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High Versatility of IPP and DMAPP Methyltransferases Enables Synthesis of C6, C7 and C8 Terpenoid Building Blocks

Abstract The natural substance class of terpenoids covers an extremely wide range of different structures, although their building block repertoire is limited to the C5 compounds DMAPP and IPP. This study aims at the characterization of methyltransferases (MTases) that modify these terpene precursors and the demonstration of their suitability for biotechnological purposes. All seven enzymes tested accepted IPP as substrate and altogether five C6 compounds and six C7 compounds were formed within the reactions. A high selectivity for the deprotonation site as well as high stereoselectivity could be observed for most of the biocatalysts. Only the enzyme from Micromonospora humi also accepted DMAPP as substrate, converting it into (2R)‐2‐methyl‐IPP in vitro. In vivo studies demonstrated the production of a C8 compound and a hydride shift step within the MTase‐catalyzed reaction. Our study presents IPP/DMAPP MTases with very different catalytic properties, which provide biosynthetic access to many novel terpene‐derived structures.


S5: De novo terpenoid production in E. coli strains containing the mevalonate pathway and one of the MTases
The production strain MG1655 harbored the plasmid pMK-16 for provision of high IPP and DMAPP levels and pET-28a derivatives containing the sequence of a MTase. Pre-cultures in reaction tubes containing 5 mL LB medium with appropriate antibiotics were incubated overnight at 37°C and 180 rpm. Main cultures with 15 mL TB medium and appropriate antibiotics in 100 ml baffled shake flasks were inoculated from pre-culture to an OD600 of 0.1. After cultivation at 37°C to an OD600 value of 1, gene expression was induced with isopropylβ-D-1-thiogalactopyranoside (IPTG, 100 μM). Induced cultures had terpenoids extracted during the following 24 hours of cultivation at 30°C and 180 rpm with a magnetic adsorbent stir bar (Twister® , Gerstel). The twister was attached to the shake flask with the help of a magnet fixated to the external part of the flask, thus preventing the twister from being loose on the culture medium. If indicated, unlabeled L-methionine, 13 C-methyl-L-methionine or (methyl-d3)-L-methionine were supplemented (cfinal = 3 g/L) to the pre-and main cultures.

S6: HS-SPME-GC/MS analysis
Volatile compounds in the headspace of in vitro assays and cultures were analyzed by extraction with an 85 μm stableflex SPME fiber composed of PDMS and Carboxen. The SPME fibre placed in a fibre holder (Gerstel) and attached to a stainless steel plunger sheathed by a protective needle (Supelco) was exposed in the headspace of each assay and culture and then inserted into the injection port of a GC-MS-QP2010 (Shimadzu) containing a DB-5 (5 %phenyl)-methylpolysiloxane column with 30 m length and 0.25 mm thickness. Measurements were conducted as follows: helium as carrier gas, splitless injections at 250 °C, 1 minute sampling time, and column flow of 1.1 mL/min. The column temperature was programmed to start with 40 °C for 1.5 minute then rising in steps of 10°C/min until 250 °C followed by rising in 20 °C/min steps up to 300°C. Compounds were identified via comparison of mass spectra and retention indices (RI) to the ones of reference substances or mass spectra of the NIST mass spectral library (v14).
For chiral analysis of the product of DMAPP methylation by humMT, an Astec CHIRALDEX B-DA cyclodextrin stationary phase column connected to an inactivated guard column was employed in a GC-MS QP2010 (Shimadzu) system. An SPME fibre was used for extraction of volatiles on the headspace of the in vitro assays with a final step of phosphatase reaction, as described in the paragraph above. To elucidate product chirality, the two enantiomers ( 2R)-2methyl-IPP and (2S)-2-methyl-IPP (synthesis described on Supplementary Information S3) were analyzed separately, by adding each synthetic prenyl pyrophosphate at a final concentration of 6 µM to a solution containing 7,5 mg/mL acid phosphatase. After incubation for 30 min at 40 o C, an SPME fibre was used to explore the headspace of the assay for 10 min at 40 o C. The retention times of both enantiomers were then compared to the one from the product generated by enzymatic assay. SPME-GC-MS measurements were conducted as follows: helium as carrier gas, splitless injections at 260 °C, 1 minute sampling time, and column flow of 0.84 mL/min. The column temperature was programmed to start with 40 °C for 2 minutes then rising in steps of 5 °C/min until 120 °C, hold for 40 minutes followed by rising in 10 °C/min steps up to 180 °C, hold for 5 minutes. Temperature was then decreased at a rate of -10 °C/min until 130 °C, hold for 1 minute and then decreased at a rate of -5 °C until 40 °C. Compounds were identified via comparison of mass spectra and retention indices (RI) to the ones of reference substances.  Figure S1 SDS-PAGE analysis of purified enzymes. 15 µg of purified protein was used per lane. Gels were stained with Coomassie Brilliant Blue. Page ruler Prestained protein ladder, 10 -180 kDa (Thermo Fischer Scientific) was used as protein size reference. Lanes were spliced to show the purified protein sample beside the respective protein ladder from the same gel, which is indicated by the black lines.

Supplementary
Supplementary Figure S2 Graphic representation of genes encoding putative SAMdependent prenyl pyrophosphate MTases and flanking genes. The red arrows represent the putative prenyl pyrophosphate MTase-encoding genes selected for further analysis. Putative functions of the proteins encoded upstream and downstream of the MTase gene were identified by the search for specific protein domains and by the search for similar protein sequences with known function.
Supplementary Figure S3. GC chromatograms (TIC) of the reference compounds which could be assigned to MTase products. Red dots in the respective structures indicate the positions of the additional methyl groups. In case of 4,5-dimethyl-isoprenol it is possible to see two peaks corresponding to the two isomers present in the mixture, although it is unclear, which peak represents which isomer.
Supplementary Figure S4. Mass spectra of all compounds detected in in vivo or in vitro experiments and of respective reference compounds. In all cases, in which an enzymatically or microbially synthesized compound could be clearly assigned to one of the reference compounds, both mass spectra are shown as comparison. In the case of unknown C7 compound 6 only the mass spectrum of detected compound is shown.