Engineering Pseudomonas putida for lignin bioconversion into cis-cis muconic acid

Bioconversion of lignin into valuable chemicals could promote both lignin valorization and biomass-based economy. However, the heterogeneity of lignin and the lack of an effective conversion route limit the efficiency of lignin utilization. In this study, the cell factory of Pseudomonas putida KT2440 was successfully designed for converting three types of lignin into cis, cis-muconic acid (CCMA). Heterologous engineering of the native O-demethylation system enabled the metabolism of S-type lignin by P. putida KT2440. The biosynthesis pathway of CCMA was designed to simultaneously funnel three types of lignin-derived aromatics, achieving a remarkable titer and yield of 13.1 mM and 99.5 %, respectively. The metabolic regulation of NADPH/NADP+ co-factor and PobA dependent co-factor further improved the bioconversion of lignin-derived aromatics and CCMA production. Implementing a co-feeding strategy substantially increased NADPH availability, resulting in enhanced CCMA production of up to 46.5 mM with a molar yield of 96.9 %. The molecular mechanism analysis demonstrated that the designed cell factory exhibited superior ligninolytic capacity in cleaving β-O-4 linkage and potentially degrading S- and G-type lignin. It showcased outstanding conversion capacity of actual lignin hydrolysates by consuming all aromatic compounds and more hydrophilic lignin molecules, producing 9.6 mM CCMA in a fed-batch fermentation mode. Therefore, the P. putida KT2440 cell factory is a promising bacterial chassis for both biological lignin valorization and production of valuable chemicals.