Enhanced Triacylglycerol Production With Genetically Modified Trichosporon oleaginosus.
ABSTRACT: Mitochondrial pyruvate dehydrogenase (PDH) is important in the production of lipids in oleaginous yeast, but other yeast may bypass the mitochondria (PDH bypass), converting pyruvate in the cytosol to acetaldehyde, then acetate and acetyl CoA which is further converted to lipids. Using a metabolic model based on the oleaginous yeast Yarrowia lipolytica, we found that introduction of this bypass to an oleaginous yeast should result in enhanced yield of triacylglycerol (TAG) on substrate. Trichosporon oleaginosus (formerly Cryptococcus curvatus) is an oleaginous yeast which can produce TAGs from both glucose and xylose. Based on the sequenced genome, it lacks at least one of the enzymes needed to complete the PDH bypass, acetaldehyde dehydrogenase (ALD), and may also be deficient in pyruvate decarboxylase and acetyl-CoA synthetase under production conditions. We introduced these genes to T. oleaginosus in various combinations and demonstrated that the yield of TAG on both glucose and xylose was improved, particularly at high C/N ratio. Expression of a phospholipid:diacyltransferase encoding gene in conjunction with the PDH bypass further enhanced lipid production. The yield of TAG on xylose (0.27 g/g) in the engineered strain approached the theoretical maximum yield of 0.289 g/g. Interestingly, TAG production was also enhanced compared to the control in some strains which were given only part of the bypass pathway, suggesting that these genes may contribute to alternative routes to cytoplasmic acetyl CoA. The metabolic model indicated that the improved yield of TAG on substrate in the PDH bypass was dependent on the production of NADPH by ALD. NADPH for lipid synthesis is otherwise primarily supplied by the pentose phosphate pathway (PPP). This would contribute to the greater improvement of TAG production from xylose compared to that observed from glucose when the PDH bypass was introduced, since xylose enters metabolism through the non-oxidative part of the PPP. Yield of TAG from xylose in the engineered strains (0.21-0.27 g/g) was comparable to that obtained from glucose and the highest so far reported for lipid or TAG production from xylose.
Project description:In a recent study, it has been shown that biosynthesis of triacylglycerol (TAG) in the oleaginous green alga Chlorella desiccata is preceded by a large increase in acetyl-coenzyme A (Ac-CoA) levels and by upregulation of plastidic pyruvate dehydrogenase (ptPDH). It was proposed that the capacity to accumulate high TAG critically depends on enhanced production of Ac-CoA. In this study, two alternative Ac-CoA producers-plastidic Ac-CoA synthase (ptACS) and ATP citrate lyase (ACL)-are shown to be upregulated prior to TAG accumulation under nitrogen deprivation in the oleaginous species C. desiccata, but not in the moderate TAG accumulators Dunaliella tertiolecta and Chlamydomonas reinhardtii. Measurements of endogenous acetate production and of radiolabelled acetate incorporation into lipids are consistent with the upregulation of ptACS, but suggest that its contribution to the overall TAG biosynthesis is negligible. Induction of ACS and production of endogenous acetate are correlated with activation of alcohol dehydrogenase, suggesting that the upregulation of ptACS is associated with activation of PDH-bypass in C. desiccata. It is proposed that activation of the PDH-bypass in C. desiccata is needed to enable a high rate of lipid biosynthesis under nitrogen deprivation by controlling the level of pyruvate reaching ptPHD and/or mtPDH. This may be an important parameter for massive TAG accumulation in microalgae.
Project description:<h4>Background</h4>Oleaginous yeasts are promising microbial platforms for sustainable, bio-based production of biofuels and oleochemical building blocks. Bio-based residues provide sustainable and cost-effective carbon sources for fermentative yeast oil production without land-use change. Considering the regional abundancy of different waste streams, we chose complex biomass residue streams of marine origin; macroalgae hydrolysate, and terrestrial origin; wheat straw hydrolysate in the presence, and absence of corn steep liquor as a complex nitrogen source. We investigated the biomass and lipid yields of an array of well-described oleaginous yeasts; R. glutinis, T. asahii, R. mucilaginosa, R. toruloides, C. oleaginosus growing on these hydrolysates. Furthermore, their sugar utilization, fatty acid profile, and inhibitory effect of the hydrolysates on yeast growth were compared. For correlative reference, we initially performed comparative growth experiments for the strains on individual monomeric sugars separately. Each of these monomeric sugars was a dominant carbon source in the complex biomass hydrolysates evaluated in this study. In addition, we evaluated N-acetylglucosamine, the monomeric building block of chitin, as a low-cost nitrogen and carbon source in yeast fermentation.<h4>Results</h4>C. oleaginosus provided the highest biomass and lipid yields. In the wheat straw and brown algae hydrolysates, this yeast strain gained 7.5 g/L and 3.8 g/L lipids, respectively. Cultivation in algae hydrolysate resulted in a higher level of unsaturated fatty acids in the lipids accumulated by all yeast strains. R. toruloides and C. oleaginosus were able to effectively co-utilize mannitol, glucose, and xylose. Growth rates on wheat straw hydrolysate were enhanced in presence of corn steep liquor.<h4>Conclusions</h4>Among the yeast strains investigated in this study, C. oleaginosus proved to be the most versatile strain in terms of substrate utilization, productivity, and tolerance in the complex media. Various fatty acid profiles obtained on each substrate encourage the manipulation of culture conditions to achieve the desired fatty acid composition for each application. This could be accomplished by combining the element of carbon source with other formerly studied factors such as temperature and oxygen. Moreover, corn steep liquor showed promise for enhancement of growth in the oleaginous strains provided that carbon substrate is available.
