55°C, was obtained. It was found capable of completely utilizing 20% (w/v) glucose at 45–48°C, producing 9.95% (w/v) ethanol, and tolerating 25% (v/v) ethanol stress.

Genome shuffling to improve thermotolerance, ethanol tolerance and ethanol productivity of Saccharomyces cerevisiae

Due to the health and environment impacts of fossil fuels utilization, biofuels have been investigated as a potential alternative renewable source of energy. Bioethanol is currently the most produced biofuel, mainly of first generation, resulting in food-fuel competition. Second generation bioethanol is produced from lignocellulosic biomass, but a costly and difficult pretreatment is required. The pulp and paper industry has the biggest income of biomass for non-food-chain production, and, simultaneously generates a high amount of residues. According to the circular economy model, these residues, rich in monosaccharides, or even in polysaccharides besides lignin, can be utilized as a proper feedstock for second generation bioethanol production. Biorefineries can be integrated in the existing pulp and paper industrial plants by exploiting the high level of technology and also the infrastructures and logistics that are required to fractionate and handle woody biomass. This would contribute to the diversification of products and the increase of profitability of pulp and paper industry with additional environmental benefits. This work reviews the literature supporting the feasibility of producing ethanol from Kraft pulp, spent sulfite liquor, and pulp and paper sludge, presenting and discussing the practical attempt of biorefineries implementation in pulp and paper mills for bioethanol production.

Second Generation Bioethanol Production: On the Use of Pulp and Paper Industry Wastes as Feedstock

https://scielo.conicyt.cl/pdf/ejb/v19n3/art13.pdf

Thermotolerant fermenting yeasts for simultaneous saccharification fermentation of lignocellulosic biomass

A major challenge for the production of ethanol from biomass-derived feedstocks is to develop yeasts that can sustain growth under the variety of inhibitory conditions present in the production process, e.g., high osmolality, high ethanol titers, and/or elevated temperatures (>40 degrees C). Using adaptive laboratory evolution, we previously isolated seven Saccharomyces cerevisiae strains with improved growth at 40 degrees C. Here, we show that genetic adaptations to high temperature caused a growth trade-off at ancestral temperatures, reduced cellular functions, and improved tolerance of other stresses. Thermotolerant yeast strains showed horizontal displacement of their thermal reaction norms to higher temperatures. Hence, their optimal and maximum growth temperatures increased by about 3 degrees C, whereas they showed a growth trade-off at temperatures below 34 degrees C. Computational analysis of the physical properties of proteins showed that the lethal temperature for yeast is around 49 degrees C, as a large fraction of the yeast proteins denature above this temperature. Our analysis also indicated that the number of functions involved in controlling the growth rate decreased in the thermotolerant strains compared with the number in the ancestral strain. The latter is an advantageous attribute for acquiring thermotolerance and correlates with the reduction of yeast functions associated with loss of respiration capacity. This trait caused glycerol overproduction that was associated with the growth trade-off at ancestral temperatures. In combination with altered sterol composition of cellular membranes, glycerol overproduction was also associated with yeast osmotolerance and improved tolerance of high concentrations of glucose and ethanol. Our study shows that thermal adaptation of yeast is suitable for improving yeast resistance to inhibitory conditions found in industrial ethanol production processes. IMPORTANCE Yeast thermotolerance can significantly reduce the production costs of biomass conversion to ethanol. However, little information is available about the underlying genetic changes and physiological functions required for yeast thermotolerance. We recently revealed the genetic changes of thermotolerance in thermotolerant yeast strains (TTSs) generated through adaptive laboratory evolution. Here, we examined these TTSs' physiology and computed their proteome stability over the entire thermal niche, as well as their preadaptation to other stresses. Using this approach, we showed that TTSs exhibited evolutionary trade-offs in the ancestral thermal niche, as well as reduced numbers of growth functions and preadaptation to other stresses found in ethanol production processes. This information will be useful for rational engineering of yeast thermotolerance for the production of biofuels and chemicals.

/pdf/thermotolerant-yeast-strains-adapted-by-laboratory-evolution-2p2xp2ckkp.pdf

Araque Edgardo

Papers

Selection of thermotolerant yeast strains Saccharomyces cerevisiae for bioethanol production