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Proteome Analysis of Xylose Metabolism in Rhodotorula Toruloides During Lipid Production Ievgeniia A

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Proteome Analysis of Xylose Metabolism in Rhodotorula Toruloides During Lipid Production Ievgeniia A Tiukova et al. Biotechnol Biofuels (2019) 12:137 https://doi.org/10.1186/s13068-019-1478-8 Biotechnology for Biofuels RESEARCH Open Access Proteome analysis of xylose metabolism in Rhodotorula toruloides during lipid production Ievgeniia A. Tiukova1,2* , Jule Brandenburg2, Johanna Blomqvist2,3, Sabine Sampels2, Nils Mikkelsen2, Morten Skaugen4, Magnus Ø. Arntzen4, Jens Nielsen1, Mats Sandgren2 and Eduard J. Kerkhoven1 Abstract Background: Rhodotorula toruloides is a promising platform organism for production of lipids from lignocellulosic substrates. Little is known about the metabolic aspects of lipid production from the lignocellolosic sugar xylose by oleaginous yeasts in general and R. toruloides in particular. This study presents the frst proteome analysis of the metabolism of R. toruloides during conversion of xylose to lipids. Results: Rhodotorula toruloides cultivated on either glucose or xylose was subjected to comparative analysis of its growth dynamics, lipid composition, fatty acid profles and proteome. The maximum growth and sugar uptake rate of glucose-grown R. toruloides cells were almost twice that of xylose-grown cells. Cultivation on xylose medium resulted in a lower fnal biomass yield although fnal cellular lipid content was similar between glucose- and xylose-grown cells. Analysis of lipid classes revealed the presence of monoacylglycerol in the early exponential growth phase as well as a high proportion of free fatty acids. Carbon source-specifc changes in lipid profles were only observed at early exponential growth phase, where C18 fatty acids were more saturated in xylose-grown cells. Proteins involved in sugar transport, initial steps of xylose assimilation and NADPH regeneration were among the proteins whose levels increased the most in xylose-grown cells across all time points. The levels of enzymes involved in the mevalonate pathway, phospholipid biosynthesis and amino acids biosynthesis difered in response to carbon source. In addition, xylose-grown cells contained higher levels of enzymes involved in peroxisomal beta-oxidation and oxidative stress response compared to cells cultivated on glucose. Conclusions: The results obtained in the present study suggest that sugar import is the limiting step during xylose conversion by R. toruloides into lipids. NADPH appeared to be regenerated primarily through pentose phosphate pathway although it may also involve malic enzyme as well as alcohol and aldehyde dehydrogenases. Increases in enzyme levels of both fatty acid biosynthesis and beta-oxidation in xylose-grown cells was predicted to result in a futile cycle. The results presented here are valuable for the development of lipid production processes employing R. toruloides on xylose-containing substrates. Keywords: Rhodotorula toruloides, Proteome, Xylose, Lipid production *Correspondence: [email protected] 1 Systems and Synthetic Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tiukova et al. Biotechnol Biofuels (2019) 12:137 Page 2 of 17 Background production of oil substitutes to replace cocoa but- Successful replacement of fossil diesel fuels with bio- ter [19]. Te mono-unsaturated 18-carbon FA oleic logical oils currently faces a number of challenges such acid (C18:1 n − 9) is the major FA and is accumulated as competing technologies, including electric vehicles, to ~ 55% of total FA in R. toruloides Y4. Less than half as well as unsustainable biological oil production prac- of this amount, ~ 20% composes palmitic acid (C16:0, tices such as clearing rainforests for palm oil cultiva- saturated). Stearic (C18:0) and linoleic (C18:2 n − 6) tion [1, 2]. Tere is also an increasing competition for acid can each reach up to 10% of total FA [11, 18, 20]. lipids from the food sector due to a growing population, A number of R. toruloides strains have been subjected increasing standards of human nutrition, and sustain- to genome sequencing [21–25], genetic engineering ability concerns. Lipids are required in animal feeds, for protocols are established [26–30] and the yeast is rec- instance oils from wild pelagic fsh are used to feed fsh in ognized as a novel platform organism for production of aquaculture. So-called “single cell oils” (SCOs) are lipids oleochemicals [31, 32]. A number of cultivation