Project description:<h4>Background</h4>Cutaneotrichosporon oleaginosus ATCC 20509 is a fast-growing oleaginous basidiomycete yeast that is able to grow in a wide range of low-cost carbon sources including crude glycerol, a byproduct of biodiesel production. When glycerol is used as a carbon source, this yeast can accumulate more than 50% lipids (w/w) with high concentrations of mono-unsaturated fatty acids.<h4>Results</h4>To increase our understanding of this yeast and to provide a knowledge base for further industrial use, a FAIR re-annotated genome was used to build a genome-scale, constraint-based metabolic model containing 1553 reactions involving 1373 metabolites in 11 compartments. A new description of the biomass synthesis reaction was introduced to account for massive lipid accumulation in conditions with high carbon-to-nitrogen (C/N) ratio in the media. This condition-specific biomass objective function is shown to better predict conditions with high lipid accumulation using glucose, fructose, sucrose, xylose, and glycerol as sole carbon source.<h4>Conclusion</h4>Contributing to the economic viability of biodiesel as renewable fuel, C. oleaginosus ATCC 20509 can effectively convert crude glycerol waste streams in lipids as a potential bioenergy source. Performance simulations are essential to identify optimal production conditions and to develop and fine tune a cost-effective production process. Our model suggests ATP-citrate lyase as a possible target to further improve lipid production.
Project description:UNLABELLED:Microbial fermentation of agro-industrial waste holds great potential for reducing the environmental impact associated with the production of lipids for industrial purposes from plant biomass. However, the chemical complexity of many residues currently prevents efficient conversion into lipids, creating a high demand for strains with the ability to utilize all energy-rich components of agricultural residues. Here, we present results of genome and transcriptome analyses of Trichosporon oleaginosus. This oil-accumulating yeast is able to grow on a wide variety of substrates, including pentoses and N-acetylglucosamine, making it an interesting candidate for biotechnological applications. Transcriptomics shows specific changes in gene expression patterns under lipid-accumulating conditions. Furthermore, gene content and expression analyses indicate that T. oleaginosus is well-adapted for the utilization of chitin-rich biomass. We also focused on the T. oleaginosus mating type, because this species is a member of the Tremellomycetes, a group that has been intensively analyzed as a model for the evolution of sexual development, the best-studied member being Cryptococcus neoformans. The structure of the T. oleaginosus mating-type regions differs significantly from that of other Tremellomycetes and reveals a new evolutionary trajectory paradigm. Comparative analysis shows that recruitment of developmental genes to the ancestral tetrapolar mating-type loci occurred independently in the Trichosporon and Cryptococcus lineages, supporting the hypothesis of a trend toward larger mating-type regions in fungi. IMPORTANCE:Finite fossil fuel resources pose sustainability challenges to society and industry. Microbial oils are a sustainable feedstock for biofuel and chemical production that does not compete with food production. We describe genome and transcriptome analyses of the oleaginous yeast Trichosporon oleaginosus, which can accumulate up to 70% of its dry weight as lipids. In contrast to conventional yeasts, this organism not only shows an absence of diauxic effect while fermenting hexoses and pentoses but also effectively utilizes xylose and N-acetylglucosamine, which are building blocks of lignocellulose and chitin, respectively. Transcriptome analysis revealed metabolic networks that govern conversion of xylose or N-acetylglucosamine as well as lipid accumulation. These data form the basis for a targeted strain optimization strategy. Furthermore, analysis of the mating type of T. oleaginosus supports the hypothesis of a trend toward larger mating-type regions in fungi, similar to the evolution of sex chromosomes in animals and plants.
