A selection platform for carbon chain elongation using the CoA-dependent pathway to produce linear higher alcohols Hidevaldo B. Machado a , Yasumasa Dekishima d , Hao Luo a , Ethan I. Lan a,c , James C. Liao a,b,c,n a Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, 5531 Boelter Hall, Los Angeles, California 90095-1570, USA b Institute for Genomics and Proteomics, University of California, Los Angeles, 5531 Boelter Hall, Los Angeles, California 90095-1570, USA c Biomedical Engineering Interdisciplinary Program, University of California, Los Angeles, 5531 Boelter Hall, Los Angeles, California 90095-1570, USA d Mitsubishi Chemical Group Science and Technology Research Center, Inc., Yokohama 227-8502, Japan article info Article history: Received 8 February 2012 Received in revised form 1 June 2012 Accepted 9 July 2012 Available online 20 July 2012 Keywords: n-hexanol n-octanol Biofuel Protein directed evolution Hexanoic acid 3-hydroxyacyl-CoA dehydrogenase abstract Production of green chemicals and fuels using metabolically engineered organisms has been a promising alternative to petroleum-based production. Higher chain alcohols (C4–C8) are of interest because they can be used as chemical feedstock as well as fuels. Recently, the feasibility of n-hexanol synthesis using Escherichia coli has been demonstrated by extending the modified Clostridium CoA-dependent n-butanol synthesis pathway, thereby elongating carbon chain length via reactions in reversed b-oxidation, (or b-reduction). Here, we developed an anaerobic growth selection platform that allows selection or enrichment of enzymes for increased synthesis of C6 and C8 linear alcohols. Using this selection, we were able to improve the carbon flux towards the synthesis of C6 and C8 acyl-CoA intermediates. Replacement of the original enzyme Clostridium acetobutylicum Hbd with Ralstonia eutropha homologue PaaH1 increased production of n-hexanol by 10-fold. Further directed evolution by random mutagenesis of PaaH1 improved n-hexanol and n-octanol production. This anaerobic growth selection platform may be useful for selecting enzymes for production of long-chain alcohols and acids using this CoA-dependent pathway. & 2012 Elsevier Inc. All rights reserved. 1. Introduction With the prospect of unstable and rising price for petroleum, there has been an increasing interest on the development of sustainable manufacturing processes to supply chemicals and fuels. Production of biofuels, in particular, has been the focus of many groups with successful outcomes (for review see Alper and Stephanopoulos, 2009; Jang et al., in press; Mainguet and Liao, 2010; Yan and Liao, 2009). The development of successful biofuels production requires utilization of alternative substrates, increased tolerance to product toxicity (Nicolaou et al., 2010; Fischer et al., 2008), and discovery of new fuel molecules. Several metabolic pathways have been engi- neered to produce higher alcohols (Atsumi et al., 2008a, 2008b; Nielsen et al., 2009), alkane (Schirmer et al., 2010), and biodiesel (Steen et al., 2010) in Escherichia coli as well as in others hosts such as Corynebacterium glutamicum (Smith et al., 2010), Clostridium cellulo- lyticum (Higashide et al., 2011), Bacillus subtilis (Li et al., 2011), cyanobacteria (Atsumi et al., 2009; Lan and Liao, 2011; Lan and Liao, 2012), and Ralstonia eutropha (Li et al., 2012). In addition, the production of biofuels using Clostridium producers has continued to make significant strides (Lee et al., 2009; Wang and Blaschek, 2011; Lehmann and Lutke-Eversloh, 2011). The synthesis of n-butanol in Clostridium species is based on coenzyme A (CoA)-dependent Claisen condensation of two acetyl- CoA followed by reduction, dehydration, and hydrogenation. This sequence of reactions follows the chemistry of b-oxidation in reverse with minor exceptions. Recently, this CoA-dependent b-reduction type linear alcohol synthesis has been extended to increase the alcohol carbon chain length (Dekishima et al., 2011; Dellomonaco et al., 2011). Although many enzymes of this CoA- dependent pathway are reversible, a key step, trans-enoyl-CoA reduction, is catalyzed by an irreversible enzyme acting in the synthesis (hydrogenation) direction, rather than the degradation (dehydrogenation) direction (Bond-Watts et al., 2011; Dekishima et al., 2011; Dellomonaco et al., 2011; Shen et al., 2011). Exten- sion of chain length (Fig. 1) requires another round of condensa- tion, reduction, dehydration, and hydrogenation. It has been shown that expression of b-ketothiolase (BktB) from Ralstonia eutropha, 3-hydroxybutyryl-CoA dehydrogenase (Hbd) and croto- nase (Crt) from Clostridium acetobutylicum, and trans-enoyl-CoA reductase (Ter) from Euglena gracilis (Egl.Ter) or Treponema denticola (Tde.Ter) were able to extend the CoA-dependent Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/ymben Metabolic Engineering 1096-7176/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ymben.2012.07.002 n Corresponding author at: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, 5531 Boelter Hall, Los Angeles, California 90095-1570, USA. Fax: þ1310 206 4107. E-mail address: liaoj@seas.ucla.edu (J.C. Liao). Metabolic Engineering 14 (2012) 504–511