Transporter Engineering for Improved Microbial Tolerance against Biofuel Molecules

Binbin Chen1,2, Jee Loon Foo1,2, Susanna Su Jan Leong1,2,3 and Matthew Wook

Chang1,2

1 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, 117599, Singapore

2 Synthetic Biology Research Consortium, National University of Singapore, 28

Medical Drive, 117456, Singapore

3 Singapore Institute of Technology, 10 Dover Drive, 138683, Singapore

Hydrocarbon alkanes, components of major fossil fuels, are considered as next- generation biofuels because their biological production has recently been shown to be possible. However, high-yield alkane production requires robust host cells that are tolerant against alkanes, which exhibit cytotoxicity. To tackle this toxicity issue, we first investigated the mechanisms of cellular response to alkane biofuels at a system level through transcriptome analyses in Saccharomyces cerevisiae, a model eukaryotic host of industrial significance. Among hypothesized mechanisms, we were interested in identifying plasma membrane transporters which could possibly aid in alkane secretion, leading to improved tolerance. In support of this hypothesis, we expressed the hypothesized native and heterologous transporters, and demonstrated that the expression of transporters significantly improved cell tolerance against alkanes through maintaining lower intracellular alkane level. Here, we demonstrated that transporter engineering - identification and expression of transporters led to significantly improved tolerance against alkane biofuels in S. cerevisiae. Further, using directed evolution, we isolated AcrB mutants with improved efflux efficiency in Escherichia coli for hydrocarbon-based biofuels. The utilization of such optimized native efflux pumps will increase productivity of biofuels synthesis and alleviate toxicity and difficulties in production scale-up in current microbial platforms. We believe that our results laid the groundwork for developing robust microbial cells through transporter engineering, which will greatly aid in next- generation biofuel production and recovery.