Construction Of S. Cerevisiae Strains For Very High Gravity Bioethanol Fermentation By Enome Shuffling

Compared with traditional ethanol fermentation process, very-high-gravity bioethanol fermentation has the advantages of high ethanol productivity, low demand for energy and water, and less yet to be treated sewage, thus can improve economy and social benefits and appreciated by ethanol industry, scientific and technology organization, becoming one of the most hottest research topic. Due to environmental stress bought by high concentrations of initial sugar, general Saccharomyces cerevisiae yeast strain would suffer from fermentation sluggish and fermentation stuck, leading to insufficient fermentation of substrate. Thus, bredding S. cerevisiae yeast strain that suit to very high gravity ethanol fermentation is of great significance in enhancing ethanol production rate, shortening fermentation cycle, improving comprehensive economic benefit. Whole genome shuffling, which can achieve gene recombination throughout the whole genome, is an efficient approach to breed S. cerevisiae yeast strains of this type.Here, to construct ADY strains for very high gravity (VHG) ethanol fermentation, the ethanol production performence of some widely used industrial S. cerevisiae yeast strains were first compared. Strain Z1showed the highest ethanol production among these strains, its performance under very high gravity fermentation conditions still need to be improved. So Z1was chosen as the original strain to undergo three rounds of whole genome shuffling. After three rounds of whole genome shuffling and multi-stress screening, the best performed strain ZY01was obtained with102.1g/L and126.96g/L ethanol production under regular conditions and very-high-gravity conditions separately. Compared to the parent strain Z1, ZY01showed1.9%and5.1%higher ethanol yield under regular and very high gravity conditions.Further investigate found that plasma membrane damage was main reason of the poor performance under very high gravity conditions, and high ethanol production capacity of ZY01was related to its stronger membrane integrity. Based on previous research, the present study elaborated the mechanism of membrane stress tolerance of S. cerevisiae yeast during VHG ethanol fermentation process. As is revealed, compared to Z1, the enhancement of productivity and survivability of the shuffled strain ZY01result from its improvement of hyperosmotic tolerance and oxidative tolerance. Under severe osmotic stress as the early stage of VHG fermentation, ZY01shows stronger adaptability and lower levels of membrane permeability. Under serious oxidative stress conditions as the later stage of VHG fermentation, ZY01maintain better membrane stability, preventing severe membrane peroxidation. Subsequently, analysis of factors related to stress tolerance showed ZY01accumulates more intracellular trehalose which is helpful to maintain active properties of hydro-membrane. Thus the shuffled strain can well handle the physical damage brought by osmotic perturbation. Meanwhile, lower fatty acid index and higher levels of glutathione enable ZY01to cope with oxidative stress during dry process.