ABSTRACT
Furfural and hydroxy-methyl-furfural (HMF) are produced by lignocellulosic biomass during heat or acid pretreatment and are toxic to yeast. Aldehyde reductase is the main enzyme to reduce furfural and HMF. To improve the conversion efficiency of lignocellulosic biomass into ethanol, we constructed Saccharomyces cerevisiae with overexpression of aldehyde reductase (encoded by ari1). The gene of aldehyde reductase (encoded by ari1) was cloned via polymerase chain reaction (PCR) and ligated with the expression vector pGAPZαC. Western blot coupled with anti-His tag confirmed overexpression of the ari1 gene. The growth curves of the wild and ari1-overexpressed strain in the YPD medium were found to be almost identical. Compare to the ari1-overexpressed strain, the wild strain showed a longer doubling time and lag phase in the presence of 20 mM furfural and 60 mM HMF, respectively. The real-time PCR results showed that furfural was much more potent than HMF in stimulating ari1 expression, but the cell growth patterns showed that 60 mM HMF was more toxic to yeast than 20 mM furfural. S. cerevisiae with ari1 overexpression appeared to confer higher tolerance to aldehyde inhibitors, thereby increasing the growth rate and ethanol production capacity of S. cerevisiae in an aldehyde-containing environment.
ABSTRACT
Lignocellulosic biomass is an attractive low-cost feedstock for bioethanol production. During bioethanol production, Saccharomyces cerevisiae, the common used starter, faces several environmental stresses such as aldehydes, glucose, ethanol, high temperature, acid, alkaline and osmotic pressure. The aim of this study was to construct a genetic recombinant S. cerevisiae starter with high tolerance against various environmental stresses. Trehalose-6-phosphate synthase gene (tps1) and aldehyde reductase gene (ari1) were co-overexpressed in nth1 (coded for neutral trehalase gene, trehalose degrading enzyme) deleted S. cerevisiae. The engineered strain exhibited ethanol tolerance up to 14% of ethanol, while the growth of wild strain was inhibited by 6% of ethanol. Compared with the wild strain, the engineered strain showed greater ethanol yield under high stress condition induced by combining 30% glucose, 30 mM furfural and 30 mM 5-hydroxymethylfurfural (HMF).
Subject(s)
Ethanol/metabolism , Genetic Enhancement/methods , Glucose/metabolism , Metabolic Engineering/methods , Multienzyme Complexes/genetics , Saccharomyces cerevisiae/physiology , Stress, Physiological/genetics , Ethanol/isolation & purification , Up-Regulation/geneticsABSTRACT
A genetic recombinant Saccharomyces cerevisiae starter with high ethanol tolerance capacities was constructed. In this study, the gene of trehalose-6-phosphate synthase (encoded by tps1), which catalyzes the first step in trehalose synthesis, was cloned and overexpressed in S. cerevisiae. Moreover, the gene of neutral trehalase (encoded by nth1, trehalose degrading enzyme) was deleted by using a disruption cassette, which contained long flanking homology regions of nth1 gene (the upstream 0.26 kb and downstream 0.4 kb). The engineered strain increased its tolerance against ethanol and glucose stress. The growth of the wild strain was inhibited when the medium contained 6 % or more ethanol, whereas growth of the engineered strain was affected when the medium contained 10 % or more ethanol. There was no significant difference in the ethanol yield between the wild strain and the engineered strain when the fermentation broth contained 10 % glucose (p > 0.05). The engineered strain showed greater ethanol yield than the wild type strain when the medium contained more than 15 % glucose (p < 0.05). Higher intracellular trehalose accumulation by overexpression of tps1 and deletion of nth1 might provide the ability for yeast to protect against environmental stress.
