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

Abstract

BackgroundSeveral 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.ResultsSince 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.ConclusionsIn 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0440-8) contains supplementary material, which is available to authorized users.

Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii

“…11-Hydroxy-13 R -manoyl oxide ( 3d ) is observed only in extracts expressing the RoFS1 and SfFS1 genes. Presence of 11-hydroxy-13 R -manoyl oxide was verified by comparison to an authentic standard (Ignea et al, 2016b) while identification of 11-oxo-13 R -manoyl oxide was confirmed by comparison of m/z spectra with a previously characterized compound ( 2 ). The results show Ro CYP76AH4 has an activity similar to Cf CYP76AH15, able to convert efficiently and specifically 13 R -manoyl oxide to 2 .…”

Advances in Metabolic Engineering of Saccharomyces cerevisiae for the Production of Industrially and Clinically Important Chemicals

“…Glutathione Overexpression of YAP1 [58] Manipulation of the sulphate assimilation pathway by overexpressing MET14 and MET16 [59] Improved oxidized glutathione production by overexpression of GSH1, GSH2, and ERV1 and the deletion of GLR1 [60] Adaptive laboratory evolution in the presence of increasing levels of acrolein and screening for enhanced glutathione production [61] Whole-genome engineering via genome shuling and screening for enhanced glutathione production [62] Artemisinin/artemisinic acid Reconstruction of the complete biosynthetic pathway of artemisinic acid, including the three-step oxidation of amorphadiene to artemisinic acid by expression of CYP71AV1, CPR1, CYB5, ADH1 and ALDH1 from Artemisia annua [48] Taxol/taxadiene Expression of a truncated version of the endogenous tHMG1 and GGPPS from Taxus chinensis or Sulfolobus acidocaldarius together with TDC1 from T. chinensis [66] Prediction of the eiciency of diferent GGPPS enzymes via computer aided protein modelling [67] Forskolin Expression of a promiscuous cytochrome P450 from Salvia pomifera [68] Polyketides Heterologous expression of 6-MSA synthase gene from Penicillium patulum together with PPTases from either Bacillus subtilis or Aspergillus nidulans [69] Construction of polyketide precursor pathways by expressing prpE from Salmonella typhimurium and PCC pathway from Streptomyces coelicolor [70] Enhanced cofactor supply by expressing 2-PS from Gerbera hybrida [71] Resveratrol Reconstruction of a de novo pathway by expressing TAL from Herpetosiphon aurantiacus, 4-CL1 from Arabidopsis thaliana and VST1 from Vitis vinifera [49] Expression of 4CL1 from A. thaliana and STS from Arachis hypogaea [73] Expression of PAL from Rhodosporidium toruloides, C4H and 4-CL1 from A. thaliana, and STS from A. hypogaea [74] Expression of 4-coumaroyl-coenzyme A ligase (4CL1) from A. thaliana and stilbene synthase (STS) from V. vinifera [75] Overexpression of the resveratrol biosynthesis pathway, enhancement of P450 activity, increasing the precursor supply for resveratrol synthesis via phenylalanine pathway [76] Dihydrochalcones Expression of the heterologous pathway genes in a TSC13-overexpressing S. cerevisiae strain [78] Alkaloids Expression of 14 monoterpene indole alkaloid pathway genes from Catharanthus roseus and enhanced secondary metabolism to produce strictosidine de novo [79] Construction of the complete de novo biosynthetic pathway to norcoclaurine by expressing a mammalian TyrH enzyme and DODC from Pseudomonas putida, along with four genes required for biosynthesis of its electron carrier cosubstrate …”

“…Although the forskolin biosynthetic pathway has not been completely discovered yet, a promiscuous cytochrome P450 from Salvia pomifera was identiied as a replacement to achieve the synthesis of the forskolin precursor. This study can provide a basis for the biosynthesis of various tricyclic (8,13)-epoxy-labdanes [68].…”

Overcoming the plasticity of plant specialized metabolism for selective diterpene production in yeast

“…Starting from the common precursor geranylgeranyl diphosphate (GGPP), different class II diTPSs synthesize copalyl diphosphate (CPP) or 8-hydroxy-copalyl diphosphate (8-OH-CPP). Miltiradiene synthase (MilS) can accept either CPP or 8-OH-CPP to produce miltiradiene or manoyl oxide, respectively (refs 11 , 20 ). Subsequently, the promiscuous enzyme, CYP76AH24, oxidizes miltiradiene, abietatriene, ferruginol and manoyl oxide, to synthesize the corresponding oxygenated molecules in position C-12 or C-11 (the biosynthetic pathways are described in details in the main text).…”