Modular and Selective Biosynthesis of Gasoline-Range Alkanes
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Modular and selective biosynthesis of gasoline-range alkanes The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Sheppard, Micah J., Aditya M. Kunjapur, and Kristala L.J. Prather. “Modular and Selective Biosynthesis of Gasoline-Range Alkanes.” Metabolic Engineering 33 (January 2016): 28–40. As Published http://dx.doi.org/10.1016/j.ymben.2015.10.010 Publisher Elsevier Version Author's final manuscript Citable link http://hdl.handle.net/1721.1/108077 Terms of Use Creative Commons Attribution-NonCommercial-NoDerivs License Detailed Terms http://creativecommons.org/licenses/by-nc-nd/4.0/ 1 Title: Modular and selective biosynthesis of gasoline-range alkanes 2 Authors: Micah J. Sheppard1, 2†, Aditya M. Kunjapur1, 3†, Kristala L. J. Prather1, 3* 3 Affiliations: 4 1. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, 5 USA 6 2. Present Address: Ginkgo BioWorks, 27 Drydock Avenue, 8th floor, Boston, Massachusetts 02210, 7 USA 8 3. Synthetic Biology Engineering Research Center (SynBERC), Massachusetts Institute of Technology, 9 Cambridge, MA 02139, USA 10 11 †These authors contributed equally to this work. 12 * Corresponding author: 13 Department of Chemical Engineering 14 77 Massachusetts Avenue 15 Room E17-504G 16 Cambridge, MA 02139 17 Phone: 617.253.1950 18 Fax: 617.258.5042 19 Email: [email protected] 20 Keywords: alkanes, gasoline, biofuel, E. coli, metabolic engineering, synthetic biology 21 1 22 Abstract: 23 Typical renewable liquid fuel alternatives to gasoline are not entirely compatible with current 24 infrastructure. We have engineered Escherichia coli to selectively produce alkanes found in gasoline 25 (propane, butane, pentane, heptane, and nonane) from renewable substrates such as glucose or glycerol. 26 Our modular pathway framework achieves carbon-chain extension by two different mechanisms. A fatty 27 acid synthesis route is used to generate longer chains heptane and nonane, while a more energy efficient 28 alternative, reverse-β-oxidation, is used for synthesis of propane, butane, and pentane. We demonstrate 29 that both upstream (thiolase) and intermediate (thioesterase) reactions can act as control points for chain- 30 length specificity. Specific free fatty acids are subsequently converted to alkanes using a broad- 31 specificity carboxylic acid reductase and a cyanobacterial aldehyde decarbonylase (AD). The selectivity 32 obtained by different module pairings provides a foundation for tuning alkane product distribution for 33 desired fuel properties. Alternate ADs that have greater activity on shorter substrates improve observed 34 alkane titer. However, even in an engineered host strain that significantly reduces endogenous 35 conversion of aldehyde intermediates to alcohol byproducts, AD activity is observed to be limiting for 36 all chain lengths. Given these insights, we discuss guiding principles for pathway selection and potential 37 opportunities for pathway improvement. 38 2 39 1 Introduction 40 The United States relies heavily on gasoline to fulfill transportation needs. The U.S. consumes 41 roughly 4 billion barrels of gasoline annually, which amounts to 40% of total annual domestic petroleum 42 usage and 47% of all gasoline produced worldwide (International Energy Statistics, 2014; Fichman et 43 al., 2012). Vast gasoline infrastructure exists to facilitate its usage, and prevailing renewable liquid fuel 44 alternatives have limited compatibility. Ethanol requires a different distribution system than gasoline 45 because of its hygroscopicity, corrosivity, and biodegradability (Strogen and Horvath, 2013). Due to 46 dissimilar fuel performance characteristics such as energy density and research octane number (RON) 47 (Table S1), renewable gasoline alternatives are blended with gasoline for use in conventional 48 automobile engines. One approach towards addressing the compatibility of renewable fuels is to 49 metabolically engineer a microbe that converts sugars into a product that mimics the composition of 50 gasoline. An added benefit of such a process would be streamlined product separation from the aqueous 51 phase due to increased product hydrophobicity (Table S1). 52 We began the pathway design process by first looking at published chemical composition studies 53 of gasoline. Typical regular unleaded gasoline is a blend of over 30 aliphatic and aromatic hydrocarbons 54 (Fig. 1A) (Cline et al., 1991; Johansen et al., 1983; Sanders and Maynard, 1968). C3 (propane) to C9 55 (nonane) are the most common alkane components in gasoline with C5 (pentane) species predominating. 