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Non-conventional yeasts are an excellent option for a number of different industrial bioprocesses. They possess beneficial natural phenotypes, which translates to several fermentation advantages when compared to traditional hosts, like Saccharomyces cerevisiae. The non-conventional yeasts Yarrowia lipolytica, Trichosporon oleaginosus, Kluyveromyces marxianus, Dekkera bruxellensis, Pichia kudriavzevii, Debaryomyces hansenii and Hansenula polymorpha, are considered desirable industrial hosts due to their natural characteristics, including tolerance to several by-products and inhibitors, thermotolerance, salt resistance or osmo- and xerotolerance. Therefore, they are a great alternative for the industrial production of bioethanol, fine chemicals, lipids and recombinant proteins, among others. In this review, we summarize the best natural characteristics of those seven non-conventional yeasts and their use in industrial biotechnology, as well as the molecular/synthetic biology tools available for their genetic modification. Moreover, possible limitations regarding their performance in industrial fermentations and a list of challenges to overcome in the future are also discussed.

Abstract Background The oleaginous yeast, Trichosporon oleaginosus, has been extensively studied for its ability to metabolize non-conventional feedstocks. These include phenol-containing waste streams, such as distillery wastewater, or streams consisting of non-conventional sugars, such as hydrolyzed biomass and various bagasse. An initial BLAST search suggests this yeast has putative aromatic metabolizing genes. Given the desirability to valorize underutilized feedstocks such as lignin, we investigated the ability of T. oleaginosus to tolerate and metabolize lignin-derived aromatic compounds. Results Trichosporon oleaginosus can tolerate and metabolize model lignin monoaromatics and associated intermediates within funneling pathways. Growth rates and biomass yield were similar to glucose when grown in 4-hydroxybenzoic acid (pHBA) and resorcinol, but had an increased lag phase when grown in phenol. Oleaginous behavior was observed using resorcinol as a sole carbon source. Fed-batch feeding resulted in lipid accumulation of 69.5% on a dry weight basis. Conclusions Though the exact pathway of aromatic metabolism remains to be determined for T. oleaginosus, the results presented in this work motivate use of this organism for lignin valorization and phenolic wastewater bioremediation. Trichosporon oleaginosus is the first yeast shown to be oleaginous while growing on aromatic substrates, and shows great promise as a model industrial microbe for biochemical and biofuel production from depolymerized lignin.

Abstract Cutaneotrichosporon oleaginosus ATCC 20509, previously known as Trichosporon oleaginosus, Cryptococcus curvatus, Apiotrichum curvatum or Candida curvata D is an oleaginous yeast with several favorable qualities: it is fast growing, accumulates high amounts of lipid and has a very broad substrate spectrum. Its resistance to hydrolysis byproducts and genetic accessibility make it a promising cell factory for custom tailored microbial oils. However, literature about this organism is of varying degree of quality. Moreover, due to numerous changes of the species name, reports are highly scattered and poorly cited. This led to a poor integration of the findings into a unified body of knowledge. Particularly, errors in strain name usage and consequently citation are found even in most recent literature. To simplify future work, this review provides an overview of published studies and main findings regarding the metabolic capacities of C. oleaginosus.

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.

Trichosporon oleaginosus is one of the most promising hosts for microbial lipid production owing to its high-productivity; In an effort to develop an economical production process, we engineered T. oleaginosus towards high-lipid production from volatile fatty acids (VFA) derived from anaerobic fermentation of food waste. First, we established a method for labeling intracellular lipid with lipophilic BODIPY fluorescent dye. Next, a random library was constructed by treatment with a chemical mutagen, and high-lipid producers were screened using fluorescence-activated cell sorting. Subsequently, one clone, N14, was successfully isolated, which exhibited 3-fold higher lipid production (19.4%) in VFA (6 g/L) media than the wild-type strain, and also showed increased lipid production in higher concentrations of VFA (18 or 24 g/L). Based on fatty acid methyl ester (FAME) analysis, N14 contained higher stearic acid (C18:0) and oleic acid (C18:1) content compared with those of the wild-type strain. [ABSTRACT FROM AUTHOR]

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.

