Synthetic bacterial cell factory for highly efficient protein secretion and consolidated lignin bioconversion to lipid

Lignin is the second most abundant biopolymer on earth, yet the utilization of lignin for fungible fuels and chemicals has become a bottleneck for biorefining. Some bacteria show high capacity in of aromatic compounds catabolism, like Rhodococcus opacus PD630. However, their lignin bioconversion efficiency was significantly hindered by their low lignin depolymerization capacity, where the bacteria lack efficient extracellular secreted lignin-degrading enzymes. Despite extensive research, secretory production of heterologous protein in bacteria remains highly challenging. The challenge is particularly true for the lignin degradation enzymes with high-redox potential like laccase. We hereby demonstrated that proteomics-guided engineering could enable efficient heterologous secretion with a total protein yield at 13.7g/L by balancing the processes among transcription, translation, secretion, and protein folding of ligninolytic laccase. The engineered secretory laccase in R. opacus PD630 well complemented its biochemical limits on lignin depolymerization. Further proteomics analysis revealed the key factor of efficient lipid biosynthesis for the R. opacus PD630, where a distinct multi-unit fatty acid synthase I drove the carbon partition to storage lipid. The discovery guided the design of efficient lipid conversion from lignin and carbohydrate. The integration of laccase-secretion based lignin depolymerization module and enhanced FASI lipid biosynthese module enabled a high titer (2.54 g/L) in converting lignin-enriched biorefinery waste to lipid. The fundamental mechanisms, engineering components, and design principle could empower transformative platforms for biomanufacturing and biorefining.