title: A novel strategy to construct yeast Saccharomyces cerevisiae strains for very high gravity fermentation.
abstract: Very high gravity (VHG) fermentation is aimed to considerably increase both the fermentation rate and the ethanol concentration, thereby reducing capital costs and the risk of bacterial contamination. This process results in critical issues, such as adverse stress factors (ie., osmotic pressure and ethanol inhibition) and high concentrations of metabolic byproducts which are difficult to overcome by a single breeding method. In the present paper, a novel strategy that combines metabolic engineering and genome shuffling to circumvent these limitations and improve the bioethanol production performance of Saccharomyces cerevisiae strains under VHG conditions was developed. First, in strain Z5, which performed better than other widely used industrial strains, the gene GPD2 encoding glycerol 3-phosphate dehydrogenase was deleted, resulting in a mutant (Z5ΔGPD2) with a lower glycerol yield and poor ethanol productivity. Second, strain Z5ΔGPD2 was subjected to three rounds of genome shuffling to improve its VHG fermentation performance, and the best performing strain SZ3-1 was obtained. Results showed that strain SZ3-1 not only produced less glycerol, but also increased the ethanol yield by up to 8% compared with the parent strain Z5. Further analysis suggested that the improved ethanol yield in strain SZ3-1 was mainly contributed by the enhanced ethanol tolerance of the strain. The differences in ethanol tolerance between strains Z5 and SZ3-1 were closely associated with the cell membrane fatty acid compositions and intracellular trehalose concentrations. Finally, genome rearrangements in the optimized strain were confirmed by karyotype analysis. Hence, a combination of genome shuffling and metabolic engineering is an efficient approach for the rapid improvement of yeast strains for desirable industrial phenotypes.
authors.affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang Province, People's Republic of China.
authors.forename: Xianglin
authors.initials: X
authors.lastname: Tao
authors.forename: Daoqiong
authors.initials: D
authors.lastname: Zheng
authors.forename: Tianzhe
authors.initials: T
authors.lastname: Liu
authors.forename: Pinmei
authors.initials: P
authors.lastname: Wang
authors.forename: Wenpeng
authors.initials: W
authors.lastname: Zhao
authors.forename: Muyuan
authors.initials: M
authors.lastname: Zhu
authors.forename: Xinhang
authors.initials: X
authors.lastname: Jiang
authors.forename: Yuhua
authors.initials: Y
authors.lastname: Zhao
authors.forename: Xuechang
authors.initials: X
authors.lastname: Wu
mesh_terms: D000222:Adaptation, Physiological / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D015825:Chromosomes, Fungal / Q000235:genetics; D032621:DNA Shuffling / Q000379:methods*; D016521:Electrophoresis, Gel, Pulsed-Field; D004875:Ergosterol / Q000378:metabolism; D000431:Ethanol / Q000494:pharmacology; D005227:Fatty Acids / Q000378:metabolism; D005285:Fermentation / Q000187:drug effects / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D015321:Gene Rearrangement / Q000187:drug effects; D005800:Genes, Fungal / Q000235:genetics; D005820:Genetic Testing; D042822:Genomic Instability; D018471:Hypergravity; D007218:Industrial Microbiology; D007621:Karyotyping; D060847:Metabolic Engineering / Q000379:methods*; D009154:Mutation / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D013312:Stress, Physiological / Q000187:drug effects; D014199:Trehalose / Q000378:metabolism
chemical_list: D005227:Fatty Acids; D000431:Ethanol; D014199:Trehalose; D004875:Ergosterol