Production of the forskolin precursor 11β-hydroxy-manoyl oxide in yeast using surrogate enzymatic activities

Abstract

Background: Several plant diterpenes have important biological properties. Among them, forskolin is a complex labdane-type diterpene whose biological activity stems from its ability to activate adenylyl cyclase and to elevate intracellular cAMP levels. As such, it is used in the control of blood pressure, in the protection from congestive heart failure, and in weight-loss supplements. Chemical synthesis of forskolin is challenging, and production of forskolin in engineered microbes could provide a sustainable source. To this end, we set out to establish a platform for the production of forskolin and related epoxy-labdanes in yeast.

Results: Since the forskolin biosynthetic pathway has only been partially elucidated, and enzymes involved in terpene biosynthesis frequently exhibit relaxed substrate specificity, we explored the possibility of reconstructing missing steps of this pathway employing surrogate enzymes. Using CYP76AH24, a Salvia pomifera cytochrome P450 responsible for the oxidation of C-12 and C-11 of the abietane skeleton en route to carnosic acid, we were able to produce the forskolin precursor 11β-hydroxy-manoyl oxide in yeast. To improve 11β-hydroxy-manoyl oxide production, we undertook a chassis engineering effort involving the combination of three heterozygous yeast gene deletions (mct1/MCT1, whi2/WHI2, gdh1/GDH1) and obtained a 9.5-fold increase in 11β-hydroxy-manoyl oxide titers, reaching 21.2 mg L(-1).

Conclusions: In this study, we identify a surrogate enzyme for the specific and efficient hydroxylation of manoyl oxide at position C-11β and establish a platform that will facilitate the synthesis of a broad range of tricyclic (8,13)-epoxy-labdanes in yeast. This platform forms a basis for the heterologous production of forskolin and will facilitate the elucidation of subsequent steps of forskolin biosynthesis. In addition, this study highlights the usefulness of using surrogate enzymes for the production of intermediates of complex biosynthetic pathways. The combination of heterozygous deletions and the improved yeast strain reported here will provide a useful tool for the production of numerous other isoprenoids.

Fig. 1

Schematic representation of isoprenoid biosynthesis via the mevalonic acid pathway. The mevalonate pathway provides the substrates for the different terpene classes [dimethylallyl diphosphate (DMAPP) for hemiterpenes (C₅), geranyl diphosphate (GPP) for monoterpenes (C₁₀), farnesyl diphosphate (FPP) for sterols, sesquiterpenes (C₁₅) and triterpenes (C₃₀), geranylgeranyl diphosphate (GGPP) for diterpenes (C₂₀) and carotenoids (C₄₀)]. Labdane-type diterpene biosynthesis from GGPP, via 8OH-CPP, is described using manoyl oxide as the example. Whi2p, previously identified as positive genetic interactor of HMG2 [38], and pathways competing for substrates involved in terpene biosynthesis, such as the mitochondrial fatty acid biosynthesis pathway or glutamate biosynthesis, are also indicated. Enzyme names correspond to the S. cerevisiae proteins. Steps downregulated in this study by heterozygous deletion of the corresponding gene are indicated by gray arrows

