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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

Quantification of ROS levels in BYL13 expressing Pdr transcription factors. a and b ROS levels upon exposure to C10 and C11 alkanes. The relative ROS levels of BYL13 without alkane were set to 1. c Comparison of fluorescence images, where stronger green fluorescence indicates higher ROS levels. AU arbitrary unit. Asterisk significant difference (one-tailed Student t test, p < 0.05); error bars SD from three biological replicates
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Fig5: Quantification of ROS levels in BYL13 expressing Pdr transcription factors. a and b ROS levels upon exposure to C10 and C11 alkanes. The relative ROS levels of BYL13 without alkane were set to 1. c Comparison of fluorescence images, where stronger green fluorescence indicates higher ROS levels. AU arbitrary unit. Asterisk significant difference (one-tailed Student t test, p < 0.05); error bars SD from three biological replicates

Mentions: ROS levels were quantified to investigate the effect of Pdr transcription factor expression on ROS production in the presence of alkanes. Figure 5a, b shows that C10 alkane enhanced ROS levels by more than fourfold whereas C11 alkane increased ROS levels by 1.5-fold. Further, in comparison to BYL13 carrying pESC-Ura, intracellular ROS was reduced by 53 % in BYL13 expressing Pdr1mt1 + Pdr3mt in the presence of C10 alkane, and reduced by 21 % in BYL13 expressing Pdr3wt in the presence of C11 alkane. The reduction of ROS in BYL13 expressing the Pdr transcription factors was further supported by our microscopy results. Figure 5c shows that, upon exposure to C10 alkane, over 90 % of the cells with pESC-Ura fluoresced in green, and only about 30 % of the cells with Pdr1mt1 + Pdr3mt fluoresced in green. On the other hand, upon exposure to C11 alkane, 15 % of the cells with pESC-Ura fluoresced in green, and no cells with Pdr3wt fluoresced in green. Here, more green cells and higher fluorescence intensities represent more ROS. The results of ROS quantification and microscopy suggest significant reduction of ROS in BYL13 expressing the Pdr transcription factors in the presence of C10 and C11 alkanes.Fig. 5


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)

Quantification of ROS levels in BYL13 expressing Pdr transcription factors. a and b ROS levels upon exposure to C10 and C11 alkanes. The relative ROS levels of BYL13 without alkane were set to 1. c Comparison of fluorescence images, where stronger green fluorescence indicates higher ROS levels. AU arbitrary unit. Asterisk significant difference (one-tailed Student t test, p < 0.05); 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

Fig5: Quantification of ROS levels in BYL13 expressing Pdr transcription factors. a and b ROS levels upon exposure to C10 and C11 alkanes. The relative ROS levels of BYL13 without alkane were set to 1. c Comparison of fluorescence images, where stronger green fluorescence indicates higher ROS levels. AU arbitrary unit. Asterisk significant difference (one-tailed Student t test, p < 0.05); error bars SD from three biological replicates
Mentions: ROS levels were quantified to investigate the effect of Pdr transcription factor expression on ROS production in the presence of alkanes. Figure 5a, b shows that C10 alkane enhanced ROS levels by more than fourfold whereas C11 alkane increased ROS levels by 1.5-fold. Further, in comparison to BYL13 carrying pESC-Ura, intracellular ROS was reduced by 53 % in BYL13 expressing Pdr1mt1 + Pdr3mt in the presence of C10 alkane, and reduced by 21 % in BYL13 expressing Pdr3wt in the presence of C11 alkane. The reduction of ROS in BYL13 expressing the Pdr transcription factors was further supported by our microscopy results. Figure 5c shows that, upon exposure to C10 alkane, over 90 % of the cells with pESC-Ura fluoresced in green, and only about 30 % of the cells with Pdr1mt1 + Pdr3mt fluoresced in green. On the other hand, upon exposure to C11 alkane, 15 % of the cells with pESC-Ura fluoresced in green, and no cells with Pdr3wt fluoresced in green. Here, more green cells and higher fluorescence intensities represent more ROS. The results of ROS quantification and microscopy suggest significant reduction of ROS in BYL13 expressing the Pdr transcription factors in the presence of C10 and C11 alkanes.Fig. 5

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