Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides

Abstract

Background: Rhodosporidium toruloides has emerged as a promising host for the production of bioproducts from lignocellulose, in part due to its ability to grow on lignocellulosic feedstocks, tolerate growth inhibitors, and co-utilize sugars and lignin-derived monomers. Ent-kaurene derivatives have a diverse range of potential applications from therapeutics to novel resin-based materials. Results: The Design, Build, Test, and Learn (DBTL) approach was employed to engineer production of the non-native diterpene ent-kaurene in R. toruloides. Following expression of kaurene synthase (KS) in R. toruloides in the first DBTL cycle, a key limitation appeared to be the availability of the diterpene precursor, geranylgeranyl diphosphate (GGPP). Further DBTL cycles were carried out to select an optimal GGPP synthase and to balance its expression with KS, requiring two of the strongest promoters in R. toruloides, ANT (adenine nucleotide translocase) and TEF1 (translational elongation factor 1) to drive expression of the KS from Gibberella fujikuroi and a mutant version of an FPP synthase from Gallus gallus that produces GGPP. Scale-up of cultivation in a 2 L bioreactor using a corn stover hydrolysate resulted in an ent-kaurene titer of 1.4 g/L. Conclusion: This study builds upon previous work demonstrating the potential of R. toruloides as a robust and versatile host for the production of both mono-and sesquiterpenes, and is the first demonstration of the production of a non-native diterpene in this organism.

Further Engineering of R. Toruloides for the Production of Terpenes From Lignocellulosic Biomass

“…Ultimate 3000 system UHPLC (Agilent Technologies) using an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) and Thermo Scienti c™ RefractoMax 520 Refractive Index Detector (RID) (Thermo Fisher Scienti c, Waltham, MA, USA) as described previously [5].…”

“…Promoters for these synthases were selected from native R. toruloides genes. Three promoters, ANT, GAPDH, and TEF1 were selected based on their relative strength, constitutive expression pro les, and utility in prior work [2,3,5,15]. P ANT and P GAPDH were used to express HYP3 from Hypoxylon sp.…”

“…A number of C 15 sesquiterpenes such as α-bisabolene [3] and farnesene [4] can function as diesel fuels, following reduction to their respective alkanes by hydrogenation. Other terpenes such as the C 20 diterpene, ent-kaurene may provide bio-based replacements for petrochemicals with a variety of industrial applications [5].…”

“…Production of α-bisabolene was reported in S. cerevisiae and R. toruloides at titers of 900 mg/L and 680 mg/L, respectively [3,6], with the latter produced from lignocellulosic hydrolysate. Somewhat surprisingly, the diterpene ent-kaurene was produced at higher titers (1.4 g/L) from lignocellulosic hydrolysate in R. toruloides, perhaps owing to the fact that the diterpene precursor, geranylgeranyl pyrophosphate (GGPP) was enhanced by expression of a heterologous GGPP synthase in the engineered strain [5]. In contrast, production of α-bisabolene and 1,8cineole has thus far relied on native levels of their respective precursors, farnesyl pyrophosphate (FPP) and geranyl pyrophosphate (GPP).…”

Rhodosporidium toruloides - A potential red yeast chassis for lipids and beyond

“…The diterpene ent -kaurene was produced in a 2-L bioreactor at a titer of 1.4 g/L from corn stover hydrolysates by engineered R. toruloides strain that expressed genes encoding the kaurene synthase from Gibberella fujikuroi and a mutated farnesyl diphosphate synthase from Gallus gallus for enhanced geranylgeranyl diphosphate supply (Geiselman et al . 2020 ).…”