56 With such targets in mind, we next examined previously observed natural and engineered enzymes 57 involved in alkane synthesis. Initial work on microbial alkane production elucidated natural routes to 58 C15-C17 alkanes produced via fatty acid synthesis (FAS) (Schirmer et al., 2010). In cyanobacteria, acyl- 59 acyl carrier protein (acyl-ACP) reductases (AAR) and aldehyde decarbonylases (ADs, also known as 60 aldehyde-deformylating oxygenases or ADOs (Aukema et al., 2013)) convert growing acyl-ACP chains 61 to long-chain alkanes. In a natural progression, fatty acid reductases and ADs have been co-expressed 3 62 with acyl-ACP thioesterases in order to terminate FAS at medium-chain lengths and convert the free 63 acids to alkanes (Akhtar et al., 2013; Andre et al., 2013; Harger et al., 2012; Howard et al., 2013; 64 Schirmer et al., 2010). The C12 and longer alkanes reported may serve as alternatives for diesel fuels but 65 not for gasoline. 66 Recent efforts have also targeted the production of short-chain alkanes (< C10). Using a similar 67 FAS termination strategy, introduction of a plant AD, CER1At from Arabidopsis thaliana, resulted in 68 production of alkanes as short as nonane from glucose in E. coli (Choi and Lee, 2013). Nonane and the 69 longer alkanes produced collectively constitute less than one weight percent of typical regular unleaded 70 gasoline. Other short-chain acyl-ACP thioesterases have been described (Jing et al., 2011), and recently 71 two thioesterases were used to synthesize heptane and propane via FAS (Kallio et al., 2014). Synthesis 72 of these alkanes relied on the use of an AD from Prochlorococcus marinus MIT9313 (ADPm), which has 73 a solved crystal structure (Andre et al., 2013; Khara et al., 2013). Kallio and colleagues achieved 74 increases in titer from < 1 mg/L to 32 mg/L propane by varying process conditions to improve oxygen 75 availability. Like nonane, propane also constitutes less than one weight percent of typical regular 76 unleaded gasoline. 77 Although the report detailing propane production describes a notable achievement in the ability 78 to produce short alkanes, there are drawbacks that provide motivation for additional approaches. One is 79 the limited ability of propane to serve as a drop-in or blended gasoline replacement. As shown in Fig. 80 1A, the range of alkanes bounded in carbon chain length between propane and nonane (C4-C8) 81 constitute a much greater fraction of gasoline. All gasoline-range alkanes (C3-C9) can separate rapidly 82 from aqueous cultures due to their hydrophobicity and volatility, but alkanes longer than propane have 83 greater energy density (Table S1). Longer alkanes also require less energy than propane to store and 84 transport in the liquid phase. Given that the number of vehicles that can be powered solely on propane is 4 85 significantly smaller than the gasoline powered automobile fleet, production of gasoline-range alkanes 86 addresses a larger problem in the transportation fuels sector. 87 Previously published approaches to alkane synthesis have also been dependent on FAS for 88 carbon chain elongation as compared to the use of CoA-dependent, reverse β-oxidation (RBO) pathways 89 (Dekishima et al., 2013; Dellomonaco et al., 2011). RBO routes provide greater theoretical efficiency 90 than FAS for alkane production, in terms of co-factor balancing and energy consumption. The vast 91 majority of FAS reductases that have been described and predicted are likely to strongly prefer an 92 NADPH cofactor (Handke et al., 2011; Javidpour et al., 2014; Ratledge, 2004). In contrast, canonical 93 CoA-dependent pathways contain beta-oxidation enzymes, which are NADH dependent (Kunau et al., 94 1995; Schulz, 1991). Barring the use of an efficient transhydrogenase to rapidly interconvert reducing 95 equivalents, the increased requirement for NADPH in FAS-based chain elongation necessitates greater 96 carbon flux to the pentose phosphate pathway, which lowers carbon efficiency of alkane production. 97 Furthermore, the carboxylation of acetyl-CoA to produce malonyl-CoA in FAS pathways leads to a 98 higher net ATP consumption than in RBO pathways (Handke et al., 2011) (see Discussion for a more 99 detailed analysis). 100 In addition to pathway energetics, the reliance of RBO on the universal CoA molecule rather 101 than the reliance of FAS on organism specific ACPs provides RBO platforms with increased 102 transferability into alternative host organisms. Every reaction in the FAS pathway, including the release 103 of a free acid by the thioesterase, requires the enzyme to interact with an acyl-ACP substrate. While 104 ACP is highly conserved across many organisms, sequence diversity exists which may influence the 105 efficiency of interactions between ACP and FAS enzymes derived from different sources (Byers and 106 Gong, 2007; De Lay and Cronan, 2007). This may already be manifested in the differing performance 107 of thioesterases expressed in E. coli (Jing et al., 2011; Lennen and Pfleger, 2012). Differences in 5 108 productivity may depend to some degree on the efficiency of interaction between E. coli acyl-ACP and 109 the given plant acyl-ACP thioesterase. Additionally, transfer of such a pathway to a yeast strain is 110 complicated by the fused structure of the type I FAS of yeast which includes an ACP domain 111 (Schweizer and Hofmann, 2004). Growing acyl chains are linked to the complex and released via a 112 transferase as palmitoyl-CoA. Instead, RBO pathways rely on a universal cofactor, CoA.