Multi-layered metabolic remodeling of Pseudomonas putida for efficient conversion of lignocellulosic sugars to the precursors of advanced aviation fuel

Isoprenyl acetate, a volatile ester derived from isoprenol, is a key biosynthetic intermediate for the advanced aviation fuel candidate, 1,4-dimethylcyclooctane. Here, we engineered Pseudomonas putida KT2440 for the production of isoprenyl acetate from mixed sugar substrates. We first generated isoprenyl acetate by introducing a heterologous alcohol acetyltransferase (ATF1) and deleting three promiscuous native esterases to reduce product degradation. Then, we engineered efficient glucose and xylose co-utilization by integrating a heterologous xylose isomerase pathway and deleting global regulators crc and hexR to alleviate catabolite repression. Additionally, intracellular acetyl-CoA flux was reinforced through the expression of auxiliary carbon-conserving routes, including non-oxidative glycolysis and acetate assimilation. Culture conditions were systematically optimized by adjusting medium composition, induction, and overlay solvent to maximize product yields and titers. These cumulative efforts achieved isoprenyl acetate titers of 1.5 g/L in shake flasks and 1.9 g/L in fed-batch bioreactor cultures from mixed sugars, corresponding to a yield of 0.067 g/g of total sugar consumed. Our work demonstrates the potential of P. putida as a robust microbial chassis for scalable biosynthesis of ester-based biofuels from lignocellulosic feedstocks. • Isoprenyl acetate biosynthesis is demonstrated in Pseudomonas putida • Product-degrading endogenous esterases are identified and deleted in P. putida • Co-utilization of glucose and xylose is engineered by relieving catabolite repression • Co-expression of Acs and Xfpk enhances acetyl-CoA flux and boosts ester yields • Fed-batch cultivation led to 1.9 g/L isoprenyl acetate from lignocellulosic sugar mixture