Metabolic Engineering of Methanotrophic Bacteria for De Novo Production of Taxadiene from Methane

The growing demand for natural products in pharmaceutical applications has prompted a focus on more sustainable methods to produce high-value terpenoids using microbial cell factories. Due to the characteristics of high abundance, renewable, and low cost, methane has emerged as a promising feedstock for biomanufacturing. In this study, the methanotrophic bacterium Methylotuvimicrobium buryatenese 5GB1C, known for its industrial potential, was metabolically engineered to synthesize taxadiene, a crucial precursor in paclitaxel production. The biosynthesis of taxadiene from methane was first established by employing an endogenous strong promoter to enhance the expression of heterologous taxadiene synthase in M. buryatenese 5GB1C. To further optimize the metabolic flux, rate-limiting enzymes (Dxs and IspA) were upregulated in the methylerythritol phosphate pathway while complementing the native pathway with the essential idi gene that was originally deficient. Coupled with a 2.1-fold improvement in the NADPH/NADP⁺ ratio, these modifications collectively boosted taxadiene production from 2.58 to 22.97 mg/L in serum vials. Ultimately, a temperature-controlled two-stage cultivation was implemented in 3 L bioreactors, which achieved a remarkable titer of 104.88 mg/L, representing the highest reported titer for diterpenoid biosynthesis from methane. This work demonstrates the potential of utilizing methane for the sustainable production of advanced terpenoids with reduced environmental impact.