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Integrative transcriptomic and proteomic analysis of the mutant lignocellulosic hydrolyzate-tolerant Rhodosporidium toruloides.

ABSTRACT: The oleaginous yeast Rhodosporidium toruloides has been considered as an economical lipid producer because it transforms carbohydrates from lignocellulosic hydrolyzate into triglycerides; however, R. toruloides cannot survive in hydrolyzate due to the inhibitors co-produced by hydrolysis. We have previously reported a plasma mutagenesis-generated mutant strain M18 that had strong tolerance for the stress environments of hydrolyzate. Here, we applied transcriptomic and proteomic approaches to analyze the global metabolic responses to the stress in hydrolyzate of R. toruloides and elucidate the tolerant mechanism of the mutant strain. The results showed that 57% genes matched and correlated well with their corresponding proteins. Five hundred and seven genes and 366 proteins had their transcription and expression levels changed, respectively, and 39 key genes with significantly changed transcription and expression levels (?5-fold changes) were identified. The results demonstrated that four cellular processes and their key genes are likely related to the mechanism of tolerance of M18 strain. Enhanced expression of the key genes in R. toruloides could improve the cellular stress tolerance to lignocellulosic hydrolyzate, while the altered expression of most key genes is probably not caused by mutagenesis, but induced by stressful environments of the hydrolyzate.

SUBMITTER: Qi F

PROVIDER: S-EPMC6999448 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Integrative transcriptomic and proteomic analysis of the mutant lignocellulosic hydrolyzate-tolerant Rhodosporidium toruloides.

Qi Feng F Zhao Xuebing X Kitahara Yuki Y Li Tian T Ou Xianjin X Du Wei W Liu Dehua D Huang Jianzhong J

Engineering in life sciences 20160329 3

The oleaginous yeast Rhodosporidium toruloides has been considered as an economical lipid producer because it transforms carbohydrates from lignocellulosic hydrolyzate into triglycerides; however, R. toruloides cannot survive in hydrolyzate due to the inhibitors co-produced by hydrolysis. We have previously reported a plasma mutagenesis-generated mutant strain M18 that had strong tolerance for the stress environments of hydrolyzate. Here, we applied transcriptomic and proteomic app ...[more]

PMID: 32624772

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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:Background:Lipid accumulation by oleaginous microorganisms is of great scientific interest and biotechnological potential. While nitrogen limitation has been routinely employed, low-cost raw materials usually contain rich nitrogenous components, thus preventing from efficient lipid production. Inorganic phosphate (Pi) limitation has been found sufficient to promote conversion of sugars into lipids, yet the molecular basis of cellular response to Pi limitation and concurrent lipid accumulation remains elusive. Results:Here, we performed multi-omic analyses of the oleaginous yeast Rhodosporidium toruloides to shield lights on Pi-limitation-induced lipid accumulation. Samples were prepared under Pi-limited as well as Pi-repleted chemostat conditions, and subjected to analysis at the transcriptomic, proteomic, and metabolomic levels. In total, 7970 genes, 4212 proteins, and 123 metabolites were identified. Results showed that Pi limitation facilitates up-regulation of Pi-associated metabolism, RNA degradation, and triacylglycerol biosynthesis while down-regulation of ribosome biosynthesis and tricarboxylic acid cycle. Pi limitation leads to dephosphorylation of adenosine monophosphate and the allosteric activator of isocitrate dehydrogenase key to lipid biosynthesis. It was found that NADPH, the key cofactor for fatty acid biosynthesis, is limited due to reduced flux through the pentose phosphate pathway and transhydrogenation cycle and that this can be overcome by over-expression of an endogenous malic enzyme. These phenomena are found distinctive from those under nitrogen limitation. Conclusions:Our data suggest that Pi limitation activates Pi-related metabolism, RNA degradation, and TAG biosynthesis while inhibits ribosome biosynthesis and TCA cycle, leading to enhanced carbon fluxes into lipids. The information greatly enriches our understanding on microbial oleaginicity and Pi-related metabolism. Importantly, systems data may facilitate designing advanced cell factories for production of lipids and related oleochemicals.

Dataset Information

Integrative transcriptomic and proteomic analysis of the mutant lignocellulosic hydrolyzate-tolerant Rhodosporidium toruloides.

Publications

Integrative transcriptomic and proteomic analysis of the mutant lignocellulosic hydrolyzate-tolerant <i>Rhodosporidium toruloides</i>.

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