Optimization of a heterologous pathway for the production of flavonoids from glucose Christine Nicole S. Santos a , Mattheos Koffas b , Gregory Stephanopoulos a,n a Department of Chemical Engineering, Massachusetts Institute of Technology, Room 56-469, Cambridge, MA 02139, USA b Department of Chemical and Biological Engineering, University at Buffalo, State University of New York, 303 Clifford C. Furnas Hall, Buffalo, NY 14260, USA article info Article history: Received 23 November 2010 Received in revised form 1 February 2011 Accepted 4 February 2011 Available online 12 February 2011 Keywords: Flavonoids Naringenin Escherichia coli Pathway balancing Metabolic engineering abstract The development of efficient microbial processes for the production of flavonoids has been a metabolic engineering goal for the past several years, primarily due to the purported health-promoting effects of these compounds. Although significant strides have been made recently in improving strain titers and yields, current fermentation strategies suffer from two major drawbacks—(1) the requirement for expensive phenylpropanoic precursors supplemented into the media and (2) the need for two separate media formulations for biomass/protein generation and flavonoid production. In this study, we detail the construction of a series of strains capable of bypassing both of these problems. A four-step heterologous pathway consisting of the enzymes tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI) was assembled within two engineered L-tyrosine Escherichia coli overproducers in order to enable the production of the main flavonoid precursor naringenin directly from glucose. During the course of this investigation, we discovered that extensive optimization of both enzyme sources and relative gene expression levels was required to achieve high quantities of both p-coumaric acid and naringenin accumulation. Once this metabolic balance was achieved, however, such strains were found to be capable of producing 29 mg/l naringenin from glucose and up to 84 mg/l naringenin with the addition of the fatty acid enzyme inhibitor, cerulenin. These results were obtained through cultivation of E. coli in a single minimal medium formulation without additional precursor supplementation, thus paving the way for the development of a simple and economical process for the microbial production of flavonoids directly from glucose. & 2011 Elsevier Inc. All rights reserved. 1. Introduction Flavonoids comprise a highly diverse family of plant secondary polyphenols which possess biochemical properties (estrogenic, antioxidant, antiviral, antibacterial, antiobesity, and anticancer) that are useful for the treatment of several human patho- logies (Forkmann and Martens, 2001; Fowler and Koffas, 2009; Harborne and Williams, 2000; Hollman and Katan, 1998; Knekt et al., 1996). Despite this broad range of pharmaceutical indica- tions, however, their widespread use and availability are cur- rently limited by inefficiencies in both their chemical synthesis and extraction from natural plant sources. As a result, the development of strains and processes for the microbial produc- tion of flavonoids has emerged recently as an interesting and commercially attractive challenge for metabolic engineering. As shown in Fig. 1, only four catalytic steps are required for the conversion of the aromatic amino acid L-tyrosine to the main flavanone precursor, naringenin. This process begins with the conversion of L-tyrosine to the phenylpropanoic acid p-coumaric acid through the action of the enzyme tyrosine ammonia lyase (TAL). Once p-coumaric acid has been generated, 4-coumarate: CoA ligase (4CL) mediates the formation of its corresponding CoA ester, coumaroyl-CoA. This compound is subsequently condensed with three malonyl-CoA units by the sequential action of the type III polyketide synthase, chalcone synthase (CHS), and, in the final step, the resulting naringenin chalcone is stereospecifically iso- merized by chalcone isomerase (CHI) to form the (2S)-flavanone naringenin. This compound is the starting point for the synthesis of a variety of other flavonoid molecules, which are created through the combined actions of functionalizing enzymes which hydroxylate, reduce, alkylate, oxidize, and glucosylate this phe- nylpropanoid core structure (Fowler and Koffas, 2009; Kaneko et al., 2003). Although previous studies have already made significant gains in demonstrating the feasibility of microbial naringenin Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/ymben Metabolic Engineering 1096-7176/$ - see front matter & 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ymben.2011.02.002 n Corresponding author. Fax: + 1 617 253 3122. E-mail addresses: cnsantos@mit.edu (C.N. Santos), mkoffas@buffalo.edu (M. Koffas), gregstep@mit.edu (G. Stephanopoulos). Metabolic Engineering 13 (2011) 392–400