Dynamic Control of ERG20 and ERG9 Expression for Improved Casbene Production in Saccharomyces cerevisiae.

Production of plant metabolites in microbial hosts represents a promising alternative to traditional chemical-based methods. Diterpenoids are compounds with interesting applications as pharmaceuticals, fragrances and biomaterials. Casbene, in particular, serves as a precursor to many complex diterpenoids found in plants from the Euphorbiaceae family that have shown potential therapeutic effects. Here, we engineered the budding yeast Saccharomyces cerevisiae for improved biosynthesis of the diterpene casbene. We first expressed, in yeast, a geranylgeranyl diphosphate synthase from Phomopsys amygdali in order to boost the geranylgeranyl diphosphate pool inside the cells. The enzyme uses isopentenyl diphosphate and dimethylallyl diphosphate to directly generate geranylgeranyl diphosphate. When co-expressing a casbene synthase from Ricinus communis the yeast was able to produce casbene in the order of 30 mg/L. Redirecting the flux from FPP and sterols, by means of the ergosterol sensitive promoter of ERG1, allowed for plasmid-based casbene production of 81.4 mg/L. Integration of the target genes into the yeast genome, together with the replacement of the promoter regions of ERG20 and ERG9 with combinations of ergosterol- and glucose-sensitive promoters, generated a titer of 108.5 mg/L of casbene. We here succeeded to engineer an improved route for geranylgeranyl diphosphate synthesis in yeast. Furthermore, we showed that the concurrent dynamic control of ERG20 and ERG9 expression, using ergosterol and carbon source regulation mechanisms, could substantially improve diterpene titer. Our approach will pave the way for a more sustainable production of GGPP- and casbene-derived products.

Biosynthesis of angelyl-CoA in Saccharomyces cerevisiae.

BACKGROUND: The angelic acid moiety represents an essential modification in many biologically active products. These products are commonly known as angelates and several studies have demonstrated their therapeutic benefits, including anti-inflammatory and anti-cancer effects. However, their availability for use in the development of therapeutics is limited due to poor extraction yields. Chemical synthesis has been achieved but its complexity prevents application, therefore microbial production may offer a promising alternative. Here, we engineered the budding yeast Saccharomyces cerevisiae to produce angelyl-CoA, the CoA-activated form of angelic acid. RESULTS: For yeast-based production of angelyl-CoA we first expressed genes recently identified in the biosynthetic cluster ssf of Streptomyces sp. SF2575 in S. cerevisiae. Exogenous feeding of propionate and heterologous expression of a propionyl-CoA synthase from Streptomyces sp. were initially employed to increase the intracellular propionyl-CoA level, resulting in production of angelyl-CoA in the order of 5 mg/L. Substituting the Streptomyces sp. propionyl-CoA carboxylase with a carboxylase derived from Streptomyces coelicolor resulted in angelyl-CoA levels up to 6.4 mg/L. In vivo analysis allowed identification of important intermediates in the pathway, including methyl-malonyl-CoA and 3-hydroxyl-2-methyl-butyryl-CoA. Furthermore, methyl-malonate supplementation and expression of matB CoA ligase from S. coelicolor allowed for methyl-malonyl-CoA synthesis and supported, together with parts of the ssf pathway, angelyl-CoA titres of approximately 1.5 mg/L. Finally, feeding of angelic acid to yeasts expressing acyl-CoA ligases from plant species led to angelyl-CoA production rates of approximately 40 mg/L. CONCLUSIONS: Our results demonstrate the biosynthesis of angelyl-CoA in yeast from exogenously supplied carboxylic acid precursors. This is the first report on the activity of the ssf genes. We envision that our approach will provide a platform for a more sustainable production of the pharmaceutically important compound class of angelates.

Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast.

