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.

Keywords: Diterpene; Geranylgeranyl pyrophosphate synthase; Metabolic engineering; Mevalonate pathway; Mutant farnesyl pyrophosphate synthase; Rhodotorula.

Fig. 1

Production of ent-kaurene from acetyl-CoA via the mevalonate pathway. Genes expressed in R. toruloides are listed in Table 1. Mutant FPP synthases employed in this study are designed to alter prenyl phosphate product chain length, resulting in enzymes that generate mainly GGPP from IPP and DMAPP, instead of the native FPP product

Fig. 2

Expression of kaurene synthase from Gibberella fujikuroi (GfKS) in R. toruloides. Ent-kaurene titer at 9 days is shown for the three highest-titer strains transformed with constructs 1 (P_ANT-GfKS) and 2 (P_GAPDH-GfKS). (N = 3, data shown as average ± standard deviation, from a single experiment in YPD₁₀ medium. ABFPUB identification numbers are listed in Table 1.)

Fig. 3

Transcript level, protein level and copy number of GfKS. Average relative abundance of GfKS transcript (ΔCt), protein, and copy number on day 3. Samples are designated by their ABFPUB identification number and description. (N = 3, data shown as average ± standard deviation, from a single experiment in YPD₁₀ medium.)

Fig. 4

Co-expression of kaurene synthase from Gibberella fujikuroi (GfKS) and various GGPP synthases in R. toruloides. Strains were constructed by transformation of strain ABFPUB_16 (P_GAPDH-GfKS) with constructs 3 to 6. Ent-kaurene titer at 9 days is shown for the three highest-titer strains for each construct. (N = 3, data shown as average ± standard deviation, from a single experiment in YPD₁₀ medium. ABFPUB identification numbers are listed in Table 1.)

Fig. 5

Co-transformation of kaurene synthase from Gibberella fujikuroi (GfKS) and various GGPP synthases into WT R. toruloides (constructs 7–10, Table 1). Ent-kaurene titer at 9 days is shown for the three highest-titer strains for each construct. (N = 3, data shown as average ± standard deviation, from a single experiment in YPD₁₀ medium. ABFPUB identification numbers are listed in Table 1.)

Fig. 6

Average relative abundance of transcript (ΔCt), protein and copy number of a GfKS and b GfFPS(F112A) on day 3. Samples are designated by their ABFPUB identification number. (N = 3, data shown as average ± standard deviation, from a single experiment in YPD₁₀ medium.)

Fig. 7

Sugar concentration, OD₆₀₀, and ent-kaurene titer data for strain ABFPUB_26 cultivated in a 2 L bioreactor containing 75% DMR-EH hydrolysate, supplemented with 10 g/L yeast extract