(620am) Improving Thermotolerance of Industrial Saccharomyces Cerevisiae TSH3 through an Optimized Strategy for Genome Shuffling and Evolutionary Engineering

As a sugarcane-like non-food energy crop, sweet sorghum can accumulate high level of fermentable sugars within the stalks up to 18 wt%, and has superior agronomic advantages over sugarcane. Unfortunately, the potential of sweet sorghum is not fully harnessed as the feedstock for fuel ethanol production so far, due to quite low sugar recovery from juice pressing process that makes conventional liquid-state fermentation (LSF) not suitable for sweet sorghum ethanol production. Recently, advanced solid-state fermentation (SSF) developed by Tsinghua University was applied to fuel ethanol production using sweet sorghum, which exhibited a higher cost-competitive and lower environmental footprint over LSF, such as simpler process configuration and fewer sewage disposal. However, relatively low heat transfer efficiency of SSF at large scale always traps reaction heat within solid substrates during the process of fermentation, causing a high-temperature SSF, sometimes up to 40 ℃. As a result, higher temperature of SSF inhibits cell metabolism, consequently reducing ethanol productivity. Meanwhile, among industrial ethanol producers, model strain Saccharomyces cerevisiae exhibits optimal growth between 28 and 30℃, and there are also very few engineered S. cerevisiae that can grow with relatively high ethanol productivity at temperature higher than 41℃. Therefore, all these factors mentioned above stymie the broad deployment of SSF at large scale, particularly in subtropical and tropical zones.  In this study, a optimized genome shuffling and evolutionary engineering methods were adopted to improve thermotolerance of S. cerevisiae strain, TSH3, which was isolated from the sweet sorghum stalks and exhibits excellent ethanol fermentative capacity of SSF. Recombinant yeast strains were acquired by recursive DNA shuffling between the entire genome of Trophcalis Candida (CICC 32626) and S. cerevisiae. After two rounds of genome shuffling and screening, one potential recombinant yeast strain S2-1 was obtained. It was able to grow at 45℃, and produce ethanol at 43℃ under ~100 g/l of glucose with 60.2% of theoretical ethanol yield during 48h. Using a long-term adaptation strategy, the recombinant yeast S2-1 produced ethanol more rapidly than the parental strain, and showed an improved ethanol yield (70%) at high temperature. In addition, the recombinant yeast S2-1 also obtained the capability of xylose utilization. Adaptation evolution work is still carried out and further improved strains might be obtained. In order to find out the key regulatory genes and networks of thermo-tolerance of S2-1 strain, genome and transcriptome sequencing were carried out. Some interesting variation of genome profiles were found. Our study gives insight into the mechanism of thermotolerant yeast at genetic level, and might provide a novel strategy of rational genetic modification for obtaining engineering yeast strains that can make bioethanol production by SSF from sweet sorghum much more cost-competitive.

Key words:  genome shuffling, thermotolerance, Saccharomyces cerevisiae, ethanol, solid-state fermentation