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Arch Microbiol (2014) 196:853–861 DOI 10.1007/s00203-014-1024-9 ORIGINAL PAPER Carbon partitioning to the terpenoid biosynthetic pathway enables heterologous β-phellandrene production in Escherichia coli cultures Cinzia Formighieri · Anastasios Melis Received: 27 June 2014 / Revised: 24 July 2014 / Accepted: 1 August 2014 / Published online: 13 August 2014 © Springer-Verlag Berlin Heidelberg 2014 Abstract Escherichia coli was used as a microbial sys- Keywords β-Phellandrene · Isoprenoid biosynthetic tem for the heterologous synthesis of β-phellandrene, a pathway · Metabolic engineering · Monoterpene monoterpene of plant origin with several potential com- biosynthesis mercial applications. Expression of Lavandula angusti- folia β-phellandrene synthase (PHLS), alone or in com- Abbreviations bination with Picea abies geranyl-diphosphate synthase DCW Dry cell weight in E. coli, resulted in no β-phellandrene accumulation, DMAPP Dimethylallyl-diphosphate in sharp contrast to observations with PHLS-transformed GPPS Geranyl-diphosphate synthase cyanobacteria. Lack of β-phellandrene biosynthesis in E. IPP Isopentenyl-diphosphate coli was attributed to the limited endogenous carbon par- IPTG Isopropyl β-D-1-thiogalactopyranoside titioning through the native 2-C-methylerythritol-4-phos- MEP 2-C-methyl-erythritol-4-phosphate phate (MEP) pathway. Heterologous co-expression of the MVA Mevalonic acid mevalonic acid pathway, enhancing cellular carbon parti- OD Optical density tioning and flux toward the universal isoprenoid precur- β-PHL β-Phellandrene sors, isopentenyl-diphosphate and dimethylallyl-diphos- PHLS β-Phellandrene synthase phate, was required to confer β-phellandrene production. TIR Translation initiation region Differences in endogenous carbon flux toward the synthe- sis of isoprenoids between photosynthetic (Synechocystis) and non-photosynthetic bacteria (E. coli) are discussed Introduction in terms of differences in the regulation of carbon parti- tioning through the MEP biosynthetic pathway in the two The monoterpene β-phellandrene (C10H16) is naturally syn- systems. thesized from geranyl-diphosphate (GPP) by a number of plant species as a constituent of their essential oils. It has commercial value as a key ingredient in medical, cosmetic and cleaning products, and potentially as a fuel (Martin et al. 2012; Bentley et al. 2013; Zerbe and Bohlmann 2014). Communicated by Erko Stackebrandt. Microbial production of β-phellandrene through metabolic engineering can provide a sustainable alternative by which Electronic supplementary material The online version of this to meet increasing product demand from the commercial article (doi:10.1007/s00203-014-1024-9) contains supplementary material, which is available to authorized users. sector. Microbial systems are suitable for large-scale pro- duction, as they are cultivated in bioreactors that satisfy C. Formighieri · A. Melis (*) containment and product sequestration requirements. Department of Plant and Microbial Biology, University Heterologous biosynthesis of β-phellandrene was of California, 111 Koshland Hall, Berkeley, CA 94720-3102, USA first engineered in a photosynthetic microorgan- e-mail: [email protected] ism, the cyanobacterium Synechocystis (Bentley et al. 1 3 854 Arch Microbiol (2014) 196:853–861 2013), taking advantage of the cell’s photosynthesis- Recent work on the synthesis of monoterpenes in E. coli, driven metabolism. A positive aspect of microbial such as limonene, pinene, and sabinene, employed heter- β-phellandrene production is the spontaneous and quan- ologous expression of the MVA pathway in combination titative separation of the molecule from the biomass with the heterologous expression of a geranyl-diphosphate and the liquid culture (Bentley et al. 2013; Formigh- synthase (GPPS), resulting in widely variable yields of ieri and Melis 2014). Floating of the β-phellandrene on the various monoterpenes (Alonso-Gutierrez et al. 2013; the surface of the liquid culture facilitates segregation Sarria et al. 2014; Zhang et al. 2014). and harvesting, a parameter that weighs heavily on the In the present work, we investigated aspects of the syn- economics of a microbial production system. Further, thesis of β-phellandrene in the chemoheterotrophic, faculta- β-phellandrene separation from the biomass alleviates tive aerobic bacterium E. coli. Heterologous co-expression potential inhibitory or toxic effects of the molecule on of Lavandula angustifolia β-phellandrene synthase (PHLS) cellular metabolism (Lindberg et al. 2010; Dunlop et al. along with Picea abies GPPS did not yield measurable 2011). However, although Synechocystis is a well-stud- amounts of β-phellandrene. The additional heterologous ied model microorganism, over-expression