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

Abstract

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.

Keywords: ERG20; ERG9; casbene; diterpene; dynamic control; metabolic engineering; mevalonate pathway; yeast.

Figure 1

Biosynthesis of casbene in yeast and examples of casbene derived compounds. Schematic overview of casbene biosynthesis based on engineered mevalonate pathway in S. cerevisiae. The new biosynthetic branch starts with PaGGPPS, for production of GGPP using IPP and DMAPP as sole substrates. RcCBS mediates casbene synthesis (in red). Endogenous genes and metabolites are shown in black. Overexpressed genes are shown in blue. Genes regulated through dynamic control (ERG20 and ERG9) and genes from the steroid biosynthetic pathway are shown in gray. The truncated endogenous gene tHMG1 and the heterologous genes PaGGPPS (encoding a truncated version of the fusicoccadiene synthase from Phomopsis amygdali serving as GGPP synthase) and RcCBS (encoding a truncated version of the casbene synthase from Ricinus communis) were overexpressed to improve casbene production. The native promoter of ERG20 was replaced with P_ERG1 or P_HXT1, respectively. The native promoter of ERG9 was replaced with P_ERG1. HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate.

Figure 2

Casbene is the precursor of many diterpenoids identified in plants from the Euphorbiaceae family. Euphorbia factor L2 (Euphorbia lathyris), resiniferatoxin (Euphorbia resinifera), ingenol-3-angelate (Euphorbia peplus), prostratin (Homolanthus nutans), and jatrophone (Jatropha gossypifolia L.).

Figure 3

Growth and production of casbene and GGOH in engineered strains expressing PaGGPPS and RcCBS. Time course of production of casbene (A) and GGOH (B) in strains CAS2 (tHMGR), CAS6 (tHMGR, P_ERG1-ERG20), CAS7 (tHMGR, P_ERG1-ERG9), and CAS8 (tHMGR, P_ERG1-ERG20, P_ERG1-ERG9). All strains episomally expressed PaGGPPS and RcCBS. (C) Growth profile of the strains determined using optical density data recorded at 600 nm (OD₆₀₀). All strains were grown in selective medium for maximally 120 h. Represented are the average and standard deviation of three independent experiments.

Figure 4

Production of casbene and GGOH in engineered strains containing integrated PaGGPPS and RcCBS. Casbene (green bars) and GGOH (blue bars) produced by strains CAS11 (tHMGR), CAS12 (tHMGR, P_ERG1-ERG20), CAS13 (tHMGR, P_HXT1-ERG20), CAS14 (tHMGR, P_ERG1-ERG20, P_ERG1-ERG9), and CAS15 (tHMGR, P_HXT1-ERG20, P_ERG1-ERG9). The corresponding OD₆₀₀ values are represented by filled circles. Engineered strains were incubated in selective SC medium for 96 h (all data: mean ± SD, n = 3).