Project description:Cocoa butter (CB) extracted from cocoa beans is the main raw material for chocolate production. However, growing chocolate demands and limited CB production has resulted in a shortage of CB supply. CB is mainly composed of three different kinds of triacylglycerols (TAGs), POP (C16:0-C18:1-C16:0), POS (C16:0-C18:1-C18:0), and SOS (C18:0-C18:1-C18:0). The storage lipids of yeasts, mainly TAGs, also contain relative high-level of C16 and C18 fatty acids and might be used as CB-like lipids (CBL). In this study, we cultivated six different yeasts, including one non-oleaginous yeast strain, Saccharomyces cerevisiae CEN.PK113-7D, and five oleaginous yeast strains, Trichosporon oleaginosus DSM11815, Rhodotorula graminis DSM 27356, Lipomyces starkeyi DSM 70296, Rhodosporidium toruloides DSM 70398, and Yarrowia lipolytica CBS 6124, in nitrogen-limited medium and compared their CBL production ability. Under the same growth conditions, we found that TAGs were the main lipids in all six yeasts and that T. oleaginosus can produce more TAGs than the other five yeasts. Less than 3% of the total TAGs were identified as potential SOS in the six yeasts. However, T. oleaginosus produced 27.8% potential POP and POS at levels of 378 mg TAGs/g dry cell weight, hinting that this yeast may have potential as a CBL production host after further metabolic engineering in future.
Project description:In the last decades, microbial oils have been extensively investigated as a renewable platform for biofuel and oleochemical production. Offering a potent alternative to plant-based oils, oleaginous microorganisms have been the target of ongoing metabolic engineering aimed at increasing growth and lipid yields, in addition to specialty fatty acids. Discovery proteomics is an attractive tool for elucidating lipogenesis and identifying metabolic bottlenecks, feedback regulation, and competing biosynthetic pathways. One prominent microbial oil producer is Cutaneotrichosporon oleaginosus, due to its broad feedstock catabolism and high lipid yield. However, this yeast has a recalcitrant cell wall and high cell lipid content, which complicates efficient and unbiased protein extraction for downstream proteomic analysis. Optimization efforts of protein sample preparation from C. oleaginosus in the present study encompasses the comparison of 8 lysis methods, 13 extraction buffers, and 17 purification methods with respect to protein abundance, proteome coverage, applicability, and physiochemical properties (pI, MW, hydrophobicity in addition to COG, and GO analysis). The optimized protocol presented in this work entails a one-step extraction method utilizing an optimal lysis method (liquid homogenization), which is augmented with a superior extraction buffer (50 mM Tris, 8/2 M Urea/Thiourea, and 1% C7BzO), followed by either of 2 advantageous purification methods (hexane/ethanol or TCA/acetone), depending on subsequent applications and target studies. This work presents a significant step forward towards implementation of efficient C. oleaginosus proteome mining for the identification of potential targets for genetic optimization of this yeast to improve lipogenesis and production of specialty lipids. Graphical abstract.
Project description:There is growing interest in using oleaginous yeast for the production of a variety of fatty acids and fatty acid-derived oleochemicals. This is motivated by natural propensity for high flux through lipid biosynthesis that has naturally evolved, making them a logical starting point for additional genetic engineering to improve titers and productivities. Much of the academic and industrial focus has centered on yeast that have significant genetic engineering tool capabilities, such as Yarrowia lipolytica, and those that have naturally high lipid accumulation, such as Rhodosporidium toruloides and Lipomyces starkeyi; however, there are oleaginous yeast with phenotypes better aligned with typically inhibitory process conditions, such as high salt concentrations and lignocellulosic derived inhibitors. This review addresses the foundational work in characterizing two emerging oleaginous yeast of interest: Debaryomyces hansenii and Trichosporon oleaginosus. We focus on the physiological and metabolic properties of these yeast that make each attractive for bioprocessing of lignocellulose to fuels and chemicals, discuss their respective genetic engineering tools and highlight the critical barriers facing the broader implementation of these oleaginous yeast.
