2-03: Biodesign of Rhodococci for Lignin Fuel: A Path from Systems to Synthetic Biology

We are employing the systems biology-guided biodesign to address one of the most challenging and imminent tasks in lignocellulosic biofuel production: the utilization of lignin for fungible fuels. Systems biology and synthetic design was carried out in Rhodococcus opacus PD630 to exploit and enhance the aromatic compound utilization and lipid production capacity toward converting lignin into lipid. Proteomics and molecular network analyses helped to reveal several key operons for aromatic compound catabolism and important genes regulating biosynthesis. In addition, the secretome proteomics and systems modeling has enabled the biodesign of three expression systems for secretive expression, constitute over-expression and inducible over-expression, respectively. These sophisticated expression systems allow us to integrate the components discovered in systems biology analysis into developing three functional modules toward biodesign of lignin-to-lipid conversion. First, we engineered functional modules in R. opacus PD630 by over-expressing catABC operons to significantly enhance the aromatic compound catabolism.  Second, we engineered strains with significantly increased lipid production on both sugar and aromatic compound carbon source by over-expressing the key lipid biosynthesis genes. Third, we are designing and optimizing lignin depolymerization functional modules with enzymes from white rot fungus and termite using the secretive enzyme expression system. Three functional modules are being integrated to achieve a complete route of lignin-to-lipid conversion. Overall, our initial analysis indicated that systems biology modeling can effectively guide the biodesign of building blocks for synthetic biology in rhodococci. Moreover, the functional modules have already enhanced aromatic compound utilization and lipid biosynthesis toward a complete ligni-to-lipid route.