Identification and characterization of a novel class of self-sufficient cytochrome P450 hydroxylase involved in cyclohexanecarboxylate degradation in Paraburkholderia terrae strain KU-64

ABSTRACT Cytochrome P450 monooxygenases play important roles in metabolism. Here, we report the identification and biochemical characterization of P450CHC, a novel self-sufficient cytochrome P450, from cyclohexanecarboxylate-degrading Paraburkholderia terrae KU-64. P450CHC was found to comprise a [2Fe-2S] ferredoxin domain, NAD(P)H-dependent FAD-containing reductase domain, FCD domain, and cytochrome P450 domain (in that order from the N terminus). Reverse transcription–polymerase chain reaction results indicated that the P450CHC-encoding chcA gene was inducible by cyclohexanecarboxylate. chcA overexpression in Escherichia coli and recombinant protein purification enabled functional characterization of P450CHC as a catalytically self-sufficient cytochrome P450 that hydroxylates cyclohexanecarboxylate. Kinetic analysis indicated that P450CHC largely preferred NADH (Km = 0.011 m m) over NADPH (Km = 0.21 m m). The Kd, Km, and kcat values for cyclohexanecarboxylate were 0.083 m m, 0.084 m m, and 15.9 s−1, respectively. The genetic and biochemical analyses indicated that the physiological role of P450CHC is initial hydroxylation in the cyclohexanecarboxylate degradation pathway.

Transcriptomic analysis of Rhodococcus opacus R7 grown on polyethylene by RNA-seq

Plastic waste management has become a global issue. Polyethylene (PE) is the most abundant synthetic plastic worldwide, and one of the most resistant to biodegradation. Indeed, few bacteria can degrade polyethylene. In this paper, the transcriptomic analysis unveiled for the first time Rhodococcus opacus R7 complex genetic system based on diverse oxidoreductases for polyethylene biodegradation. The RNA-seq allowed uncovering genes putatively involved in the first step of oxidation. In-depth investigations through preliminary bioinformatic analyses and enzymatic assays on the supernatant of R7 grown in the presence of PE confirmed the activation of genes encoding laccase-like enzymes. Moreover, the transcriptomic data allowed identifying candidate genes for the further steps of short aliphatic chain oxidation including alkB gene encoding an alkane monooxygenase, cyp450 gene encoding cytochrome P450 hydroxylase, and genes encoding membrane transporters. The PE biodegradative system was also validated by FTIR analysis on R7 cells grown on polyethylene.

Synthesis of tobacco-derived cembratriene-ol and cembratriene-diol in yeast using engineered enzymes

Background: Cembranoids are one kind of diterpenoids with multiple biological activities, and the tobacco cembatriene-ol (CBT-ol) and cembatriene-diol (CBT-diol) have high anti-insect and anti-fungal activities, which is attracting great attention for their potential usage in sustainable agriculture. Cembranoids have been supposed to be formed through the 2-C-methyl-D-erythritol-4- phosphate (MEP) pathway, yet the involvement of mevalonate (MVA) pathway in their synthesis remains unclear. Exploring the roles of MVA pathway in cembranoid synthesis could contribute not only to the technical approach but also to the molecular mechanismfor cembranoid biosynthesis. Results: We constructed a vector to express an engineered protein fusion of cembranol synthase (CBTS1) and the GAL4 AD domain as a N-terminal translation leader. Eventually, the engineered enzyme AD-CTBS1 was successfully expressed, which further resulted in the production of CBT-ol in yeast with an optimized MVA pathway for geranyl-geranyl diphosphate (GGPP) production, but not in other yeast strains with low GGPP supply. Subsequently, CBT-diol was also synthesized by co-expression of engine-ered cembranol synthase (CBTS1) and cytochrome P450 hydroxylase (CYP450) in the yeast enhanced MVA pathway. Conclusions: We demonstrated that yeast could be applied to the production of tobacco-derived CBT-ol and CBT-diol, which are anti-fungal compounds. And, established a new way to produce the tobacco-derived CBT-ol and CBT-diol in yeast with optimized MVA pathway for GGPP production. Thus, this study established a feasibility for cembranoid production via the MVA pathway and provided an alternative bio-approach for the production of cembranoids in microbes.