title: Engineering transcription factors to improve tolerance against alkane biofuels in Saccharomyces cerevisiae.
abstract: BACKGROUND
  Biologically produced alkanes can be used as 'drop in' to existing transportation infrastructure as alkanes are important components of gasoline and jet fuels. Despite the reported microbial production of alkanes, the toxicity of alkanes to microbial hosts could pose a bottleneck for high productivity. In this study, we aimed to improve the tolerance of Saccharomyces cerevisiae, a model eukaryotic host of industrial significance, to alkane biofuels.
  
  
  RESULTS
  To increase alkane tolerance in S. cerevisiae, we sought to exploit the pleiotropic drug resistance (Pdr) transcription factors Pdr1p and Pdr3p, which are master regulators of genes with pleiotropic drug resistance elements (PDREs)-containing upstream sequences. Wild-type and site-mutated Pdr1p and Pdr3p were expressed in S. cerevisiae BY4741 pdr1Δ pdr3Δ (BYL13). The point mutations of PDR1 (F815S) and PDR3 (Y276H) in BYL13 resulted in the highest tolerance to C10 alkane, and the expression of wild-type PDR3 in BYL13 led to the highest tolerance to C11 alkane. To identify and verify the correlation between the Pdr transcription factors and tolerance improvement, we analyzed the expression patterns of genes regulated by the Pdr transcription factors in the most tolerant strains against C10 and C11 alkanes. Quantitative PCR results showed that the Pdr transcription factors differentially regulated genes associated with multi-drug resistance, stress responses, and membrane modifications, suggesting different extents of intracellular alkane levels, reactive oxygen species (ROS) production and membrane integrity. We further showed that (i) the expression of Pdr1mt1 + Pdr3mt reduced intracellular C10 alkane by 67 % and ROS by 53 %, and significantly alleviated membrane damage; and (ii) the expression of the Pdr3wt reduced intracellular C11 alkane by 72 % and ROS by 21 %. Alkane transport assays also revealed that the reduction of alkane accumulation was due to higher export (C10 and C11 alkanes) and lower import (C11 alkane).
  
  
  CONCLUSIONS
  We improved yeast's tolerance to alkane biofuels by modulating the expression of the wild-type and site-mutated Pdr1p and Pdr3p, and extensively identified the correlation between Pdr transcription factors and tolerance improvement by analyzing gene patterns, alkane transport, ROS, and membrane integrity. These findings provide valuable insights into manipulating transcription factors in yeast for improved alkane tolerance and productivity.
authors.affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, 117597 Singapore, Singapore ; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 28 Medical Drive, 117456 Singapore, Singapore.
authors.forename: Hua
authors.initials: H
authors.lastname: Ling
authors.affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, 117597 Singapore, Singapore ; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 28 Medical Drive, 117456 Singapore, Singapore.
authors.forename: Nina Kurniasih
authors.initials: NK
authors.lastname: Pratomo Juwono
authors.affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, 117597 Singapore, Singapore ; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 28 Medical Drive, 117456 Singapore, Singapore.
authors.forename: Wei Suong
authors.initials: WS
authors.lastname: Teo
authors.affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, 117597 Singapore, Singapore ; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 28 Medical Drive, 117456 Singapore, Singapore.
authors.forename: Ruirui
authors.initials: R
authors.lastname: Liu
authors.affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, 117597 Singapore, Singapore ; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 28 Medical Drive, 117456 Singapore, Singapore ; Singapore Institute of Technology, 10 Dover Drive, 138683 Singapore, Singapore.
authors.forename: Susanna Su Jan
authors.initials: SS
authors.lastname: Leong
authors.affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, 117597 Singapore, Singapore ; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 28 Medical Drive, 117456 Singapore, Singapore.
authors.forename: Matthew Wook
authors.initials: MW
authors.lastname: Chang
keywords: Alkanes; Biofuels; Pleiotropic drug resistance; Saccharomyces cerevisiae; Site mutagenesis; Tolerance; Transcription factors