of trans- expression of the MVA biosynthetic pathway was required genic proteins, to a level that can be visually detected in for β-phellandrene accumulation in the culture. Results are Coomassie-stained SDS-PAGE of total protein extract, discussed in terms of the regulation of carbon flux through is rarely achieved. This is an important consideration, as the bacterial MEP isoprenoid biosynthetic pathway, and low concentration of the transgenic terpene synthase is a also in terms of the plasmid- versus chromosomal-based factor limiting β-phellandrene production rate and yield expression of transgenes in production-type photosynthetic (Formighieri and Melis 2014). (e.g., Synechocystis) and chemoheterotrophic (E. coli) Different biological systems employ two independent bacteria. metabolic pathways by which to generate the universal terpenoid precursors isopentenyl-diphosphate (IPP) and dimethylallyl-diphosphate (DMAPP). The 2-C-methyler- Materials and methods ythritol-4-phosphate (MEP) pathway, which is of prokary- otic origin and active in most bacteria and plant plastids, Escherichia coli strains and plasmids requires pyruvate and glyceraldehyde 3-phosphate as initial substrates for IPP and DMAPP biosynthesis. The The L. angustifolia (lavender) codon-optimized PHLS gene mevalonic acid (MVA) pathway uses acetyl-CoA and (Bentley et al. 2013; Formighieri and Melis 2014, see also operates in the cytosol of eukaryotes and archaea. Escher- Supplemental materials) was cloned via NdeI and BglII ichia coli possesses the MEP pathway for the synthesis restriction enzymes in the pJF plasmid (AmpR) under the of primary terpenoids essential for cellular function, in control of the Ptrc promoter and lac Operator. particular for the prenylation of tRNAs and the synthesis Three different types of GPPS gene have been identified of farnesyl diphosphate (FPP), which is used for quinone in plants, a heterodimeric and two homodimeric enzymes. and cell wall biosynthesis (Bouhss et al. 2008; Dumelin Picea abies (Norway spruce) harbors two GPPS genes et al. 2012). The limited number and amount of terpenoid (GPPS2 and GPPS3), each encoding a different homodi- products in E. coli suggest slow flux of endogenous car- meric enzyme. In contrast to GPPS3, GPPS2 was chosen bon through the MEP pathway and low intracellular lev- in the present study as it clusters with other gymnosperm els of C5 isoprenoid precursors (IPP and DMAPP) (Carter GPPS sequences, producing GPP as the sole in vitro prod- et al. 2003). Engineering the endogenous MEP pathway uct (Schmidt and Gershenzon 2008). The sequence encod- to enhance flux is one strategy by which to increase IPP ing for Picea abies (Norway spruce) GPPS2 was obtained and DMAPP supply (Farmer and Liao 2001; Kim and from the NCBI GenBank database (EU432047.2) and Keasling 2001; Matthews and Wurtzel 2000). However, codon optimized using the Gene Designer 2.0 software heterologous expression of the MVA pathway in E. coli from DNA2.0, upon removal of 67 amino acids predicted was reported to be a superior strategy (Martin et al. 2003; by ChloroP to constitute the chloroplast transit peptide. The Vadali et al. 2005; Yoon et al. 2009; Zurbriggen et al. resulting GPPS2 sequence (Supplemental material) was 2012; Alonso-Gutierrez et al. 2013), avoiding native cel- cloned via BglII restriction downstream of PHLS in the pJF lular regulatory mechanisms that apply breaks to the MEP plasmid in an operon configuration. The (N)6-AGGAGG- pathway (Banerjee et al. 2013). With respect to monoter- (N)6 nucleotide sequence, shown to afford efficient ribo- pene production, an additional issue is the apparent some binding in the translation of recombinant proteins in absence of a distinct GPP synthase in E. coli, further rais- E. coli (Zurbriggen et al. 2012), was used as GPPS2 trans- ing the question of the efficacy of this microbial system lation initiation region (TIR). E. coli Rosetta cells were first for the synthesis of monoterpenes (Carter et al. 2003). transformed with the pJF-PHLS-GPPS2 plasmid, while a 1 3 Arch Microbiol (2014) 196:853–861 855 second transformation event introduced the pET28a-MVA Protein analysis of cleared lysate and pellet by SDS-PAGE (KanR) plasmid. The latter drives expression of a super- and immunoblotting operon containing the seven genes for expression of a com- plete MVA pathway, as described (Zurbriggen et al. 2012), At the end of the 20-h incubation period, an aliquot of under the control of the T7 promoter and lac Operator. 50-mL culture was pelleted and resuspended in buffer (50 mM Tris–HCl pH 8, 50 mM NaCl, 10 mM CaCl2, β-Phellandrene production assay in E. coli 10 mM MgCl2) plus protease inhibitors (1 mM PMSF, 2 mM aminocaproic acid, 1 mM benzamidine). The cell A 400-mL bacterial culture, comprising