Transporter-mediated biofuel secretion

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.

[1]  C. Raetz,et al.  MsbA-dependent Translocation of Lipids across the Inner Membrane of Escherichia coli* , 2004, Journal of Biological Chemistry.

[2]  J. Keasling,et al.  Engineering microbial biofuel tolerance and export using efflux pumps , 2011, Molecular systems biology.

[3]  F. Sharom,et al.  Functional Characterization of Escherichia coli MsbA , 2008, Journal of Biological Chemistry.

[4]  D. Rees,et al.  ABC transporters: the power to change , 2009, Nature Reviews Molecular Cell Biology.

[5]  H. Nikaido,et al.  Mechanisms of RND multidrug efflux pumps. , 2009, Biochimica et biophysica acta.

[6]  Seon-Won Kim,et al.  In situ recovery of lycopene during biosynthesis with recombinant Escherichia coli. , 2008, Journal of biotechnology.

[7]  J. Keasling,et al.  Microbial engineering for the production of advanced biofuels , 2012, Nature.

[8]  J. Sheng,et al.  CO2-limitation-inducible Green Recovery of fatty acids from cyanobacterial biomass , 2011, Proceedings of the National Academy of Sciences.

[9]  A. Kester,et al.  Extractability of Carotenoid Pigments from Non-photosynthetic Bacteria with Solvents and Detergents: Implications for the Location and Binding of the Pigments , 1980 .

[10]  E. Gantt,et al.  A portfolio of plasmids for identification and analysis of carotenoid pathway enzymes: Adonis aestivalis as a case study , 2007, Photosynthesis Research.

[11]  M. Mathews,et al.  INTRACELLULAR LOCATION OF CAROTENOID PIGMENTS AND SOME RESPIRATORY ENZYMES IN SARCINA LUTEA , 1959, Journal of bacteriology.

[12]  Zhang Yu-zhong,et al.  Developments in oil extraction from microalgae , 2012 .

[13]  H. V. van Veen,et al.  Dissection of the conformational cycle of the multidrug/lipidA ABC exporter MsbA , 2010, Proteins.

[14]  Stephen G. Aller Structure of P-Glycoprotein Reveals a Molecular Basis for Poly-Specific Drug Binding , 2010 .

[15]  A. Herrmann,et al.  Function of prokaryotic and eukaryotic ABC proteins in lipid transport. , 2005, Biochimica et biophysica acta.

[16]  D. Crunkleton,et al.  Catalytic cracking of squalene to gasoline-range molecules , 2011 .

[17]  Durga Madhab Mahapatra,et al.  Milking Diatoms for Sustainable Energy: Biochemical Engineering versus Gasoline-Secreting Diatom Solar Panels , 2009 .

[18]  Robert E. Jinkerson,et al.  Genetic Engineering of Algae for Enhanced Biofuel Production , 2010, Eukaryotic Cell.

[19]  M. Dunlop Engineering microbes for tolerance to next-generation biofuels , 2011, Biotechnology for biofuels.

[20]  J. Keasling,et al.  Engineering a mevalonate pathway in Escherichia coli for production of terpenoids , 2003, Nature Biotechnology.

[21]  C. Largeau,et al.  Botryococcus braunii: a rich source for hydrocarbons and related ether lipids , 2005, Applied Microbiology and Biotechnology.

[22]  C. Higgins,et al.  ABC transporters: from microorganisms to man. , 1992, Annual review of cell biology.

[23]  Geoffrey Chang,et al.  Understanding polyspecificity of multidrug ABC transporters: closing in on the gaps in ABCB1. , 2010, Trends in biochemical sciences.

[24]  Jay D. Keasling,et al.  Identification and microbial production of a terpene-based advanced biofuel , 2011, Nature communications.

[25]  C. Higgins,et al.  A bacterial antibiotic-resistance gene that complements the human multidrug-resistance P-glycoprotein gene , 1998, Nature.

[26]  R. Jetter,et al.  Plant Cuticular Lipid Export Requires an ABC Transporter , 2004, Science.

[27]  T. Janvilisri,et al.  The ATP Binding Cassette Multidrug Transporter LmrA and Lipid Transporter MsbA Have Overlapping Substrate Specificities* , 2003, Journal of Biological Chemistry.

[28]  C. Raetz,et al.  Periplasmic orientation of nascent lipid A in the inner membrane of an Escherichia coli LptA mutant , 2008, Proceedings of the National Academy of Sciences.

[29]  D. Birdsell,et al.  Production and Ultrastructure of Lysozyme and Ethylenediaminetetraacetate-Lysozyme Spheroplasts of Escherichia coli , 1967, Journal of bacteriology.

[30]  Geoffrey Chang,et al.  Flexibility in the ABC transporter MsbA: Alternating access with a twist , 2007, Proceedings of the National Academy of Sciences.

[31]  J. Keasling Manufacturing Molecules Through Metabolic Engineering , 2010, Science.