The Ethylmalonyl-Coa Pathway For Methane-Based Biorefineries: A Case Study Of Using Methylosinus Trichosporium Ob3b, An Alpha-Proteobacterial Methanotroph, For Producing 2-Hydroxyisobutyric Acid And 1,3-Butanediol From Methane

The ethylmalonyl-CoA pathway is one of three known anaplerotic pathways that replenish tricarboxylic acid cycle intermediates and plays a major role in the carbon metabolism of many alpha-proteobacteria including Methylosinus trichosporium OB3b. Despite being less popular than the ribulose monophosphate pathway from gamma-proteobacterial methanotrophs, theoretically, a functional EMC pathway coupled with the serine cycle can achieve higher carbon usage efficiency, wherein one CO2 and one HCO3- are re-assimilated into biomass. Therefore, it is an attractive pathway for biomass-derived products such as biopolymers. However, efforts to harness the ethylmalonyl-CoA pathway in alpha-proteobacterial methanotrophs for biosynthesizing chemical compounds have been limited. As groundwork for future studies, in this study, various exploratory tools were employed to evaluate the regulatory nodes of this pathway and identify potential ethylmalonyl-CoA pathway-derived products in Methylosinus trichosporium OB3b. Based on transcriptomic analysis coupled with genome-scale metabolic model prediction, the production of 2-hydroxyisobutyric acid and 1,3-butanediol from methane was chosen and demonstrated. These two compounds are derived from the dynamic flux branching node of the ethylmalonyl-CoA pathway and poly(3-hydroxybutyrate) pathways. Furthermore, starting from acetyl-CoA there is no carbon loss. By knocking out phaC that encodes polyhydroxybutyrate synthase, and inducing the PHB overflow mechanism, 2HIBA and 1,3-BDO titers were improved to 29.99 +/- 1.827 mg L-1 and 60.51 +/- 3.805 mg L-1, respectively. The current work demonstrated the first proof of concept for the production of EMC-derived products from methane.