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Directed evolution of an E. coli inner membrane transporter for improved efflux of biofuel molecules.

ABSTRACT: 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 ?-pinene. Two mutants exhibiting increased efflux efficiency for n-octane and ?-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 ?-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.

SUBMITTER: Foo JL

PROVIDER: S-EPMC3680313 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Directed evolution of an E. coli inner membrane transporter for improved efflux of biofuel molecules.

Foo Jee Loon JL Leong Susanna Su Jan SS

Biotechnology for biofuels 20130521 1

<h4>Background</h4>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 e ...[more]

PMID: 23693002

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Project description:Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ?19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ?25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with biological membrane surfaces that we are only beginning to decipher.

Dataset Information

Directed evolution of an E. coli inner membrane transporter for improved efflux of biofuel molecules.

Publications

Directed evolution of an E. coli inner membrane transporter for improved efflux of biofuel molecules.

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