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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.

Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides