Correlation Analysis of Targeted Proteins and Metabolites to Assess and Engineer Microbial Isopentenol Production Kevin W. George, 1,2 Amy Chen, 1,3 Aakriti Jain, 1,4 Tanveer S. Batth, 1,2 Edward E.K. Baidoo, 1,2 George Wang, 1,2 Paul D. Adams, 1,2,3 Christopher J. Petzold, 1,2 Jay D. Keasling, 1,2,3,4 Taek Soon Lee 1,2 1 Joint BioEnergy Institute, Emeryville, California 94608; telephone: þ1-510-495-2469; fax: þ1-510-495-2437; e-mail: tslee@lbl.gov 2 Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 3 Department of Bioengineering, University of California, Berkeley, California 4 Department of Chemical & Biomolecular Engineering, University of California, Berkeley, California ABSTRACT: The ability to rapidly assess and optimize heterologous pathway function is critical for effective metabolic engineering. Here, we develop a systematic approach to pathway analysis based on correlations between targeted proteins and metabolites and apply it to the microbial production of isopentenol, a promising biofuel. Starting with a seven-gene pathway, we performed a correlation analysis to reduce pathway complexity and identified two pathway proteins as the primary determinants of efficient isopentenol production. Aided by the targeted quantification of relevant pathway intermediates, we con- structed and subsequently validated a conceptual model of isopentenol pathway function. Informed by our analysis, we assembled a strain which produced isopentenol at a titer 1.5 g/L, or 46% of theoretical yield. Our engineering approach allowed us to accurately identify bottlenecks and determine appropriate pathway balance. Paired with high-throughput cloning techniques and analytics, this strategy should prove useful for the analysis and optimization of increasingly complex heterologous pathways. Biotechnol. Bioeng. 2014;111: 1648–1658. ß 2014 Wiley Periodicals, Inc. KEYWORDS: biofuel; correlation analysis; isopentenol; proteomics; metabolic engineering; isoprenoid Introduction Metabolic engineering has the potential to produce a large variety of chemicals—many of which are currently derived from limited resources—from simple, renewable starting materials (Keasling, 2010). In the last decade, the heterolo- gous over-production of pharmaceuticals, commodity chemicals, alternative transportation fuels, and other natural products in microbial hosts has been convincingly demon- strated with a variety of pathways (Ajikumar et al., 2010; Atsumi et al., 2008; Dellomonaco et al., 2011; Martin et al., 2013; Peralta-Yahya et al., 2011; Ro et al., 2006; Yim et al., 2011). Unfortunately, progress towards the large-scale production of these compounds in microbial biorefineries (Kamm and Kamm, 2004) has been slow. Although advances in systems and synthetic biology have greatly expanded the tools available to metabolic engineers (Boyle and Silver, 2012; Mukhopadhyay et al., 2008), the identification of pathway bottlenecks and establishment of appropriate pathway balance remains challenging due to confounding factors such as feedback regulation, product toxicity, and strain instability. The continued development of systematic methods to assess and engineer heterologous pathways (Yadav et al., 2012) is necessary to address these challenges and improve the efficacy of metabolic engineering. The collection of metabolomics and proteomics data provides essential insight into microbial metabolism and complex physiological behavior. On the genome scale, large sets of “omics” data have facilitated continued advances in Author contributions: K.W.G., J.D.K., and T.S.L. designed the experiments. K.W.G., A.C., A.J., T.S.B., E.B., G.W., P.D.A., C.J.P. performed the experiments. K.W.G., J.D.K., and T.S.L. wrote the manuscript. Competing financial interests: J.D.K. has financial interest in Amyris, LS9, and Lygos. Correspondence to: T.S. Lee Contract grant sponsor: US Department of Energy Received 22 November 2013; Revision received 23 January 2014; Accepted 18 February 2014 Accepted manuscript online 25 February 2014; Article first published online 1 May 2014 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.25226/abstract). DOI 10.1002/bit.25226 ARTICLE 1648 Biotechnology and Bioengineering, Vol. 111, No. 8, August, 2014 ß 2014 Wiley Periodicals, Inc.