Construction of pDiv and pRSFDuet-DivMT

Four DNA pieces containing portions of the div pathway with putative biosynthetic genes codon-optimized were synthesized by Integrated DNA Technologies (IDT) and pre-assembled into two larger pieces by overlapping PCR (Supplementary Table 3). Pieces 1a and 2 were joined via an initial reaction with 15 cycles of denaturing (98°C, 15 s), annealing (62°C, 30 s), and elongation (72°C, 1 min) using Phusion® DNA polymerase (New England BioLabs). This was followed by another 30 cycles of denaturing (98°C, 15 s), annealing (62°C, 15 s), and elongation (72°C, 2 min) with primers div1a-fwd and div2-rvs, using the first reaction directly as the template (Supplementary Table 4). Similarly, DNA pieces 3 and 1b were joined using primers div3-fwd and div1b-rev. The final products were gel purified using Ultrafree-DA Centrifugal Filters (EMD Millipore). The vector backbone was obtained by digestion of pPat with SacII and KpnI and gel purification of the resulting 4450 bp fragment.²³

The backbone was transformed into the uracil auxotroph Saccharomyces cerevisiae BY4741 along with the PCR-joined 1a-2 and 3-1b DNA pieces for assembly by yeast recombination. The resulting colonies, grown on uracil-deficient SD agar, were combined and grown overnight in liquid uracil-deficient SD. Plasmid DNA was purified from the overnight culture using a QIAprep Spin Miniprep kit (Qiagen) with protocol modifications to enhance cell lysis: the cells were resuspended in buffer P1 with 0.5 mm glass beads and mixed by vortex on high for 10 min. Yeast plasmid was transformed into DH10β E. coli and the resulting colonies screened for the presence of div genes by sequencing. Additional plasmid was generated from one of the div-positive clones and fully sequenced using primers 5 through 15, confirming the desired sequence of pDiv.

Expression of pDiv in E. coli did not yield any 1-like products, so modifications were made to the vector to induce production. We had previously observed expression of soluble, recombinant DivM only with the native gene sequence (data not shown), and reasoned that the codon-optimization of divM might have resulted in poor expression or incorrect protein folding, rendering the initial biosynthetic step catalyzed by DivM nonfunctional in pDiv.⁵⁴ The native sequence divM gene was cloned by PCR from E11-036 metagenomic DNA using primers intergenic-divM-native-fwd and intergenic-divM-native-rvs with Platinum® Taq DNA Polymerase High Fidelity (ThermoFisher Scientific) and 35 cycles of denaturation (94°C, 30 s), annealing (72°C, 30 s), and elongation (68°C, 3 min 30 s) to yield a faint band (Supplementary Figure 2). The product was diluted 100-fold and amplified by PCR with Phusion® with the same primers by 30 cycles of denaturation (98°C, 15 s), annealing (65/72°C gradient, 30 s), and elongation (72°C, 3 min 30 s) and purified using a QIAquick PCR Purification kit (Qiagen) (Supplementary Figure 2). pDiv was digested with MluI-HF and AflII to remove the majority of the codon-optimized divM gene and the resulting 8355 bp band was gel purified using a Qiaex II Gel Extraction kit (Qiagen). The native divM gene and linearized pDiv were assembled by yeast homologous recombination as previously described. Yeast lysis was further enhanced by incubation with buffer P2 at 55°C for 10 min after pulverization with glass beads by vortex. Plasmids were extracted from individual E. coli colonies transformed with yeast DNA and digested with SpeI and NdeI to confirm replacement of the codon-optimized divM with the native gene, then sequenced with primers 17–22 to verify the desired sequence of divM in pDiv-2.

The pDiv-3 expression vector for 2 was constructed in two separate phases, with replacement of divA in pDiv with a divA sequence encoding 2, followed by replacement of codon-optimized divM with the native divM gene. First, pDiv was digested with ClaI and NheI restriction enzymes. The product was then gel purified using a centrifugal filter as described previously. The linearized vector was recombined with synthetic DNA divA-divamideB (IDT; Supplementary Table 3) using yeast as described above. Plasmids obtained from pDiv-containing E. coli colonies transformed with extracted yeast DNA were sequenced with primer 1, confirming the altered sequence of divA with a single noncoding C to G mutation, the position of which is underlined in the sequence shown in Supplementary Table 3. The native divM gene was then introduced as described for the construction of pDiv-2, using the divamide B-encoding pDiv as a template for recombination. During the restriction digest of pDiv-2, XhoI was used in addition to AflII and MluI-HF to cut the codon-optimized divM gene and prevent reinsertion.

To construct the methyltransferase expression plasmid pRSFDuet-DivMT, the codon-optimized divMT gene was PCR amplified using pDiv as a template with primers divMT-MCS1-fwd and divMT-MCS1-rvs, Platinum® Taq Hi-Fi, and 2% DMSO by 30 cycles of denaturation (94°C, 30 s), annealing (45.5°C, 30 s), and elongation (68°C, 2 min). The PCR product was purified as described above. Both purified PCR product and the empty pRSFDuet vector were then digested with SacI and NotI restriction enzymes. Reaction mixtures were heat inactivated and the linearized pRSFDuet was dephosphorylated using Antarctic Phosphatase (New England BioLabs). The two pieces were then ligated together using T4 DNA ligase (New England BioLabs). The ligation reaction was transformed into DH10β E. coli and the transformed cells plated on LB kanamycin selection media. Individual colonies were picked and grown overnight in liquid culture. Cells were harvested and plasmids purified using the QIAprep Spin Miniprep kit. Plasmids were triple digested with ScaI, MluI, and PstI to verify the expected band sizes, and positive plasmids were sequenced for confirmation.