Dataset Information

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.

SUBMITTER: Geiselman GM

PROVIDER: S-EPMC7003354 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides.

Geiselman Gina M GM Zhuang Xun X Kirby James J Tran-Gyamfi Mary B MB Prahl Jan-Philip JP Sundstrom Eric R ER Gao Yuqian Y Munoz Munoz Nathalie N Nicora Carrie D CD Clay Derek M DM Papa Gabriella G Burnum-Johnson Kristin E KE Magnuson Jon K JK Tanjore Deepti D Skerker Jeffrey M JM Gladden John M JM

Microbial cell factories 20200205 1

<h4>Background</h4>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.<h4>Results</h4>The Design, Build, Test, and Learn (DBTL) approach was employed to engineer production ...[more]

PMID: 32024522

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Project description:BackgroundDue to their high energy density and compatible physical properties, several monoterpenes have been investigated as potential renewable transportation fuels, either as blendstocks with petroleum or as drop-in replacements for use in vehicles (both heavy and light-weight) or in aviation. Sustainable microbial production of these biofuels requires the ability to utilize cheap and readily available feedstocks such as lignocellulosic biomass, which can be depolymerized into fermentable carbon sources such as glucose and xylose. However, common microbial production platforms such as the yeast Saccharomyces cerevisiae are not naturally capable of utilizing xylose, hence requiring extensive strain engineering and optimization to efficiently utilize lignocellulosic feedstocks. In contrast, the oleaginous red yeast Rhodosporidium toruloides is capable of efficiently metabolizing both xylose and glucose, suggesting that it may be a suitable host for the production of lignocellulosic bioproducts. In addition, R. toruloides naturally produces several carotenoids (C40 terpenoids), indicating that it may have a naturally high carbon flux through its mevalonate (MVA) pathway, providing pools of intermediates for the production of a wide range of heterologous terpene-based biofuels and bioproducts from lignocellulose.ResultsSixteen terpene synthases (TS) originating from plants, bacteria and fungi were evaluated for their ability to produce a total of nine different monoterpenes in R. toruloides. Eight of these TS were functional and produced several different monoterpenes, either as individual compounds or as mixtures, with 1,8-cineole, sabinene, ocimene, pinene, limonene, and carene being produced at the highest levels. The 1,8-cineole synthase HYP3 from Hypoxylon sp. E74060B produced the highest titer of 14.94 ± 1.84 mg/L 1,8-cineole in YPD medium and was selected for further optimization and fuel properties study. Production of 1,8-cineole from lignocellulose was also demonstrated in a 2L batch fermentation, and cineole production titers reached 34.6 mg/L in DMR-EH (Deacetylated, Mechanically Refined, Enzymatically Hydorlized) hydrolysate. Finally, the fuel properties of 1,8-cineole were examined, and indicate that it may be a suitable petroleum blend stock or drop-in replacement fuel for spark ignition engines.ConclusionOur results demonstrate that Rhodosporidium toruloides is a suitable microbial platform for the production of non-native monoterpenes with biofuel applications from lignocellulosic biomass.

Project description:BackgroundAs the circular economy advocates a near total waste reduction, the industry has shown an increased interest toward the exploitation of various residual biomasses. The origin and availability of biomass used as feedstock strongly affect the sustainability of biorefineries, where it is converted in energy and chemicals. Here, we explored the valorization of Camelina meal, the leftover residue from Camelina sativa oil extraction. In fact, in addition to Camelina meal use as animal feed, there is an increasing interest in further valorizing its macromolecular content or its nutritional value.ResultsCamelina meal hydrolysates were used as nutrient and energy sources for the fermentation of the carotenoid-producing yeast Rhodosporidium toruloides in shake flasks. Total acid hydrolysis revealed that carbohydrates accounted for a maximum of 31 ± 1.0% of Camelina meal. However, because acid hydrolysis is not optimal for subsequent microbial fermentation, an enzymatic hydrolysis protocol was assessed, yielding a maximum sugar recovery of 53.3%. Separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), and SSF preceded by presaccharification of Camelina meal hydrolysate produced 5 ± 0.7, 16 ± 1.9, and 13 ± 2.6 mg/L of carotenoids, respectively. Importantly, the presence of water-insoluble solids, which normally inhibit microbial growth, correlated with a higher titer of carotenoids, suggesting that the latter could act as scavengers.ConclusionsThis study paves the way for the exploitation of Camelina meal as feedstock in biorefinery processes. The process under development provides an example of how different final products can be obtained from this side stream, such as pure carotenoids and carotenoid-enriched Camelina meal, can potentially increase the initial value of the source material. The obtained data will help assess the feasibility of using Camelina meal to generate high value-added products.

Dataset Information

Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides.

Publications

Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides.

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