Appl Microbiol Biotechnol (2011) 90:1681–1690 DOI 10.1007/s00253-011-3173-y BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING High-flux isobutanol production using engineered Escherichia coli: a bioreactor study with in situ product removal Antonino Baez & Kwang-Myung Cho & James C. Liao Received: 15 October 2010 / Revised: 3 February 2011 / Accepted: 4 February 2011 / Published online: 10 March 2011 # The Author(s) 2011. This article is published with open access at Springerlink.com Abstract Promising approaches to produce higher alco- hols, e.g., isobutanol, using Escherichia coli have been developed with successful results. Here, we translated the isobutanol process from shake flasks to a 1-L bioreactor in order to characterize three E. coli strains. With in situ isobutanol removal from the bioreactor using gas strip- ping, the engineered E. coli strain (JCL260) produced more than 50 g/L in 72 h. In addition, the isobutanol production by the parental strain (JCL16) and the high isobutanol-tolerant mutant (SA481) were compared with JCL260. Interestingly, we found that the isobutanol- tolerant strain in fact produced worse than either JCL16 or JCL260. This result suggests that in situ product removal can properly overcome isobutanol toxicity in E. coli cultures. The isobutanol productivity was approxi- mately twofold and the titer was 9% higher than n-butanol produced by Clostridium in a similar integrated system. Keywords Biofuels . Isobutanol . E. coli . Gas stripping . Bioreactor A. Baez : K.-M. Cho : J. C. Liao (*) Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, 5531 Boelter Hall, 420 Westwood Plaza, Los Angeles, CA 90095, USA e-mail: liaoj@seas.ucla.edu J. C. Liao Institute for Genomics and Proteomics, University of California, Los Angeles, 5531 Boelter Hall, 420 Westwood Plaza, Los Angeles, CA 90095, USA Introduction The growing need for alternative energy calls for new strategies to produce liquid fuels such as alcohols, alkanes, and fatty acid esters (or biodiesel) from renewable sources (Dellomonaco et al. 2010; Peralta-Yahya and Keasling 2010; Solomon 2010). Compared to ethanol, higher alcohols (e.g., 1-propanol, isobutanol, n-butanol, 2- methyl-1-butanol, 3-methyl-1-butanol) have higher energy density and lower hygroscopicity, which make them better candidates as gasoline additives or substitutes. However, no native organisms have been identified to produce these advanced biofuels in appreciable quantities (Clomburg and Gonzalez 2010). For example, isobutanol was identified as a by-product in beer fermentation, but with a titer as low as 16 mg/L (Garcia et al. 1994). Since the feasibility of a bioprocess depends critically on the final product titer (Stephanopoulos 2007), a synthetic approach to produce higher alcohols from non- fermentative pathways in Escherichia coli was previously devised (Atsumi et al. 2008b). In addition, metabolic engineering efforts to produce these alcohols at higher titers and yields have been performed (Atsumi et al. 2008a, b; Cann and Liao 2008; Clomburg and Gonzalez 2010; Connor and Liao 2008; Jarboe et al. 2010; Shen and Liao 2008; Smith et al. 2010). This strategy uses the amino acid biosynthetic pathway and diverts its 2- ketoisovalerate for isobutanol synthesis by overexpression of 2-ketoisovalerate decarboxylase (Kivd) and alcohol dehydrogenase (AdhA). Because of the ubiquity of the amino acid pathways, this strategy is compatible with many organisms and shows significant promise for industrial applications.