Metabolic engineering of Rhodococcus opacus for the development of biorefinery platforms using waste plastics

Abstract

Increased demand for petroleum-based chemicals promotes industrial pollution while simultaneously accelerating global climate change. Furthermore, waste plastics have become a significant issue for both humans and the environment, mandating their recycling and reuse. As a result, green alternatives to fossil fuel-based chemicals should be developed while addressing the plastic issues. In this thesis, I constructed the bio-refinery platforms using waste plastics by engineering Rhodococcus opacus PD630. First, systems metabolic engineering was performed for high-level production of FFAs, fundamental precursors for numerous commodity chemicals. Free fatty acids (FFAs) are essential products for food, industrial, and energy applications. Due to the increasing demand for biodiesel, there has been much interest in producing FFAs from renewable non-edible biomass such as lignocellulosic biomass instead of plant oils such as palm oil, which causes food vs. fuel issues. Here, a strategy of combining fermentation optimization and strain development was developed to enhance the production of FFAs. Metabolic engineering together with peroxidase-based membrane engineering resulted in enhanced production of FFAs. Fermentation optimization after glucose adaptation of the resulting engineered strain allowed the production of 218.14 g/L of FFAs in 84 h. Moreover, further engineering allowed the production of FFA derivatives such as long-chain hydrocarbons (LCHCs) and medium-chain-length polyhydroxyalkanoates (MCL-PHAs). The synergistic effect of artificial lipid peroxidation and fatty acid decarboxylation with alkB-deleted super-FFA producer allowed the production of 31 g/L of LCHCs in 91 h. Also, vinyl polymer copolymerized MCL-PHAs could be newly achieved without monomer supplement by employing PHA synthesizing genes in the super-FFA producer. Finally, the capability of the super-FFA producers as the integrated microbial plastic recycling platform for polypropylene, polyethylene and polyethylene terephthalate was confirmed. The FFA overproducing system reported here will be useful for the high-level one-step fermentative production of FFAs and their derivatives from the most abundant renewable lignocellulosic biomass and the most abundant waste, plastics.