Functions of enzyme domains in 2-methylisoborneol biosynthesis and enzymatic synthesis of non-natural analogs

Two aspects of the biosynthesis of the non-canonical terpene synthase for 2-methylisoborneol have been studied. Several 2-methylisoborneol synthases have a proline-rich N-terminal domain of unknown function. The results presented here demonstrate that this domain leads to a reduced enzyme activity, in addition to its ability to increase long-term solubility of the protein. Furthermore, the substrate scope of the 2-methylisoborneol synthase was investigated through enzyme incubations with several substrate analogs, giving access to two C12 monoterpenoids. Implications on the stereochemical course of the terpene cyclisation by 2-methylisoborneol synthase are discussed.


Gene cloning
Following a published procedure [1], the target genes of domain A and domain B from Streptomyces coelicolor A3(2) were amplified from gDNA by PCR using Q5 Highfidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA) and primer pairs as listed in Table S1.Yeast homologous recombination of the PCR products with the linearised pYE-Express shuttle vector [2] were carried out through the standard protocol using LiOAc, polyethylene glycol and salmon sperm DNA [3].After yeast transformation cultures were grown on SM-URA agar (425 mg yeast nitrogen base, 1.25 g ammonium sulfate, 5 g glucose, 192.5 mg nutritional supplement minus uracil, 5 g agar, 250 mL water) at 28 °C for 3 days.The recombinant plasmids were isolated from grown yeast colonies using the Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research, Irvine, CA, USA) and subsequently used for transformation of Escherichia coli BL21(DE3) electrocompetent cells.Cells were plated on LB agar plates with kanamycin (50 µg mL −1 ) followed by incubation at 37 °C overnight.Single colonies were selected and used to inoculate LB medium (6 mL) liquid cultures with kanamycin (6 µL; 50 mg mL −1 ).After 24 h growth the plasmid DNA was isolated and checked for correct insertion of the desired gene by PCR amplifying the DNA sequence, containing the target gene, using the T7 primer pair and by sequencing.The obtained plasmids were named pYE-Domain A and pYE-Domain B. Construction of the pYE-Express plasmid containing the gene for 2MIBS from S. coelicolor A3(2) (pYE-WP_011031839) was reported before [4].

S2
Table S1.Primers used for cloning of the coding genes for 2MIBS, domain A and domain B.

Domain B_R TCTCAGTGGTGGTGGTGGTGGTGCTCGAGTGctaccagaagtcgggcaggctgtagc
[a] Primer names are composed of the accession numbers and "F" for forward or "R" for reverse complement primer.
[b] The sequence shown in capital letters are homology arms for recombination in yeast which match the terminal sequences of the linearised expression vector pYE-Express (HindIII and EcoRI digestion).

Gene expression and protein purification
Following a published procedure [1], precultures of E. coli BL21 (DE3) transformed with pYE-Domain A (Domain A), pYE-Domain B (Domain B) and pYE-WP_011031839 (2MIBS) were grown in LB medium with kanamycin (50 µg mL −1 ) overnight with shaking at 37 °C.Gene expression cultures were inoculated with the precultures (2/100) and grown in LB medium containing kanamycin (50 µg mL −1 ) with shaking at 37 °C until OD600 = 0.4-0.6 was reached.After cooling the cultures to 18 °C the enzyme expression was induced by the addition of aqueous IPTG solution (400 mM, 1/1000).
The cultures were shaken at 18 °C overnight.

Incubation of 2-Me-GPP with 2-MIBS, domain A, domain B and domains A + B
Culture conditions, protein expressions and protein purifications were performed as described above.The soluble enzyme fractions were checked for purity by SDS-PAGE.The incubations were performed in incubation buffer (50 mM Tris/HCl, 10 mM MgCl2, 10% glycerol, pH 8.2) with a final reaction volume of 1 mL.2-Me-GPP (0.3 mg, 0.8 mM) dissolved in 50 µL substrate buffer (25 mM NH4HCO3) and the enzyme(s) (2MIBS, domain A, domain B, or domain A + domain B; 0.3 µM each) were added and the final reaction volumes were adjusted to 1 mL with incubation buffer, followed by incubation with shaking at 30 °C for 12 h.The crude product was extracted with hexane (500 µL), the extract was dried with MgSO4 and directly analysed by GC-MS.
Retention indices (I) were determined from a homologous series of n-alkanes (C7-C40).

Enzyme incubation of different combinations of DMAPP derivatives and IPP derivatives with FPPS and 2MIBS
Following a published procedure [6], Culture conditions, protein expressions and protein purifications were performed as described above.The soluble enzyme fractions   S3.S4 and S5.Table S2.NMR data of 2 in C6D6 recorded at 298 K.
3    Table S5.NMR data of 5 in C6D6 recorded at 298 K. 5 Figure S1.SDS-PAGE analysis of recombinant 2MIBS, domain A and domain B (all enzymes carry N-terminal His-tags).

Table S4 .
NMR data of 4 in C6D6 recorded at 298 K.