Modular polyketide synthases (PKSs) have been proposed as promising megasynthases for retrobiosynthesis because of their fitness for rational carbon skeleton design. However, over the last three decades, all engineered PKSs produce carboxylic acids via terminal thioesterase (TE), which significantly narrows available chemical scope. We proposed the possibility of expanding PKS chemical diversity via terminal thioreductase (TR) engineering, and comprehensively characterised PKS TRs as NADPH-dependent enzymes to terminate polyketides with aldehyde. These aldehyde products can be readily converted to industrially valuable alcohols and amines, demonstrated by an engineered rimocidin PKS-TR producing 1,3-butanediol, 1,3-pentanediol, and 1,3-hexanediol with a total titer of 1008 mg/L in Streptomyces albus, or producing 554 mg/L 1-amino-3-alcohols via post-PKS transamination, both in shake flasks. Furthermore, efficient control of the product profile was achieved by coenzyme A (CoA) substrate regulation, as elevating butyryl-CoA level resulted in 1,3-hexanediol product ratio increased from 11% to 77%. We also demonstrated that PKS-TR can produce biosynthetically challenging branched-chain diols, culminating in production of 166 mg/L branched-chain 2-methyl-1,3-diols via acyltransferase exchange.

Proteins were resuspended with 100 mM ammonium bicarbonate, reduced in 5 mM tris 2-(carboxyethyl)phosphine (TCEP) and alkylated in 10 mM iodoacetamide (IAM) before subjecting to trypsin digestion. The resulting peptides were analyzed using an Agilent 1290 Infinity liquid chromatography system coupled to an Agilent 6460 QQQ mass spectrometer (Agilent Technologies, Santa Clara, CA). Peptides (~10 μg) were separated on an Ascentis Express Peptide ES-C18 column (2.7 μm particle size, 160 Å pore size, 50 x 2.1mm) fitted with a guard column (5 mm x 2.1 mm, Sigma Aldrich). The column was heated to 60°C. The mobile phase consisted of 0.1% formic acid in H2O (A) and 0.1% formic acid in acetonitrile (B). Peptides were eluted from the column by using a 3.5 minute linear gradient from 95% solvent A and 2% solvent B to 60% solvent A and 40% solvent B. Peptides were ionized using an Agilent Jet Stream ESI source operating in positive-ion mode with the following source parameters: Gas Temperature = 250°C, Gas Flow = 13 L/min, Nebulizer Pressure = 35 psi, Sheath Gas Temperature = 250°C, Sheath Gas Flow = 11 L/min, and Capillary Voltage = 3,500 V. Dwell times were set to 18ms. Data was acquired using Agilent MassHunter Data Acquisition (Version B.08.02). The MRM method for quantifying phosphopantetheine bearing peptides was built using the Skyline (version 21.2). LC-MS raw data were imported and analysed in Skyline.

PimS0 was added in 100 μL reaction mix (10% v/v glycerol, 50 mM NaCl, 20 mM HEPES pH 7.5, 5 mM ATP, 1 mM acetate + CoA/malonate + CoA/acetyl-CoA/malonyl-CoA) in 7.5 mg/mL final concentration to initiate the substrate loading reaction. At 1 h , 4 h, and 24 h, 20 uL reaction solution was quenched with 1% formic acid, flash cooled in liquid N2, and stored at -80 °C for subsequent treatment. PimS0 with no tested substrate added in the reaction mix was taken as a negative control. Proteins in the reaction samples were precipitated by addition of 1 mM NaCl and 4 x vol acetone, followed by two additional washes with 80% acetone in water