A Novel Strategy To Construct Yeast Saccharomyces Cerevisiae For Very High Gravity Ethanol Fermentation

As compared with the traditional low or normal gravity ethanol fermentation process, very high gravity (VHG) fermentation has the advantages of high usage ratio of equipment, energy-saving and emission reduction, pollutant reduction, low capital costs and high efficiency. It is a research focus of global ethanol fermentation industry in latest years. However, this process results in critic issues, such as osmotic pressure at the early stage of fermentation and ethanol inhibition at the later stage of fermentation, resulting in stuck or slugglish fermentation. Meanwhile, during the process of VHG fermentation, Saccharomyces cerevisiae cells produce more glycerol which will dramatically decrease the rate of sugar-to-ethanol conversion and then the efficiency of ethanol production. Therefore, breeding Saccharomyces cerevisiae strains with enhanced stress resistance and higher rate of sugar-to-ethanol conversion is an important approach for achieving VHG fermentation and producing economic and social profits for ethanol production. Combining metabolic engineering and genome shuffling is a new strategy to construct excellent Saccharomyces cerevisiae strains with enhanced stress resistance and high yield of ethanol.This study used industrial Saccharomyces cerevisiae strain Z5as parent strain, combined metabolic engineering and genome shuffling to breed excellent recombinants. First, we deleted glycerol-3-phosphate dehydrogenase GPD2in strain Z5, resulting in a mutant (Z5△GPD2) with20%lower glycerol yield. Then, strain Z5△GPD2was subjected to three rounds of genome shuffling. Finally, we obtained a shuffled strain SZ3-1with130.52g/L ethanol yield under VHG fermentation conditions (initial glucose concentration equals to280g/L) and enhanced ethanol stress tolerance. Compared to parent strain Z5, strain SZ3-1has9%lower glycerol yield and8%higher ethanol production. These results demonstrated that combing metabolic engineering and genome shuffling could efficiently improve complicate phenotypes of Saccharomyces cerevisiae strains and successfully construct Saccharomyces cerevisiae strains suitable for VHG fermentation.Further analysis suggested that the improved ethanol stress tolerance of strain SZ3-1contributed to its fermentative capacity. Elaborating the mechanism of ethanol tolerance has significant value for improving ethanol yield and exploring the mechanism of stress resistance in Saccharomyces cerevisiae. On the basis of previous research, this study elaborated the mechanism of ethanol tolerance on thorough discussion. Research showed that the ethanol tolerance was closely associated with long-chain unsaturated fatty acids C18:1in cell membrane. More ethanol-tolerant strain incorporated more C18:1in cell membrane. The relationship between cell membrane total unsaturated fatty acids, ergosterol and ethanol tolerance is not obvious. Strains which could accumulate more intracellular trehalose can better cope with ethanol stress. Ethanol could promote the expression of genes involved in trehalose metabolism including TPS1、TPS2、TPS3、TSL1、ATH1and NTH1. Ethanol tolerance is a complicated phenotype controlled by multiple factors rather than a single factor.