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

Biotechnology for Biofuels

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

 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.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]Fortman JL, Chhabra S, Mukhopadhyay A, Chou H, Lee TS, Steen E, Keasling JD: Biofuel alternatives to ethanol: pumping the microbial well. Trends Biotechnol 2008, 26:375-381.
• [2]Atsumi S, Liao JC: Directed evolution of Methanococcus jannaschii citramalate synthase for biosynthesis of 1-propanol and 1-butanol by Escherichia coli. Appl Environ Microbiol 2008, 74:7802-7808.
• [3]Zhang K, Sawaya MR, Eisenberg DS, Liao JC: Expanding metabolism for biosynthesis of nonnatural alcohols. Proc Natl Acad Sci USA 2008, 105:20653-20658.
• [4]Steen EJ, Kang Y, Bokinsky G, Hu Z, Schirmer A, McClure A, Del Cardayre SB, Keasling JD: Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 2010, 463:559-562.
• [5]Lee SK, Chou H, Ham TS, Lee TS, Keasling JD: Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels. Curr Opin Biotechnol 2008, 19:556-563.
• [6]Schirmer A, Rude MA, Li X, Popova E, del Cardayre SB: Microbial biosynthesis of alkanes. Science 2010, 329:559-562.
• [7]Connor MR, Cann AF, Liao JC: 3-Methyl-1-butanol production in Escherichia coli: random mutagenesis and two-phase fermentation. Appl Microbiol Biotechnol 2010, 86:1155-1164.
• [8]Tsukagoshi N, Aono R: Entry into and release of solvents by Escherichia coli in an organic-aqueous two-liquid-phase system and substrate specificity of the AcrAB-TolC solvent-extruding pump. J Bacteriol 2000, 182:4803-4810.
• [9]Dunlop MJ, Dossani ZY, Szmidt HL, Chu HC, Lee TS, Keasling JD, Hadi MZ, Mukhopadhyay A: Engineering microbial biofuel tolerance and export using efflux pumps. Mol Syst Biol 2011, 7:487.
• [10]Dunlop M, Keasling J, Mukhopadhyay A: A model for improving microbial biofuel production using a synthetic feedback loop. Syst Synth Biol 2010, 4:95-104.
• [11]Bokma E, Koronakis E, Lobedanz S, Hughes C, Koronakis V: Directed evolution of a bacterial efflux pump: adaptation of the E. coli TolC exit duct to the Pseudomonas MexAB translocase. FEBS Lett 2006, 580:5339-5343.
• [12]Niwa T, Ying BW, Saito K, Jin W, Takada S, Ueda T, Taguchi H: Bimodal protein solubility distribution revealed by an aggregation analysis of the entire ensemble of Escherichia coli proteins. Proc Natl Acad Sci USA 2009, 106:4201-4206.
• [13]Pos KM: Drug transport mechanism of the AcrB efflux pump. Biochim Biophys Acta 2009, 1794:782-793.
• [14]Yu EW, Aires JR, Nikaido H: AcrB multidrug efflux pump of Escherichia coli: composite substrate-binding cavity of exceptional flexibility generates its extremely wide substrate specificity. J Bacteriol 2003, 185:5657-5664.
• [15]Violi A, Yan S, Eddings EG, Sarofim F, Granata S, Faravelli T, Ranzi E: Experimental formulation and kinetic model for JP-8 surrogate mixtures. Combust Sci Technol 2002, 174:399-417.
• [16]Bohnert JA, Karamian B, Nikaido H: Optimized Nile Red efflux assay of AcrAB-TolC multidrug efflux system shows competition between substrates. Antimicrob Agents Chemother 2010, 54:3770-3775.
• [17]Vermue M, Sikkema J, Verheul A, Bakker R, Tramper J: Toxicity of homologous series of organic solvents for the gram-positive bacteria Arthrobacter and Nocardia Sp. and the gram-negative bacteria Acinetobacter and Pseudomonas Sp. Biotechnol Bioeng 1993, 42:747-758.
• [18]Aono R, Kobayashi M, Nakajima H, Kobayashi H: A close correlation between improvement of organic solvent tolerance levels and alteration of resistance toward low levels of multiple antibiotics in Escherichia coli. Biosci Biotechnol Biochem 1995, 59:213-218.
• [19]Asako H, Nakajima H, Kobayashi K, Kobayashi M, Aono R: Organic solvent tolerance and antibiotic resistance increased by overexpression of marA in Escherichia coli. Appl Environ Microbiol 1997, 63:1428-1433.
• [20]Hayashi S, Aono R, Hanai T, Mori H, Kobayashi T, Honda H: Analysis of organic solvent tolerance in Escherichia coli using gene expression profiles from DNA microarrays. J Biosci Bioeng 2003, 95:379-383.
• [21]Murakami S, Nakashima R, Yamashita E, Matsumoto T, Yamaguchi A: Crystal structures of a multidrug transporter reveal a functionally rotating mechanism. Nature 2006, 443:173-179.
• [22]Tamura N, Murakami S, Oyama Y, Ishiguro M, Yamaguchi A: Direct interaction of multidrug efflux transporter AcrB and outer membrane channel TolC detected via site-directed disulfide cross-linking. Biochemistry 2005, 44:11115-11121.
• [23]Husain F, Bikhchandani M, Nikaido H: Vestibules are part of the substrate path in the multidrug efflux transporter AcrB of Escherichia coli. J Bacteriol 2011, 193:5847-5849.
• [24]Husain F, Nikaido H: Substrate path in the AcrB multidrug efflux pump of Escherichia coli. Mol Microbiol 2010, 78:320-330.
• [25]Nakashima R, Sakurai K, Yamasaki S, Nishino K, Yamaguchi A: Structures of the multidrug exporter AcrB reveal a proximal multisite drug-binding pocket. Nature 2011, 480:565-569.
• [26]Schulz R, Vargiu AV, Collu F, Kleinekathofer U, Ruggerone P: Functional rotation of the transporter AcrB: insights into drug extrusion from simulations. PLoS Comput Biol 2010, 6:e1000806.
• [27]Foo JL, Jackson CJ, Carr PD, Kim HK, Schenk G, Gahan LR, Ollis DL: Mutation of outer-shell residues modulates metal ion co-ordination strength in a metalloenzyme. Biochem J 2010, 429:313-321.
• [28]Jackson CJ, Foo JL, Tokuriki N, Afriat L, Carr PD, Kim HK, Schenk G, Tawfik DS, Ollis DL: Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase. Proc Natl Acad Sci USA 2009, 106:21631-21636.
• [29]Okusu H, Ma D, Nikaido H: AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants. J Bacteriol 1996, 178:306-308.
• [30]Miller JH: A short course in bacterial genetics: a laboratory manual and handbook for Escherichia coli and related bacteria. Plainview, N.Y.: Cold Spring Harbor Laboratory Press; 1992.
• [31]Sambrook J, Russell DW: Molecular cloning: a laboratory manual. 3rd edition. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press; 2001.
• [32]Rabhi I, Guedel N, Chouk I, Zerria K, Barbouche MR, Dellagi K, Fathallah DM: A novel simple and rapid PCR-based site-directed mutagenesis method. Mol Biotechnol 2004, 26:27-34.
• [33]Schrodinger LLC: The PyMOL Molecular Graphics System, Version 1.3r1. 2010.