Metabolic Engineering of Methanotrophic Bacteria for <i>De Novo</i> 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 <i>Methylotuvimicrobium buryatenese</i> 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 <i>M. buryatenese</i> 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 <i>idi</i> 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.