Prokaryotic expression and characterization of artificial self-sufficient CYP120A monooxygenases.

Cytochrome P450 monooxygenases CYP120As are the unique non-membrane P450s, which are extensively involved in retinoid biodegradation. As the O-functionalized 1,3,3-trimethylcyclohex-1-ene moiety exists in many bioactive compounds which could only be catalyzed by Class II P450s, exploration of the catalytic repertoire of CYP120As is therefore highly attractive. However, up to date, only one bacteriogenic candidate (CYP120A1) was demonstrated for the hydroxylation of C16 and C17 of retinoic acid, by utilizing the integral membrane protein cytochrome P450 reductase redox partner for the electron transfer. Herein, we provided an efficient prokaryotic functional expression system of CYP120As in E. coli by expression of the CYP120A1 coupled with several reductase partners. Fusion redox partners to the C-terminal of the heme-domain are also working on other CYP120A members. Among them, the fusion protein of CYP120A29 and FAD/FMN reductase from Bacillus megaterium P450BM3 (CYP101A2) showed the highest expression level. Based on the available translational fusion systems, the regioselectivity and the substrate scope of the CYP120As have also been explored. This work represents a good starting point for further expanding the catalytic potential of CYP120 family. KEY POINTS: • Characterization of CYP120As in E. coli is firstly achieved by constructing fusion proteins. • The feasibility of three P450 reductase domains to CYP120As was evaluated. • Hydroxylated products of retinoic acid by six CYP120As were sorted and analyzed.

Mining methods and typical structural mechanisms of terpene cyclases.

Terpenoids, formed by cyclization and/or permutation of isoprenes, are the most diverse and abundant class of natural products with a broad range of significant functions. One family of the critical enzymes involved in terpenoid biosynthesis is terpene cyclases (TCs), also known as terpene synthases (TSs), which are responsible for forming the ring structure as a backbone of functionally diverse terpenoids. With the recent advances in biotechnology, the researches on terpene cyclases have gradually shifted from the genomic mining of novel enzyme resources to the analysis of their structures and mechanisms. In this review, we summarize both the new methods for genomic mining and the structural mechanisms of some typical terpene cyclases, which are helpful for the discovery, engineering and application of more and new TCs.