Engineering an iterative polyketide pathway in Escherichia coli results in single-form alkene and alkane overproduction Qian Liu a,c , Kaiyue Wu a,b , Yongbo Cheng a,c , Lei Lu a,c , Erting Xiao a,c , Yuchen Zhang a,c , Zixin Deng a,c , Tiangang Liu a,c,n a Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China b J1 Biotech, Co. Ltd., Wuhan 430075, China c Hubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of Biotechnology, Wuhan 430075, China article info Article history: Received 22 August 2014 Received in revised form 7 December 2014 Accepted 12 December 2014 Available online 20 December 2014 Keywords: Iterative Pks Thioesterases Alkene Alkane Precise protein regulation in vitro reconstitution Targeted engineering abstract Alkanes and alkenes are ideal biofuels, due to their high energy content and ability to be safely transported. To date, fatty acid-derived pathways for alkane and alkene bioproduction have been thoroughly explored. In this study, we engineered the pathway of the iterative Type I polyketide synthase (PKS) SgcE with the cognate thioesterase (TE) SgcE10 in Escherichia coli, with the goal of overproducing pentadecaheptaene (PDH) followed by its hydrogenation to pentadecane (PD). Based on initial in vitro titration assays, we learned that PDH production is strongly dependent on the SgcE10:SgcE ratio. Thus, we engineered a high-yield E. coli strain by fine-tuning SgcE10 expression via synthetic promoters. We analyzed engineered E. coli strains using a modified multiple reactions monitoring mass spectrometry (MRM-MS)-based targeted proteomic approach, using a chimeric SgcE10 and SgcE fusion construct to gain insight into expression levels of the two proteins. Lastly, through fed-batch fermentation followed by flow chemical hydrogenation, we obtained a PD yield of nearly 140 mg/L in single-alkane form. Thus, we not only employed a metabolic engineering approach to the iterative polyketide pathway, we highlighted the potential of PKS shunt products to play a role in the production of single-form and high-value chemicals. & 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved. 1. Introduction Microbial systems for fuel production have the potential to help respond to increasing global energy demand and growing green- house gas emissions (Kerr, 2007). Four well-established systems, the 2-keto acid-dependent pathway, the isoprenoid-based pathway, the reversed β-oxidation pathway, and the fatty acid-based pathway, have been engineered for future fuel feedstock (Dellomonaco et al., 2011; Gonzalez, 2013; Liu and Khosla, 2010). The products of these pathways are represented by butanol, farnesane, fatty alcohols, fatty acid alkyl esters, alkenes, and alkanes. Of these, alkanes and alkenes are valued as primary components of diesel fuels. As summarized in Fig. S1, thus far there have been five reported pathways for engineering alkane and alkene production derived from the fatty acid system (Beller et al., 2010; Choi and Lee, 2013; Howard et al., 2013; Rude et al., 2011; Schirmeret al., 2010; Sukovich et al., 2010), as well as a reversed β-oxidation pathway demonstrated by an in silico modeling study (Cintolesi et al., 2014). Short-chain alkanes can be produced at levels exceeding 500 mg/L through fatty acyl-acyl carrier protein (ACP) to fatty acid to fatty acyl-CoA pathway (Choi and Lee, 2013). An engineering Escherichia coli strain harboring the cyano- bacteria alkane pathway can convert fatty acyl-ACP into long-chain alkanes and alkenes, resulting in final alkane titers over 300 mg/L, with pentadecane (PD) and heptadecene as major components (Schirmer et al., 2010). After optimizing the free fatty acid-derived pathway catalyzed by OleT, the highest total hydrocarbon titer obtained was 97.6 mg/L (Liu et al., 2014). Alkane or alkene production by other fatty acid biosynthesis-derived pathways has remained under the mg/L level (Beller et al., 2010; Howard et al., 2013; Rude et al., 2011; Sukovich et al., 2010). It should be noted that all these products are in mixture form, due to the nature of the Type-II fatty acid biosynthesis mechanism (Beller et al., 2010; Howard et al., 2013; Rude et al., 2011; Sukovich et al., 2010). In addition to the pathways already reported, polyketide synthase (PKS) appears to be a promising candidate enzyme for application in Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/ymben Metabolic Engineering http://dx.doi.org/10.1016/j.ymben.2014.12.004 1096-7176/& 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved. n Corresponding author at: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharma- ceutical Sciences, Wuhan 430071. Fax: þ86 27 68755086. E-mail address: liutg@whu.edu.cn (T. Liu). Metabolic Engineering 28 (2015) 82–90