© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 Biotechnol. J. 2012, 7 DOI 10.1002/biot.201200227 www.biotechnology-journal.com 1 Introduction Microbial tolerance is a complex, multigenic, and hetero- geneous trait that is influenced by a multitude of factors [1]. Strains tolerant to specific stressors are important in bioprocessing for the production of chemicals and bio- fuels, but also in bioremediation applications [1–3]. Toler- ant phenotypes are the result of diverse mechanistic ac- tions that involve the interplay of a multitude of genes en- gaged in, among others, changes in cell-wall composition, membrane phospholipid cis–trans-isomerization, cellular- energy management, the action of molecular pumps, and the activation of stress–response systems [1, 4]. Several approaches have been engaged in screening for complex phenotypes [5]. Methods such as SCALEs [6], genome shuffling [7], genomic library enrichment [2, 8, 9], and global transcription machinery engineering (gTME) [10] can be used to “engineer” tolerant strains. Ethanol tolerance has been investigated in Escherichia coli as a model system of alcohol tolerance but also for practical applications since E. coli can be engineered to produce ethanol from sugars [11, 12]. Notable benchmarks of suc- cessful ethanol-tolerance engineering in E. coli include a recent report where tolerance, as determined by cell via- bility, was improved by approximately 65% after an 8-h exposure to 7% v/v ethanol by overexpressing murB, en- coding for an UDP-N-acetylenolpyruvoylglucosamine re- ductase, which is involved in cell wall biosynthesis [13]. The ethanol tolerant strain LY01, which was isolated by Research Article Exploring the combinatorial genomic space in Escherichia coli for ethanol tolerance Sergios A. Nicolaou, Stefan M. Gaida and Eleftherios T. Papoutsakis Molecular Biotechnology Laboratory, Department of Chemical and Biomolecular Engineering, The Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA Strain tolerance to toxic chemicals is desirable for biologically producing biofuels and chemicals. Standard genomic libraries can be screened to identify genes imparting tolerance, but cannot cap- ture interactions among proximal or distant loci. In search of ethanol tolerance determinants, we expanded the genomic space combinatorially by screening coexisting genomic libraries (CoGeLs) of fosmids (large inserts) and plasmids (smaller inserts) under increasing ethanol concentrations. Such screening led to identification of interacting genetic loci imparting ethanol tolerance. One pair of fragments ([galT, galE] and [recA, pncC, mltB]) increased survival under 50 g/L ethanol by 38% when coexpressed, but individually the fragments had no effect. Coexpression of two genomic fragments ([sfsB, murA, yrbA, mlaB, mlaC, mlaD, mlaE, mlaF, yrbG] and [yrbA, mlaB, mlaC ]) enhanced Escherichia coli survival to 50 g/L ethanol by up to 115%. A 35-kb fosmid fragment increased tolerance to 63 g/L ethanol by 160%. While the tolerance levels of these strains compare favorably to or exceed the performance of previously reported engineered strains, more signifi- cantly, this study demonstrates that combinatorial library screening and screening fosmid libraries offer new, previously unexplored tools for identifying genetic determinants of ethanol, and by extrapolation, other alcohol tolerance. Keywords: Biofuel · Gene interaction · Genomic library · Microbial tolerance · Renewable chemical Correspondence: Prof. Eleftherios T. Papoutsakis, Molecular Biotechnology Laboratory, Department of Chemical and Biomolecular Engineering, The Delaware Biotechnology Institute, University of Delaware, 15 Innova- tion Way, Newark, DE 19711, USA E-mail: epaps@udel.edu Abbreviations: bp, base pair; CFU, colony-forming unit; CoGeL, coexist- ing/coexpressing genomic libraries; gDNA, genomic DNA; gTME, global transcription machinery engineering; IPTG, isopropyl β-D-1-thiogalactopy- ranoside Received 15 JUN 2012 Revised 11 JUL 2012 Accepted 27 JUL 2012 Accepted article online 30 JUL 2012 Supporting information available online