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Experimental Procedures
Materials
All chemicals and antibiotics used in this study were obtained from Cayman, Sigma Aldrich or Sinopharm Chemical Reagent, unless otherwise specified. Fast-digest restriction endonucleases were purchased from Monad. I-5TM 2 × High-Fidelity DNA Polymerase and Trelief™ SoSoo Cloning Kit were obtained from TsingKe Biotech. Isolation of plasmid DNA from E. coli was performed using the MonPure™ Plasmid Mini Prep Kit from Monad. Purification of DNA fragments from PCR reactions or agarose gels was performed using a MonPure™ Gel & PCR Clean Kit from Monad. ClonExpress II One-Step Cloning Kit was purchased from Vazyme. Malachite Green Phosphate Assay Kit and the inorganic pyrophosphatase were obtained from Bioassay System and Sigma Aldrich, respectively. n-Alkanes (C10-C40) standard mixture was bought from O2Si. Ni-NTA SefinoseTM Resin (Settled Resin) for protein purification was purchased from Sangon Biotech. The FlexiRunTM premixed gel solution for SDS-PAGE, GelRed and Terrific broth (TB) medium were obtained from MDBio. Primer synthesis and DNA sequencing were performed by Tsingke.
Analytical methods
GC analysis was conducted using an Agilent HP-5 column (30 m × 0.32 mm × 0.25 μm) on an Agilent 7890B GC instrument with the following temperature program: 40 °C for 5 min, 40 to 300 °C at 10 °C/min, and 300 °C for 2 min. GC-MS analysis was performed using an Agilent DB-5 GC column (30 m × 0.25 mm × 0.25 μm) on an Agilent Quadruple GC-MS instrument with the temperature program as follow: 40 °C for 5 min, 40 to 300 °C at 10 °C/min, and 300 °C for 2 min. The high resolution mass spectra (HRMS) and nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Maxis UHR-TOF and a Bruker Avance III 600 MHz spectrometer, respectively. The X-ray single crystal diffraction was performed on a Bruker D8 Venture and the data was output by CIF editor (publCIF) from the IUCr.
Bioinformatic analysis
AntiSMASH analysis was carried out using the online tools available at https://antismash.secondarymetabolites.org/#!/start. Evolutionary analysis was conducted using MEGA 7.0 software. Protein sequence alignment was performed using ClustalW and the results were output by ESPript 3.0 (http://espript.ibcp.fr/ESPript/ESPript/). DNA sequence alignment was conducted using DNAman 7.0. The prediction of transmembrane helices was carried out with TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/).