condi- derived from microorganisms, which represent a poten- tions of R. toruloides have therefore been examined, e.g. tially more sustainable source of lipids to partially replace growth on mixed substrates [33] or diferent cultivation fsh-derived oils [3]. Some microorganisms can produce modes on lignocellulosic substrates [34, 35] and lipid lipids from lignocellulose-based substrates. Te fve- production at sulfate [36] or phosphate-limitation [37]. carbon (C5) monosaccharide xylose is the second-most Improved genetically modifed strains of R. toruloides abundant sugar in plant biomass and its utilization by for conversion of biomass have been generated [38]. microorganisms is regarded as a challenge to reach a sus- Te molecular physiology of R. toruloides during tainable valorization of plant biomass and commerciali- lipid production from diferent sugar substrates have zation of lignocellulosic fuels and chemicals [4]. Some been investigated using both proteomics [39, 40] and oleaginous yeasts can convert xylose to lipids up to more transcriptomics [25, 41, 42] approaches. Meanwhile, than 20% of its dry cell weight: 22% Rhodotorula glutinis the molecular physiology during conversion of xylose [5], 58% Trichosporon fermentans [6], 37% Cryptococcus into lipids in R. toruloides and in oleaginous yeasts in curvatus [7], 33% Cryptococcus albidus [8], 42% Rhodoto- general has been poorly investigated. Xylose assimila- rula toruloides [9–11]. Cultivation on xylose in combina- tion involves the enzymes NAD(P)H-dependent xylose tion with glucose was also investigated using Lipomyces reductase (XR) and NADH-dependent xylitol dehy- starkeyi with 61% lipids of dry cell weight (DCW) pro- drogenase (XDH). XR reduces xylose to xylitol, which duced [12], while engineering of Yarrowia lipolytica is then oxidized by XDH to xylulose. Xylulose is sub- strain enabled it to assimilate xylose [13, 14]. Microbial sequently phosphorylated by xylulose kinase before production of lipids from xylose was furthermore inves- entering the pentose phosphate pathway (PPP). R. tigated in flamentous fungi: Cunninghamella echinulata toruloides also possesses the enzyme phosphoketolase, (lipids constituted 58% DCW), Mortierella isabellina which cleaves xylulose-5-phosphate to acetyl phos- (lipids constituted 66% DCW) [15]. phate and glyceraldehyde-3-phosphate [43]. Unlike glu- Establishment of sustainable lucrative microbial lipid cose assimilation, xylose assimilation requires NADPH production in industrial scale includes: (i) use of complex due to the initial reduction of xylose into xylitol by substrate (generated from plant biomass and containing a XR. Te cellular NADPH pool can be replenished by a mixture of C6 and C5- sugars and lignocellulose-derived number of pathways including PPP, the malic enzyme inhibitors); (ii) energy-consuming aerated cultivation; (ME) transhydrogenase cycle or the cytoplasmatic (iii) production of oil with high value compositional fatty NADP+-dependent isocitrate dehydrogenase. While acid (FA) profle including n − 3 long-chain polyunsatu- xylose utilization by oleaginous yeasts is poorly under- rated FA; (iv) production of value-added by-products. stood, a number of studies have investigated xylose Te basidiomycete yeast R. toruloides is relatively fast metabolism in ethanol-producing yeasts: e.g. in engi- growing, can assimilate pentoses [11], is resistant to lig- neered Saccharomyces cerevisiae [44], Scheferomy- nocellulosic inhibitors [16], has a benefcial FA compo- ces stipitis [45], and Ogataea polymorpha [46]. Tese sition [9] including n − 3 linolenic acid, and produces studies reported that the carbon source afected levels carotenoids [17]. R. toruloides is one of the most promis- of proteins involved in e.g. glucose repression, sugar ing species for lipid production with reported lipid yields transport, gluconeogenesis and amino acid catabolism. of up to 65.5% DCW under high cell density cultivation In the current study, comparative whole-proteome with low-nitrogen feeding [18]. analysis was employed to study lipid formation in R. Rhodotorula toruloides was originally applied in toruloides cultivated on either glucose or xylose as sole 1980 as a production organism for industrial-scale carbon source under conditions of nitrogen limitation. Tiukova et al. Biotechnol Biofuels (2019) 12:137 Page 3 of 17 Results or 40 h) and lipid production phase (64 or 96 h), with the Batch cultivation of Rhodotorula toruloides later sampling-points referring to xylose, as indicated in Growth,
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