BAHD acyltransferase cloning, expression, and purification

Candidate ASATs were cloned into pET28b(+) (MilliporeSigma, Burlington, MA, USA). Open reading frames from genes were either synthesized by Twist Biosciences (South San Francisco, CA, USA) with or without codon-optimization for E. coli expression (Supplementary Table S39) or amplified from genomic DNA or cDNA with primers listed in Supplementary Table S40. Q5 2X Hotstart master mix (New England Biolabs, Ipswich, MA, USA) was used for cloning PCRs. The source of cloned DNA and whether a gene was codon optimized is described in Supplementary Table S39. Amplified genes were purified by agarose gel electrophoresis and extraction with the Monarch DNA Gel Extraction Kit (New England Biolabs, Ipswich, MA, USA). Both the synthesized genes and PCR amplified genes were then inserted into a doubly digested BamHI/XhoI pET28b(+) through Gibson assembly using the 2X Gibson Assembly Master Mix (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's instructions. The constructs using synthesized genes were transformed into BL21(DE3) (MilliporeSigma, Burlington, MA, USA) and constructs with PCR amplified genes were transformed into BL21 Rosetta(DE3) cells (MilliporeSigma, Burlington, MA, USA). Constructs were verified with colony PCR and Sanger sequencing using T7 terminator and promoter primers (Supplementary Table S40). Sanger sequencing was completed by the Michigan State University Research Technology Support Facility Genomics Core (East Lansing, MI, USA).

Protein expression occurred as previously described (Lou et al. 2021; Leong et al. 2022). Briefly, 50 mL cultures of picked transformation colonies were grown overnight at 37 °C, shaking at 225 rpm in Luria–Bertani (LB) media (Neogen, Lansing, MI, USA) supplemented with 1% glucose (w/v). Fifteen milliliter of the overnight cultures were inoculated into 1 L of fresh LB medium, which was incubated at 37 °C shaking at 225 rpm until an OD600 of 0.5 was reached. The cultures were incubated on ice for 25 min, after which, isopropylthio-β-galactoside was added to a final concentration of 50 to 500 µm. Then, cultures were incubated at 16 °C shaking at 180 rpm overnight for 16 h before cells were harvested by centrifugation at 4,000 rpm for 10 min at 4 °C.

S. melongena BAHDs were purified as previously described (Leong et al. 2020) with the following modifications. The extraction buffer contained 10 mm imidazole, the wash buffer contained 20 mm imidazole, and the elution buffer contained 500 mm imidazole. Protein eluent was concentrated with 30-kD Amicon Ultra centrifugal filter units (MilliporeSigma).