Identification of gene targets eliciting improved alcohol tolerance in Saccharomyces cerevisiae through inverse metabolic engineering.
The economic production of biofuels from renewable biomass using Saccharomyces cerevisiae requires tolerance to high concentrations of sugar and alcohol. Here we applied an inverse metabolic engineering approach to identify endogenous gene targets conferring improved alcohol tolerance in S. cerevisiae. After transformation with a S. cerevisiae genomic library, enrichment of the transformants exhibiting improved tolerance was performed by serial subculture in the presence of iso-butanol (1%). Through sequence analysis of the isolated plasmids from the selected transformants, four endogenous S. cerevisiae genes were identified as overexpression targets eliciting improved tolerance to both iso-butanol and ethanol. Overexpression of INO1, DOG1, HAL1 or a truncated form of MSN2 resulted in remarkably increased tolerance to high concentrations of iso-butanol and ethanol. Overexpression of INO1 elicited the highest ethanol tolerance, resulting in higher titers and volumetric productivities in the fermentation experiments performed with high glucose concentrations. In addition, the INO1-overexpressing strain showed a threefold increase in the specific growth rate as compared to that of the control strain under conditions of high levels of glucose (10%) and ethanol (5%). Although alcohol tolerance in yeast is a complex trait affected by simultaneous interactions of many genes, our results using a genomic library reveal potential target genes for better understanding and possible engineering of metabolic pathways underlying alcohol tolerance phenotypes.
Hong
Min-Eui
ME
School of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
Lee
Ki-Sung
KS
Yu
Byung Jo
BJ
Sung
Young-Je
YJ
Park
Sung Min
SM
Koo
Hyun Min
HM
Kweon
Dae-Hyuk
DH
Park
Jae Chan
JC
Jin
Yong-Su
YS
D001709:Biotechnology / Q000379:methods*; D000440:Butanols / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000431:Ethanol / Q000494:pharmacology*; D005285:Fermentation; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D007296:Myo-Inositol-1-Phosphate Synthase / Q000235:genetics / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D000440:Butanols; D004268:DNA-Binding Proteins; C076232:HAL1 protein, S cerevisiae; D047908:Intracellular Signaling Peptides and Proteins; C081935:MSN2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D000431:Ethanol; C093600:DOG2 protein, S cerevisiae; D010744:Phosphoric Monoester Hydrolases; C519641:INO1 protein, S cerevisiae; D007296:Myo-Inositol-1-Phosphate Synthase