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Engineering transcription factors to improve tolerance against alkane biofuels in Saccharomyces cerevisiae.

Ling H, Pratomo Juwono NK, Teo WS, Liu R, Leong SS, Chang MW - Biotechnol Biofuels (2015)

Bottom Line: 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 %.These findings provide valuable insights into manipulating transcription factors in yeast for improved alkane tolerance and productivity.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

Assays of membrane integrity in BYL13 expressing Pdr transcription factors. a and b RFU ratios of PI and SYTO 9 upon exposure to C10 and C11 alkanes. The relative RFU ratios of BYL13 without alkane were set to 1. c Representative fluorescent images. Error bars SD from three biological replicates
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Fig6: Assays of membrane integrity in BYL13 expressing Pdr transcription factors. a and b RFU ratios of PI and SYTO 9 upon exposure to C10 and C11 alkanes. The relative RFU ratios of BYL13 without alkane were set to 1. c Representative fluorescent images. Error bars SD from three biological replicates

Mentions: To this end, we exposed the cells to C10 and C11 alkanes and stained the exposed cells with fluorescence nucleic acid stains PI and SYTO 9. Subsequently, we measured fluorescence signals and observed the cells under microscope. Figure 6a shows that relative fluorescence unit (RFU) ratios of PI to SYTO 9 were enhanced by 16.7-fold in BYL13 with pESC-Ura, and enhanced by 6.4-fold in BYL13 expressing Pdr1mt1 + Pdr3mt, upon exposure to C10 alkane as compared with those without alkane exposure. Moreover, in the presence of C10 alkane, the RFU ratio in BYL13 expressing Pdr1mt1 + Pdr3mt was about 62 % lower than that with pESC-Ura, likely due to Ict1p-mediated membrane modifications in the presence of C10 alkane. However, Fig. 6b shows that, in the presence of C11 alkane, both BYL13 with Pdr3wt and the control cells had comparable RFU ratios, suggesting intact cell membrane in the presence of C11 alkane.Fig. 6


Engineering transcription factors to improve tolerance against alkane biofuels in Saccharomyces cerevisiae.

Ling H, Pratomo Juwono NK, Teo WS, Liu R, Leong SS, Chang MW - Biotechnol Biofuels (2015)

Assays of membrane integrity in BYL13 expressing Pdr transcription factors. a and b RFU ratios of PI and SYTO 9 upon exposure to C10 and C11 alkanes. The relative RFU ratios of BYL13 without alkane were set to 1. c Representative fluorescent images. Error bars SD from three biological replicates
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4696261&req=5

Fig6: Assays of membrane integrity in BYL13 expressing Pdr transcription factors. a and b RFU ratios of PI and SYTO 9 upon exposure to C10 and C11 alkanes. The relative RFU ratios of BYL13 without alkane were set to 1. c Representative fluorescent images. Error bars SD from three biological replicates
Mentions: To this end, we exposed the cells to C10 and C11 alkanes and stained the exposed cells with fluorescence nucleic acid stains PI and SYTO 9. Subsequently, we measured fluorescence signals and observed the cells under microscope. Figure 6a shows that relative fluorescence unit (RFU) ratios of PI to SYTO 9 were enhanced by 16.7-fold in BYL13 with pESC-Ura, and enhanced by 6.4-fold in BYL13 expressing Pdr1mt1 + Pdr3mt, upon exposure to C10 alkane as compared with those without alkane exposure. Moreover, in the presence of C10 alkane, the RFU ratio in BYL13 expressing Pdr1mt1 + Pdr3mt was about 62 % lower than that with pESC-Ura, likely due to Ict1p-mediated membrane modifications in the presence of C10 alkane. However, Fig. 6b shows that, in the presence of C11 alkane, both BYL13 with Pdr3wt and the control cells had comparable RFU ratios, suggesting intact cell membrane in the presence of C11 alkane.Fig. 6

Bottom Line: 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 %.These findings provide valuable insights into manipulating transcription factors in yeast for improved alkane tolerance and productivity.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus