Biological production of aviation fuels has a realistic impact on global warming. Important sustainable aviation fuel (SAF) targets are emerging and isoprenol is recently identified as a precursor for a promising SAF compound DMCO (1,4-dimethylcyclooctane). Isoprenol has been produced in several engineered microorganisms, and recently, Pseudomonas putida has gained interest as a promising host for isoprenol bioproduction as it can utilize carbon sources generated from inexpensive plant biomass. Here, we engineer metabolically versatile host P. putida for isoprenol production. We employ two computational modeling approaches (Bilevel optimization and Constrained Minimal Cut Sets) to predict gene knockout targets and optimize the “IPP-bypass” pathway in P. putida to maximize isoprenol production. Altogether, the highest isoprenol production titer from P. putida was achieved at 3.5 g/L under fed-batch conditions. This combination of computational and biological engineering on P. putida for an advanced biofuels production has vital significance in enabling a bioproduction process that can use renewable carbon streams.

Targeted proteomics analysis was performed on an Agilent 1290 UHPLC system coupled to an Agilent 6460 QqQ mass spectrometer according to an established protocol (dx.doi.org/10.17504/protocols.io.bf9xjr7n). Briefly, 20 g Peptides of each sample were separated on an Ascentis Express Peptide C18 column [2.7-mm particle size, 160-Å pore size, 5-cm length × 2.1-mm inside diameter (ID), coupled to a 5-mm × 2.1-mm ID guard column with the same particle and pore size, operating at 60°C; Sigma-Aldrich] operating at a flow rate of 0.4 ml/min via the following gradient: initial conditions were 98% solvent A (0.1% formic acid), 2% solvent B (99.9% acetonitrile, 0.1% formic acid). Solvent B was increased to 5% over 1 min, and was then increased to 40% over 3.5 min. It was increased to 80% over 0.5 min and held for 2.5 min at a flow rate of 0.6 mL/min, followed by a linear ramp back down to 2% B at a flow rate of 0.4 mL/min over 0.5 min where it was held for 1 min to re-equilibrate the column to original conditions. The eluted peptides were ionized via an Agilent Jet Stream ESI source operating in positive ion mode. The MS raw data were acquired using Agilent MassHunter version B.08.02, and were analyzed by Skyline software version 21.20 (MacCoss Lab Software).