Miniprep reagents

All bacterial strains used in this study are listed in Supplementary Table 1. Phusion DNA Polymerase was purchased from New England Biolabs (Ipswich, MA). Plasmid extractions were performed with QIAGEN (Valencia, CA) miniprep reagents. DNA, including oligonucleotide primers and short gBlocks, was synthesized by Integrated DNA Technologies (IDT), Inc. (San Diego, CA). Fatty acids and methyl ketone standards used in this study were purchased from Sigma-Aldrich (St. Louis, MO).
E. coli DH5α cells were used for cloning and assembling DNA molecules. Lysogeny broth (LB) was used to cultivate cells during the cloning and DNA assembly process. E. coli NHL17 (K12 MG1655 ΔaraBAD ΔfadD::trcCpFatB1*), E. coli ΔRABIJ (MG1655 ΔaraBAD ΔfadR ΔfadA ΔfadB ΔfadI ΔfadJ ) and E. coli TY34 (MG1655 ΔaraBAD ΔfadE::P_trc-BTE ΔfadAB::P_trc-BTE ΔackApta::P_trc-BTEΦ(P_trc-fadD)) were created as part of prior studies (Agnew et al., 2012 (link); Hernández Lozada et al., 2018 (link); Youngquist et al., 2013 (link)) and were used as base strains for production of 2-heptanone (NHL17), 2-nonanone (ΔRABIJ) and 2-undecanone (TY34). Pre-cultures were prepared in test tubes containing 5 mL LB and the appropriate antibiotics (final concentrations: carbenicillin, 100 μg/mL; chloramphenicol, 34 μg/mL; kanamycin 50 μg/mL).

Plasmid DNA was purified from 2 to 6 ml of E. coli DH5α or S. aureus RN4220 overnight cultures. For preparation from S. aureus cultures, cells were pelleted, re-suspended in 100 μl TSM buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 0.5 M sucrose) then treated with 5 μl lysostaphin (2 mg ml⁻¹) at 37 °C for 1 h before treatment with plasmid miniprep reagents from Qiagen. Purification used Qiagen or EconoSpin columns.