Systems Biology Guided Synthetic Design of Lignin-to-Bioplastics Production

Pseudomonas putida are of interest in value-added chemicals production from renewable biological polymer, due to their diverse spectrum of carbon catabolic metabolism and great potential in biosynthesis of biodegradable plastics (polyhydroxyalkanoate, PHA). In this study, we firstly isolated and characterized fifteen P. putida strains exhibiting diverse spectrums of carbon catabolic metabolism from various environments. From these fifteen strains, P. putida A514 was employed as the research model, for its ability to utilize a wide range of carbon sources, including hexose, pentose, aromatic compounds and lignin. In order to unravel the potential catabolic pathways for various lignin-derived aromatic compounds, whole genome of P. putida A514 was sequenced via the PacBio sequencing platform. Moreover, the global proteome profiles at late-log phase were measured via shot-gun proteomics approaches, when A514 was cultured in glucose, vanillic acid and lignin as sole carbon sources. Approximately 250 to 500 uniquely expressed proteins were identified for each state, indicating different metabolism pathways among these three substrates. Furthermore, different expression levels of enzymes involved in β-ketoadipate pathway, ferulate catabolic pathway, TCA cycle, aromatic amino acid metabolism and β-oxidation were detected. In addition, putative aromatic compounds specific transporters were also identified. Based on the systems biology analysis, the functional modules for lignin-to-PHA production are designed and implemented. The synthetic strains with aromatics catabolism module showed significantly improved growth rate and yield on aromatic compounds and lignin substrates. The synthetic strains with PHA biosynthesis module reached over 70% PHA yield on aromatic compound media. The further integration of lignin depolymerization module will enable the high yield of PHA produced from lignin. Considering the relative higher energy density of aromatic compounds, our finding paved the path for converting aromatic waste or lignin to a valuable product, which could have improve the economics and sustainability of paper pulping, biofuel and other industries.