A ‘Plug and Play’ Platform for the Production of Diverse Monoterpene Hydrocarbon Scaffolds in Escherichia coli

The terpenoids constitute one of the largest and most diverse classes of natural compounds with applications as pharmaceuticals, flavorings and fragrances, pesticides and biofuels. Synthetic biology is ideally placed to create new routes to this chemical diversity and facilitation of new compound discovery. The C10 monoterpenoids display a huge structural diversity produced from a single substrate, geranyl diphosphate, by a family of monoterpene cyclases and synthases (mTC/S). Here we employ a library of mTC/S in a single ‘plug and play’ platform system for the production of over 30 different monoterpenoids in Escherichia coli by fermentation on glucose. These products include several compounds never before produced in engineered microbes demonstrating the power of this approach to rapidly create routes to structural diversity.


Table of contents Experimental Section
Bacterial strains and media 2 Construction of plasmids 2 Monoterpene production conditions, product capture and detection 3 Figure S1: Plasmid maps of pMVA and pBbB2a-trAgGPPS(co)-trMsLS 4 Table S1: Strains used in this study 5 Table S2: Primers used in this study 6 Table S3: Monoterpene synthases used in this study 7 Table S4: Plasmids used in this study 8 Figure S2: Limonene production titres in different E. coli strains 9 Figure S3: GCMS analysis of authentic monoterpenoid standards used in this study 10 Figure Table S5: Product profiles and monoterpenoid titres of production strains 33 Table S6: Overview of all linear monoterpenoids produced 34 Table S7: Overview of all monocyclic monoterpenoids produced 35 Table S8: Overview of all bicyclic monoterpenoids produced 36 Table S9: Overview of all additional terpenoids produced 37 Figure S5: Titres produced in the platform vs. previously published titres 38 Figure S6: Monoterpenoid, geranoid and farnesol titres for each strain 39

References 40
Monoterpene production conditions, product capture and detection Expression strains were inoculated with freshly transformed colonies into 3 ml terrific broth (TB) supplemented with 0.4 % glucose and antibiotics in 28 ml glass screw capped vials. Cultures were grown for 7 h at 37 °C with shaking at 200 rpm before transferring to 30 °C and induction with 50 μM (isopropyl β-D-1-thiogalactopyranoside) IPTG and 25 nM anhydro-tetracycline (aTet) and overlaid with a 20 % n-nonane layer followed by incubation for 72 h with shaking at 200 rpm. A 600 readings were taken of the aqueous phase, and nonane layers were harvested and clarified by centrifugation (14,000 rpm, 3 min, 4°C), dried over anhydrous MgSO 4 and mixed 1:1 with ethyl acetate containing 0.1 % sec-butyl benzene.
Monoterpene products were analysed by GCMS using an Agilent Technologies 7890B GC equipped with an Agilent Technologies 5977A MSD. The products were separated on a DB-WAX column (30 m x 0.32 mm i.d., 0.25 µM film thickness, Agilent Technologies). The injector temperature was set at 240°C with a split ratio of 20:1 (1 µL injection). The carrier gas was helium with a flow rate of 1 mL/min and a pressure of 5.1 psi. The following oven program was used: 50°C (1 min hold), ramp to 68°C at 5°C/min (2 min hold), and ramp to 230°C at 25°C/min (2 min hold). The ion source temperature of the mass spectrometer (MS) was set to 230°C and spectra were recorded from m/z 50 to m/z 250. Compound identification was carried out using authentic standards and comparison to reference spectra in the NIST library of MS spectra and fragmentation patterns.
Monoterpenoids were quantified using authentic standards wherever possible, using experimentally determined relative response factors in relation to the internal standard used. In the absence of an authentic standard concentrations were estimated using a relative response factor of 1.  K-12 [6] W3110 K-12 [7] DH1 K-12 [8] MDS42 Meta K-12 Scarab Genomics Production strain DH5 (-Select) K-12 Bioline

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Additional terpenoids detected: farnesal (rt: 13.222), and farnesol (rt: 13.543). The peak at rt 14.039 is indole. overlay for each E. coli strain containing the MVA pathway and a distinct monoterpene synthase. Averages of at least 3 biological replicates per mTC/S and the corresponding standard deviations (in brackets) are shown. Highest values for each compound are highlighted in bold.