Project description:<h4>Background</h4> The oleaginous yeast Cutaneotrichosporon oleaginosus represents one of the most promising microbial platforms for resource-efficient and scalable lipid production, with the capacity to accept a wide range of carbohydrates encapsulated in complex biomass waste or lignocellulosic hydrolysates. Currently, data related to molecular aspects of the metabolic utilisation of oligomeric carbohydrates are sparse. In addition, comprehensive proteomic information for C. oleaginosus focusing on carbohydrate metabolism is not available. <h4>Results</h4> In this study, we conducted a systematic analysis of carbohydrate intake and utilisation by C. oleaginosus and investigated the influence of different di- and trisaccharide as carbon sources. Changes in the cellular growth and morphology could be observed, depending on the selected carbon source. The greatest changes in morphology were observed in media containing trehalose. A comprehensive proteomic analysis of secreted, cell wall-associated, and cytoplasmatic proteins was performed, which highlighted differences in the composition and quantity of secreted proteins, when grown on different disaccharides. Based on the proteomic data, we performed a relative quantitative analysis of the identified proteins (using glucose as the reference carbon source) and observed carbohydrate-specific protein distributions. When using cellobiose or lactose as the carbon source, we detected three- and five-fold higher diversity in terms of the respective hydrolases released. Furthermore, the analysis of the secreted enzymes enabled identification of the motif with the consensus sequence LALL[LA]L[LA][LA]AAAAAAA as a potential signal peptide. <h4>Conclusions</h4> Relative quantification of spectral intensities from crude proteomic datasets enabled the identification of new enzymes and provided new insights into protein secretion, as well as the molecular mechanisms of carbo-hydrolases involved in the cleavage of the selected carbon oligomers. These insights can help unlock new substrate sources for C. oleaginosus, such as low-cost by-products containing difficult to utilize carbohydrates. In addition, information regarding the carbo-hydrolytic potential of C. oleaginosus facilitates a more precise engineering approach when using targeted genetic approaches. This information could be used to find new and more cost-effective carbon sources for microbial lipid production by the oleaginous yeast C. oleaginosus. <h4>Supplementary Information</h4> The online version contains supplementary material available at 10.1186/s12934-021-01692-2.
Project description:Cardiolipin (CL), the signature phospholipid of mitochondrial membranes, plays an important role in mitochondrial processes and bioenergetics. CL is synthesized de novo and undergoes remodeling in the mitochondrial membranes. Perturbation of CL remodeling leads to the rare X-linked genetic disorder Barth syndrome, which shows disparities in clinical presentation. To uncover biochemical modifiers that exacerbate CL deficiency, we carried out a synthetic genetic array screen to identify synthetic lethal interactions with the yeast CL synthase mutant crd1?. The results indicated that crd1? is synthetically lethal with mutants in pyruvate dehydrogenase (PDH), which catalyzes the conversion of pyruvate to acetyl-CoA. Acetyl-CoA levels were decreased in the mutant. The synthesis of acetyl-CoA depends primarily on the PDH-catalyzed conversion of pyruvate in the mitochondria and on the PDH bypass in the cytosol, which synthesizes acetyl-CoA from acetate. Consistent with perturbation of the PDH bypass, crd1? cells grown on acetate as the sole carbon source exhibited decreased growth, decreased acetyl-CoA, and increased intracellular acetate levels resulting from decreased acetyl-CoA synthetase activity. PDH mRNA and protein levels were up-regulated in crd1? cells, but PDH enzyme activity was not increased, indicating that PDH up-regulation did not compensate for defects in the PDH bypass. These findings demonstrate for the first time that CL is required for acetyl-CoA synthesis, which is decreased in CL-deficient cells as a result of a defective PDH bypass pathway.
Project description:Previous studies reported that the use of Metschnikowia pulcherrima in sequential culture fermentation with Saccharomyces cerevisiae mainly induced a reduction of volatile acidity in wine. The impact of the presence of this yeast on the metabolic pathway involved in pyruvate dehydrogenase (PDH) bypass and glycerol production in S. cerevisiae has never been investigated. In this work, we compared acetic acid and glycerol production kinetics between pure S. cerevisiae culture and its sequential culture with M. pulcherrima during alcoholic fermentation. In parallel, the expression levels of the principal genes involved in PDH bypass and glyceropyruvic fermentation in S. cerevisiae were investigated. A sequential culture of M. pulcherrima/S. cerevisiae at an inoculation ratio of 10:1 produced 40% less acetic acid than pure S. cerevisiae culture and led to the enhancement of glycerol content (12% higher). High expression levels of pyruvate decarboxylase PDC1 and PDC5, acetaldehyde dehydrogenase ALD6, alcohol dehydrogenase ADH1 and glycerol-3-phosphate dehydrogenase PDC1 genes during the first 3 days of fermentation in sequential culture conditions are highlighted. Despite the complexity of correlating gene expression levels to acetic acid formation kinetics, we demonstrate that the acetic acid production pathway is altered by sequential culture conditions. Moreover, we show for the first time that the entire acetic acid and glycerol metabolic pathway can be modulated in S. cerevisiae by the presence of M. pulcherrima at the beginning of fermentation.