Abstract
BackgroundRhodosporidium 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.ResultsThe 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.ConclusionThis 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.
Highlights
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
In plant and bacterial systems, ent-kaurene is synthesized from geranylgeranyl diphosphate (GGPP) in a two-step process, via copalyl diphosphate (CDP), while G. fujikuroi and other fungi contain bifunctional CDP/kaurene synthase (KS) enzymes that generate ent-kaurene directly from GGPP (Fig. 1) [13]
Constructs were transformed into R. toruloides by Agrobacterium tumefaciens mediated transformation (ATMT), which results in random chromosomal integration of transgenes
Summary
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. The diverse terpenoid family contains over 70,000 unique compounds that are predominantly produced from two fundamental isoprenoid building blocks, the C5 prenyl phosphates, dimethylallyl diphosphate (DMAPP) and Diterpenes are less volatile than monoterpenes and sesquiterpenes, and have various industrial and biological applications from resin-based adhesives to potential. Diterpenes play diverse roles such as protection against pathogens and herbivores [9] and production of growth regulators such as gibberellins [10,11,12]. Besides serving as the precursor to gibberellins, ent-kaurene may be transformed into a variety of bioactive derivatives, with a range of potential applications. In traditional Chinese medicine, pharmacologically active ent-kaurene diterpenoids from Isodon plants (Lamiaceae) are used to treat inflammation and cancers [16]. Kaurene-derivatives may play a key role in the development of new renewable polymers [17]
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