Dataset Information

Transporter-mediated biofuel secretion.

ABSTRACT: Engineering microorganisms to produce biofuels is currently among the most promising strategies in renewable energy. However, harvesting these organisms for extracting biofuels is energy- and cost-intensive, limiting the commercial feasibility of large-scale production. Here, we demonstrate the use of a class of transport proteins of pharmacological interest to circumvent the need to harvest biomass during biofuel production. We show that membrane-embedded transporters, better known to efflux lipids and drugs, can be used to mediate the secretion of intracellularly synthesized model isoprenoid biofuel compounds to the extracellular milieu. Transporter-mediated biofuel secretion sustainably maintained an approximate three- to fivefold boost in biofuel production in our Escherichia coli test system. Because the transporters used in this study belong to the ubiquitous ATP-binding cassette protein family, we propose their use as "plug-and-play" biofuel-secreting systems in a variety of bacteria, cyanobacteria, diatoms, yeast, and algae used for biofuel production. This investigation showcases the potential of expressing desired membrane transport proteins in cell factories to achieve the export or import of substances of economic, environmental, or therapeutic importance.

SUBMITTER: Doshi R

PROVIDER: S-EPMC3651508 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Transporter-mediated biofuel secretion.

Doshi Rupak R Nguyen Tuan T Chang Geoffrey G

Proceedings of the National Academy of Sciences of the United States of America 20130423 19

Engineering microorganisms to produce biofuels is currently among the most promising strategies in renewable energy. However, harvesting these organisms for extracting biofuels is energy- and cost-intensive, limiting the commercial feasibility of large-scale production. Here, we demonstrate the use of a class of transport proteins of pharmacological interest to circumvent the need to harvest biomass during biofuel production. We show that membrane-embedded transporters, better known to efflux li ...[more]

PMID: 23613592

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Project description:BackgroundThe 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.ResultsWe 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.ConclusionsUsing 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.

Dataset Information

Transporter-mediated biofuel secretion.

Publications

Transporter-mediated biofuel secretion.

Similar Datasets

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