Fig. 2

Proposed biosynthetic pathway of forskolin and carnosic acid. Both pathways begin from the common diterpene precursor GGPP (1). In C. forskohlii, a class II diTPS converts GGPP to 8OH-CPP (2), which is then taken up by a class I enzyme, to form manoyl oxide (3). This, in turn, becomes oxidized at several positions (C-1, C-6, C-7, C-11), presumably by the action of specific CYPs, and eventually O-acetylated at the C-7 hydroxy to generate forskolin (6). 11β-hydroxy-manoyl oxide (4) and 11-oxo-manoyl oxide (5) are believed to be the first steps in this mechanism, although the enzyme(s) catalyzing these reactions in C. forskohlii have not yet been identified. In S. pomifera, GGPP is converted to CPP (7) and then to miltiradiene (8) by corresponding class II (CPP synthase; CDS) and class I (Miltiradiene synthase; SpMilS) diTPSs. Miltiradiene is non-enzymatically converted to abietatriene (11), the substrate of CYP76AH24. CYP76AH24 catalyzes two successive oxidation events, one on C-12 of abietatriene producing ferruginol (12), and a second one on C-11 of ferruginol producing 11-hydroxy-ferruginol (13). When provided with miltiradiene, in vitro or in yeast cells, CYP76AH24 catalyzes a two step oxidation leading to 11-keto-miltiradiene (10), via 11-hydroxy-miltiradiene (9) [26]. The dashed box encloses the reactions catalyzed by CYP76AH24. CYP76AK6 takes up 11-hydroxy-ferruginol to catalyze a three step oxidation leading to carnosic acid (14) [26]. The promiscuous class I diTPSs, SpMilS, can also accept 8OH-CPP (2) to produce manoyl oxide (3) [25] To reconstruct the first steps of the forskolin biosynthetic pathway in yeast, CcCLS was used to produce 8OH-CPP, SpMilS was employed to convert 8OH-CPP to manoyl oxide, and CYP76AH24 was exploited to oxidize manoyl oxide (3) to 11β-hydroxy-manoyl oxide (4)

Fig. 3

Diagrammatic illustration of configuration of the yeast platform for the production manoyl oxide and 11β-hydroxy-manoyl oxide. a The fusion between the class II diTPS, CcCLS, responsible for 8OH-CPP formation and the yeast variant Erg20p(F96C), engineered to synthesize GGPP, under uracil selection (U) was co-expressed in yeast cells with the class I diTPS, SpMilS, under histidine (H) selection, for the production of manoyl oxide. b Introduction of CYP76AH24 in the above system, under leucine (L) selection, enabled oxidation of manoyl oxide to 11β-hydroxy-manoyl oxide

Fig. 4

Formation of 11β-hydroxy-manoyl oxide by CYP76AH24. a Expression of CYP76AH24 in manoyl oxide-producing yeast cells resulted in the production of a new compound, which after isolation and structural analysis was identified as 11β-hydroxy-manoyl oxide. b GC–MS chromatogram of the products of an in vitro reaction using microsomal CYP76AH24 protein, manoyl oxide as substrate and NADPH as co-factor. A preparation of yeast microsomal membranes from a strain that expressed only CPR2 is used as control. Production of 11β-hydroxy-manoyl oxide was confirmed by comparison of retention time and mass spectrum with purified compound. c Mass spectrum of 11β-hydroxy-manoyl oxide standard (isolated from engineered yeast cells and characterized by NMR spectroscopy). d Mass spectrum of 11β-hydroxy-manoyl oxide produced by CYP76AH24 in an in vitro reaction

Fig. 5

Steady-state kinetic analysis of the oxidation of manoyl oxide by CYP76AH24. The enzymatic activity of CYP76AH24 was evaluated using varying concentration (1–75 μM) of manoyl oxide substrate and 80 pmol of enzyme. The produced 11β-hydroxy-manoyl oxide was quantified by CG–MS analysis using purified compound as standard. Quantification of CYP76AH24 enzyme concentration was performed by measuring the binding of CO to the reduced form of the purified enzyme (450 nm peak), according to [47]. The differential spectrum of the CO-treated enzyme preparation is shown in the inset

Fig. 6

Optimization of 11β-hydroxy-manoyl oxide production in yeast cells. a Bar chart depicting the titers of manoyl oxide and 11β-hydroxy-manoyl oxide obtained using the different strains developed in this study. b Efficiency of conversion of manoyl oxide to 11β-hydroxy-manoyl oxide by CYP76AH24 in the different yeast strains developed. The efficiency of conversion is calculated as the ratio of 11β-hydroxy-manoyl oxide titer to the sum of manoyl oxide and 11β-hydroxy-manoyl oxide titres, expressed as percentage