The development of medical applications exploiting the broad bioactivities of the diterpene therapeutic triptolide from Tripterygium wilfordii is limited by low extraction yields from the native plant. Furthermore, the extraordinarily high structural complexity prevents an economically attractive enantioselective total synthesis. An alternative production route of triptolide through engineered Saccharomyces cerevisiae (yeast) could provide a sustainable source of triptolide. A potential intermediate in the unknown biosynthetic route to triptolide is the diterpene dehydroabietic acid. Here, we report a biosynthetic route to dehydroabietic acid by transient expression of enzymes from T. wilfordii and Sitka spruce (Picea sitchensis) in Nicotiana benthamiana. The combination of diterpene synthases TwTPS9, TwTPS27, and cytochromes P450 PsCYP720B4 yielded dehydroabietic acid and a novel analog, tentatively identified as 'miltiradienic acid'. This biosynthetic pathway was reassembled in a yeast strain engineered for increased yields of the pathway intermediates, the diterpene olefins miltiradiene and dehydroabietadiene. Introduction in that strain of PsCYP720B4 in combination with two alternative NADPH-dependent cytochrome P450 reductases resulted in scalable in vivo production of dehydroabietic acid and its analog from glucose. Approaching future elucidation of the remaining biosynthetic steps to triptolide, our findings may provide an independent platform for testing of additional recombinant candidate genes, and ultimately pave the way to biotechnological production of the high value diterpenoid therapeutic.

Oxidation and cyclization of casbene in the biosynthesis of Euphorbia factors from mature seeds of Euphorbia lathyris L.

The seed oil of Euphorbia lathyris L. contains a series of macrocyclic diterpenoids known as Euphorbia factors. They are the current industrial source of ingenol mebutate, which is approved for the treatment of actinic keratosis, a precancerous skin condition. Here, we report an alcohol dehydrogenase-mediated cyclization step in the biosynthetic pathway of Euphorbia factors, illustrating the origin of the intramolecular carbon-carbon bonds present in lathyrane and ingenane diterpenoids. This unconventional cyclization describes the ring closure of the macrocyclic diterpene casbene. Through transcriptomic analysis of E. lathyris L. mature seeds and in planta functional characterization, we identified three enzymes involved in the cyclization route from casbene to jolkinol C, a lathyrane diterpene. These enzymes include two cytochromes P450 from the CYP71 clan and an alcohol dehydrogenase (ADH). CYP71D445 and CYP726A27 catalyze regio-specific 9-oxidation and 5-oxidation of casbene, respectively. When coupled with these P450-catalyzed monooxygenations, E. lathyris ADH1 catalyzes dehydrogenation of the hydroxyl groups, leading to the subsequent rearrangement and cyclization. The discovery of this nonconventional cyclization may provide the key link to complete elucidation of the biosynthetic pathways of ingenol mebutate and other bioactive macrocyclic diterpenoids.

Yeast-based assays for screening 11ß-HSD1 inhibitors.

BACKGROUND: Intracellular metabolism of glucocorticoid hormones plays an important role in the pathogenesis of metabolic syndrome and regulates, among many physiological processes, collagen metabolism in skin. At the peripheral level the concentration of active glucocorticoids is mainly regulated by the 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) enzyme, involved in the conversion of cortisone into the biologically active hormone cortisol. Cortisol interacts with the glucocorticoid receptor and regulates the expression of different classes of genes within the nucleus. Due to its implication in glucocorticoid metabolism, the inhibition of 11ß-HSD1 activity has become a dominant strategy for the treatment of metabolic syndrome. Moreover, inhibitors of this target enzyme can be used for development of formulations to counteract skin ageing. Here we present the construction of two yeast cell based assays that can be used for the screening of novel 11ß-HSD1 inhibitors. RESULTS: The yeast Saccharomyces cerevisiae is used as a host organism for the expression of human 11ß-HSD1 as well as a genetically encoded assay system that allows intracellular screening of molecules with 11ß-HSD1 inhibitory activity. As proof of concept the correlation between 11ß-HSD1 inhibition and fluorescent output signals was successfully tested with increasing concentrations of carbenoxolone and tanshinone IIA, two known 11ß-HSD1 inhibitors. The first assay detects a decrease in fluorescence upon 11ß-HSD1 inhibition, whereas the second assay relies on stabilization of yEGFP upon inhibition of 11ß-HSD1, resulting in a positive read-out and thus minimizing the rate of false positives sometimes associated with read-outs based on loss of signals. Specific inhibition of the ABC transporter Pdr5p improves the sensitivity of the assay strains to cortisone concentrations by up to 60 times. CONCLUSIONS: Our yeast assay strains provide a cost-efficient and easy to handle alternative to other currently available assays for the screening of 11ß-HSD1 inhibitors. These assays are designed for an initial fast screening of large numbers of compounds and enable the selection of cell permeable molecules with target inhibitory activity, before proceeding to more advanced selection processes. Moreover, they can be employed in yeast synthetic biology platforms to reconstitute heterologous biosynthetic pathways of drug-relevant scaffolds for simultaneous synthesis and screening of 11ß-HSD1 inhibitors at intracellular level.