Carnosic acid biosynthesis elucidated by a synthetic biology platform

Abstract

Synthetic biology approaches achieving the reconstruction of specific plant natural product biosynthetic pathways in dedicated microbial "chassis" have provided access to important industrial compounds (e.g., artemisinin, resveratrol, vanillin). However, the potential of such production systems to facilitate elucidation of plant biosynthetic pathways has been underexplored. Here we report on the application of a modular terpene production platform in the characterization of the biosynthetic pathway leading to the potent antioxidant carnosic acid and related diterpenes in Salvia pomifera and Rosmarinus officinalis.Four cytochrome P450 enzymes are identified (CYP76AH24, CYP71BE52, CYP76AK6, and CYP76AK8), the combined activities of which account for all of the oxidation events leading to the biosynthesis of the major diterpenes produced in these plants. This approach develops yeast as an efficient tool to harness the biotechnological potential of the numerous sequencing datasets that are increasingly becoming available through transcriptomic or genomic studies.

Keywords: cytochrome P450; metabolic engineering; terpene; yeast.

Fig. 1.

Chemical structures and biosynthesis of labdane-type diterpenes. (A) Chemical structures of the bioactive labdane-related diterpenes carnosic acid (1), cryptotanshinone (2), tanshinone IIA (3), tanshinone I (4), and forskolin (5) (Top). The main diterpenes isolated from S. pomifera leaves include salviol (6), pisiferic acid (7), carnosol (8), 12-methoxy-carnosic acid (9), O-methyl-pisiferic acid (10), and 2α-hydroxy-O-methyl-pisiferic acid (11) (Bottom). (B) The biosynthesis of tanshinone and carnosic acid begins with the cyclization of GGPP (12) by a class II diTPS to produce (+) copalyl diphosphate (CPP) (13), which is in turn converted to miltiradiene (14) by a class I diTPS. Spontaneous oxidation of 14 gives rise to abietatriene (15), which is oxidized to ferruginol (16) by a CYP enzyme. However, uncharacterized subsequent events lead to carnosic acid (1) and tanshinones (2–4).

Fig. 2.

Characterization of S. pomifera CYP76AH24 ortholog as a bifunctional monooxygenase. (A) Diagrammatic representation of the modular design and the platform configuration used. (a) General design of the modular terpene production platform. (b–e) Diagrammatic illustratrations of the steps taken to engineer the platform to facilitate the rapid functional characterization of CYP enzymes. (B) Expression of the CYP76AH24 ortholog in optimized miltiradiene-producing yeast cells (A, b–d) resulted in the production of 16, as main compound, and the formation of 17 and 18 minor products. The two peaks indicated by asterisks correspond to degradation products of 17. In vitro enzymatic assay using a microsomal preparation of yeast cells expressing CPR2 and the S. pomifera CYP76AH24 ortholog (pink) or R. officinalis ferruginol synthase, CYP76AH4 (teal) confirmed the hydroxylation of 16 at C-11. Microsomal preparations of cells expressing only CPR2 are used as negative control (black).

Fig. 3.

CYP71BE52 is a salviol synthase. Expression of S. pomifera CYP71BE52 (pink) as an M3b-specific part in the ferruginol-producing yeast platform resulted in the production of 6, as identified by GC-MS analysis of the TMS-derivatized solvent extract (Top). A microsomal preparation of CYP71BE52 converted 16 to 6 in the presence of NADPH as cofactor (Middle). Formation of 6 was confirmed by comparison with authentic standard (Bottom).

Fig. 4.

CYP76AK6 and its homolog, CYP76AK8, catalyze successive oxidation events at C-20. Formation of 1 by CYP76AK6 (pink) and CYP76AK8 (teal) in yeast cells coexpressing CYP76AH24 and SpMilS. TMS-derivatized extracts of yeast cultures analyzed by GC-MS revealed the formation of 1 (Top), identified by comparison with authentic standard (blue) (Bottom). Enzymatic assays containing microsomal preparations of CYP76AH24 ortholog and CYP76AK6 (pink) or CYP76AK8 (teal), 16 and NADPH revealed the formation of 1 in vitro (Middle).

Fig. 5.

Proposed mechanism for the biosynthesis of carnosic-acid–related diterpenes in S. pomifera and R. officinalis. The biosynthesis of labdane-type diterpenes in S. pomifera (black) and R. officinalis (red) is initiated by the action of bifunctional enzymes, CYP76AH24 or CYP76AH4, respectively, which are responsible for the hydroxylation of 15 initially at position C-12 to produce 16 and subsequently at position C-11 to yield 17. The same bifunctional enzymes can also catalyze two successive oxidation events on 14 to yield 18. A S. pomifera enzyme, CYP71BE52, catalyzes oxidation of 16 at position 2α to synthesize 6. CYP76AK6 or CYP76AK8 catalyze successive oxygenations at position C-20 of 16 and 17 to yield 7 via 19, and 1, respectively. In the yeast platform, 1 is further oxidized to 8 by a yet undefined mechanism. The combined activities of CYP76AH24, CYP71BE52, and CYP76AK6 on the abietatriene skeleton (Inset) are sufficient to explain the biosynthesis of the main diterpenes (1, 6-11) isolated from S. pomifera and R. officinalis.