Biotechnology for Biofuels | |
Directed evolution of an E. coli inner membrane transporter for improved efflux of biofuel molecules | |
Jee Loon Foo1  Susanna Su Jan Leong1  | |
[1] School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore | |
关键词: Transporter; Directed evolution; Biofuel; Efflux; Synthetic biology; Protein engineering; | |
Others : 798026 DOI : 10.1186/1754-6834-6-81 |
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received in 2012-10-29, accepted in 2013-05-16, 发布年份 2013 | |
【 摘 要 】
Background
The depletion of fossil fuels and the rising need to meet global energy demands have led to a growing interest in microbial biofuel synthesis, particularly in Escherichia coli, due to its tractable characteristics. Besides engineering more efficient metabolic pathways for synthesizing biofuels, efforts to improve production yield by engineering efflux systems to overcome toxicity problems is also crucial. This study aims to enhance hydrocarbon efflux capability in E. coli by engineering a native inner membrane transporter, AcrB, using the directed evolution approach.
Results
We developed a selection platform based on competitive growth using a toxic substrate surrogate, which allowed rapid selection of AcrB variants showing enhanced efflux of linear and cyclic fuel molecule candidates, n-octane and α-pinene. Two mutants exhibiting increased efflux efficiency for n-octane and α-pinene by up to 47% and 400%, respectively, were isolated. Single-site mutants based on the mutations found in the isolated variants were synthesized and the amino acid substitutions N189H, T678S, Q737L and M844L were identified to have conferred improvement in efflux efficiency. The locations of beneficial mutations in AcrB suggest their contributions in widening the substrate channel, altering the dynamics of substrate efflux and promoting the assembly of AcrB with the outer membrane channel protein TolC for more efficient substrate export. It is interesting to note that three of the four beneficial mutations were located relatively distant from the known substrate channels, thus exemplifying the advantage of directed evolution over rational design.
Conclusions
Using directed evolution, we have isolated AcrB mutants with improved efflux efficiency for n-octane and α-pinene. 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.
【 授权许可】
2013 Foo and Leong; licensee BioMed Central Ltd.
【 预 览 】
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