Gibson assembly mix

The strain carrying the kanamycin resistance cassette was obtained by PCR amplifying a plasmid obtained from the Gene Synthesis services from Genscript Inc. found in Supplementary Table S2 with primers ResistanceF and ResistanceR (Supplementary Table S1). Roughly 400 bps on either side of the AR (Antibiotic Resistance) insertion point were PCR amplified with primer pairs UpGCF and UpGCR_resistance on one hand and DownGCF_resistance and DownGCR on the other hand. Fragments were joined by using Gibson Assembly Mix (New England Biolabs). The assembled product was further amplified by the external primers, transformed into MS11 and selected to obtain the AR::Kan MS11 strain (MS11_AR::Kan).

All oligonucleotide primers used for cloning are listed in S1 Table and were obtained from Integrated DNA Technologies (Coralville, IA, USA). For plasmid construction, the vector backbone and gene fragments were amplified by PCR using Q5 DNA polymerase (New England Biolabs, Ipswich, MA, USA) following the manufacturer’s protocol. DNA fragments were purified by agarose gel electrophoresis and assembled using Gibson Assembly mix (New England Biolabs, Ipswich, MA, USA) following the manufacturer’s protocol. All plasmids used in this study were verified by DNA sequencing and restriction enzyme digestion analysis and are listed in S2 Table.
The plasmids pCel3A and pGH3 were constructed by cloning the respective glycosyl hydrolases encoded by cel3A from Cellvibrio japonicus and CC_0968 from Caulobacter crescentus in pIND4-spec (pVector). The predicted localizations of Cel3A and CC_0968 are shown in S3 Table.

For in vivo diterpenoid synthase activity, 8 TSs listed in Table 1 were amplified by pETM11-fw and pETM11-rev using a pETM11-TS as a template containing homologous sequences at both ends for Gibson assembly [33 (link)]. IspAM22 (D2G, C155G) inserts were created by amplification of the ispA gene from E. coli DH5α with primers containing the desired base changes (S1 Table). A primer extension method was used to amplify the full-length gene which was inserted into pBbA2k-ispAM22-TS (for full list see S1 Table) by annealing at 50°C using Gibson assembly mix (NEB). For mono- or sesqui-terpene production pBbb2a-GPPS plasmid backbone was used for cloning and TSs (S1 Table) were amplified by pETM11-fw and pETM11-rev using a pETM11-TS as a template containing homologous sequences at both ends for Gibson assembly.
Genes encoding selected TS from different bacteria were codon optimised for expression in E. coli (S1 Table), synthesized, and sub-cloned into pETM11 with N-terminal His-tag (GeneArt, Life Technologies). Protein sequence of the selected TSs are shown S2 Table.

TRPV4-FLAG in pcDNA3.1 was previously described^{88 (link)}. TRPV4-GFP was generated by replacing the FLAG epitope tag with EGFP amplified from pEGFP-C1 (Clontech). TRPV4-M680K, TRPV4-R232C, TRPV4-R237L, TRPV4-R269C, TRPV4-I331F, TRPV4-D333G, TRPV4 P142A/P143L, TRPV4 ¹²¹AAWAA¹²⁵, RhoA E47A, and RhoA E54A with either FLAG or EGFP tags were generated by site directed mutagenesis using the Quikchange XL-II kit (Agilent, 200521) according to manufacturer protocols. TRPV4-mScarlet was generated from TRPV4-FLAG in pcDNA3.1 using Gibson assembly mix (New England Biolabs, E2611S) and PCR amplification of the mScarlet coding sequence from pmScarlet-C1 (Addgene, 85042). Primers used for mutagenesis and subcloning are listed in Supplementary Table 2. The following additional plasmids were obtained from Addgene: WT RhoA-GFP WT (12965), T19N RhoA-GFP (12967), Q63L RhoA-GFP (12968), WT RhoA-Myc (12962), T19N RhoA-Myc (12963), Q63L RhoA-Myc (12964), Cdc42-Myc (12972), RhoA2G-mTFP-mVenus (40176), mTFP-N1 (54521), mVenus-N1 (54640), and LifeAct-mCherry (67302). Expression plasmids for p63RhoGEF-mVenus and p63RhoGEF-Myc were a generous gift from Dr. Thomas Wieland (Universität Heidelberg). The expression plasmid for PACSIN1-Myc was a generous gift from Dr. Markus Plomann (University of Cologne).