Rapid combinatorial rewiring of metabolic networks for enhanced poly(3- hydroxybutyrate) production in Corynebacterium glutamicum

Abstract Background: The disposal of plastic waste is a major environmental challenge. With recent advances in microbial genetic and metabolic engineering technologies, microbial polyhydroxyalkanoates (PHAs) are being used as next-generation biomaterials to replace petroleum-based synthetic plastics in a sustainable future. However, the relatively high production cost of bioprocesses hinders the production and application of microbial PHAs on an industrial scale. Results: Here, we describe a rapid strategy to rewire metabolic networks in an industrial microorganism, Corynebacterium glutamicum, for the enhanced production of poly(3-hydroxybutyrate) (PHB). A three-gene PHB biosynthetic pathway in Rasltonia eutropha was refactored for high-level gene expression. A fluorescence-based quantification assay for cellular PHB content using BODIPY was devised for the rapid fluorescence-activated cell sorting (FACS)-based screening of a large combinatorial metabolic network library constructed in C. glutamicum. Rewiring metabolic networks across the central carbon metabolism enabled highly efficient production of PHB up to 29% of dry cell weight, which is the highest PHB content ever reported in C. glutamicumusing a sole carbon source. Conclusions: We successfully constructed a heterologous PHB biosynthetic pathway and rapidly optimized metabolic networks across central metabolism in C. glutamicum for enhanced production of PHB using glucose or fructose as the sole carbon source in minimal medium. We expect that this FACS-based metabolic rewiring framework will accelerate strain engineering processes for the production of diverse biochemicals and biopolymers.