Monoterpene production by the carotenogenic yeast Rhodosporidium toruloides

John M Gladden,Anthe George,Xun Zhuang,Jeffrey M Skerker,Mary Bao Tran-Gymfi,Eric Monroe,Todd Pray,Mona Mirsiaghi,Masakazu Ito,Fang Liu,Oliver Kilian,Eric Sundstrom,Ryan W Davis

doi:10.1186/s12934-019-1099-8

Abstract

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.

Highlights

Due 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 replace‐ ments for use in vehicles or in aviation
The 2016 Department of Energy (DOE) Billion-Ton study reported that the US could produce at least 1 billion dry tons of lignocellulosic biomass annually by 2040, suggesting that developing lignocellulosic-derived biofuels could play a major role in the domestic production of renewable transportation fuels [5]
Codon optimized monoterpene synthases (MTS) genes were cloned into a binary vector designed for Agrobacterium tumefaciens mediated transformation (ATMT) where expression is driven by the glyceraldehyde-3-phosphate dehydrogenase promoter

Summary

Introduction

Due 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 replace‐ ments 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. The global transportation sector has high energy demands and is dependent on the availability of high energy density liquid fuels for use in light/heavy-duty vehicles, and in aviation. Lignocellulosic biomass has been identified as an abundant carbon source for the production of biofuels, and has been the subject of intensive investigation in the past decades, resulting in the development of a number of bio-based gasoline, diesel and jet fuel alternatives. The 2016 DOE Billion-Ton study reported that the US could produce at least 1 billion dry tons of lignocellulosic biomass annually by 2040, suggesting that developing lignocellulosic-derived biofuels could play a major role in the domestic production of renewable transportation fuels [5]

Methods

Results

Conclusion