PCR reagents, plasmid purification, restriction enzymes, and T4 DNA ligase were used as recommended by New England Biolabs (NEB) unless otherwise noted. Vectors used in this study included the bacterial expression vectors pCWori (43 (link)) and pET28a (Novagen). E. coli cell strains used in this study included Top10 (Invitrogen), DH5α, and BL21(DE3) (NEB). All reagents were used as received from commercial sources. Loganin (Cayman Chemical) and loganic acid (Arctom Chemicals) were 98+% pure.
LC-MS analyses of the Camptotheca SLAS mutants were conducted using a Shimadzu LC-MS2010EV system at the Institute for Genomic Biology, University of Illinois Urbana-Champaign. High resolution LC-MS analyses of the WT Camptotheca SLASs and the SLS, SLAS common ancestor were performed by Dr Alexander Ulanov in the Metabolomics Laboratory of the Roy. J. Carver Biotechnology Center at the University of Illinois Urbana-Champaign using a Dionex Ultimate 3000 series HPLC system (Thermo Scientific) with Q-Exactive MS system (Thermo Scientific). Spectroscopic measurements were recorded with a Cary UV-Vis Bio100 dual beam spectrophotometer or Molecular Devices SpectraMax M-series plate reader as appropriate.
LC-MS analyses of the Camptotheca SLAS mutants were conducted using a Shimadzu LC-MS2010EV system at the Institute for Genomic Biology, University of Illinois Urbana-Champaign. High resolution LC-MS analyses of the WT Camptotheca SLASs and the SLS, SLAS common ancestor were performed by Dr Alexander Ulanov in the Metabolomics Laboratory of the Roy. J. Carver Biotechnology Center at the University of Illinois Urbana-Champaign using a Dionex Ultimate 3000 series HPLC system (Thermo Scientific) with Q-Exactive MS system (Thermo Scientific). Spectroscopic measurements were recorded with a Cary UV-Vis Bio100 dual beam spectrophotometer or Molecular Devices SpectraMax M-series plate reader as appropriate.