Peng Shuaiying,Chu Zhongmei,Lu Jianfeng,Li Dongxiao,Wang Yonghong,Yang Shengli,Zhang Yi
The self-sufficient cytochrome P450 BM3 mutant (A74G/F87V/D168H/L188Q) can serve as a biocatalyst for whole-cell catalysis process of indigo. Nevertheless, the bioconversion yield of indigo is generally low under normal cultivation conditions (37 °C, 250 rpm). In this study, a recombinant E. coli BL21(DE3) strain was constructed to co-express the P450 BM3 mutant gene and GroEL/ES genes to investigate whether GroEL/ES can promote the indigo bioconversion yield in E. coli . The results revealed that the GroEL/ES system could significantly increase the indigo bioconversion yield, and the indigo bioconversion yield of the strain co-expressing P450 BM3 mutant and GroEL/ES was about 21-fold that of the strain only expressing the P450 BM3 mutant. In addition, the P450 BM3 enzyme content and in vitro indigo bioconversion yield were determined to explore the underlying mechanism for the improvement of indigo bioconversion yield. The results revealed that GroEL/ES did not increase indigo bioconversion yield by increasing the content of P450 BM3 enzyme and its enzymatic transformation efficiency. Moreover, GroEL/ES could improve the intracellular nicotinamide adenine dinucleotide phosphate (NADPH)/NADP + ratio. Given that NADPH is an important coenzyme in the catalytic process of indigo, the underlying mechanism for the improvement of indigo bioconversion yield is probably related to an increase in the intracellular NADPH/NADP + ratio.
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.
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