Background: The oleaginous yeast, Trichosporon oleaginosus, has been extensively studied for its ability to metabolize non-conventional feedstocks. These include phenol-containing waste streams, such as distillery wastewater, or streams consisting of non-conventional sugars, such as hydrolyzed biomass and various bagasse. An initial BLAST search suggests this yeast has putative aromatic metabolizing genes. Given the desirability to valorize underutilized feedstocks such as lignin, we investigated the ability of T. oleaginosus to tolerate and metabolize lignin-derived aromatic compounds. Results: Trichosporon oleaginosus can tolerate and metabolize model lignin monoaromatics and associated intermediates within funneling pathways. Growth rates and biomass yield were similar to glucose when grown in 4-hydroxybenzoic acid (pHBA) and resorcinol, but had an increased lag phase when grown in phenol. Oleaginous behavior was observed using resorcinol as a sole carbon source. Fed-batch feeding resulted in lipid accumulation of 69.5% on a dry weight basis. Conclusions: Though the exact pathway of aromatic metabolism remains to be determined for T. oleaginosus, the results presented in this work motivate use of this organism for lignin valorization and phenolic wastewater bioremediation. Trichosporon oleaginosus is the first yeast shown to be oleaginous while growing on aromatic substrates, and shows great promise as a model industrial microbe for biochemical and biofuel production from depolymerized lignin. [ABSTRACT FROM AUTHOR]

This study investigated the potential of corn bran as a feedstock for microbial lipid production using oleaginous yeast, Trichosporon oleaginosus ATCC20509. Different conditions (solid loading of biomass, acid loading, and pretreatment duration) were applied to optimize pretreatment processes using the Box-Behnken design. The highest sugar yield of 0.53 g/g was obtained from corn bran hydrolysates at a pretreatment condition of 5% solid loading and 1% acid loading for 30 min. Compared with synthetic media, up to 50% higher lipid accumulations in T. oleaginosus were achieved using corn bran hydrolysates during fermentation. Also, the direct effect of pretreatment condition on the lipid accumulation of T. oleaginosus was investigated using response surface methodology (RSM). Solid loading of biomass during the pretreatment process significantly affected the fermentation process for lipid accumulation of T. oleaginosus . The RSM model can provide useful information to design an integrated bioconversion platform. [ABSTRACT FROM AUTHOR]

Cutaneotrichosporon oleaginosus ATCC 20509, previously known as Trichosporon oleaginosus, Cryptococcus curvatus, Apiotrichum curvatum or Candida curvata D is an oleaginous yeast with several favorable qualities: it is fast growing, accumulates high amounts of lipid and has a very broad substrate spectrum. Its resistance to hydrolysis byproducts and genetic accessibility make it a promising cell factory for custom tailored microbial oils. However, literature about this organism is of varying degree of quality. Moreover, due to numerous changes of the species name, reports are highly scattered and poorly cited. This led to a poor integration of the findings into a unified body of knowledge. Particularly, errors in strain name usage and consequently citation are found even in most recent literature. To simplify future work, this review provides an overview of published studies and main findings regarding the metabolic capacities of C. oleaginosus. [ABSTRACT FROM AUTHOR]

The influence of cultural conditions on lipid production was investigated in two species, Trichosporon oleaginosus and Rhodosporidium azoricum . We showed that nitrogen limitation is not the main factor triggering the mechanism of lipid accumulation in T. oleaginosus . Moreover, a scarce availability of oxygen negatively affected lipid synthesis to a lesser extent in T. oleaginosus than in R. azoricum. This highlights how the importance of controlling fermentation parameters is strictly linked to the yeast species employed. We showed that these parameters affect the activity of important enzymes, influencing the metabolic fluxes into different pathways, in particular pentose phosphate pathway and cytoplasmic pyruvate bypass. Furthermore, T. oleaginosus exhibited wider substrate flexibility, faster growth and higher lipid accumulation in fed-batch cultivation. Microbial oils obtained from both yeasts proved a valuable feedstock, alternative to vegetable oils, for advanced diesel biofuel production. [ABSTRACT FROM AUTHOR]

BACKGROUND Biodiesel is an alternative to conventional fossil diesel fuel. Currently, it is mainly produced from vegetable oils and consequently it has an adverse impact on food price. Lipids from oleaginous microorganisms (e.g. microalgae, bacteria, fungi and yeasts) could be an alternative feedstock for biodiesel production and therefore the growth and lipid accumulation of Trichosporon oleaginosus was studied under different conditions. RESULTS The growth of T. oleaginosus on the xylose containing media enhanced the lipid accumulation and reduced cell growth, while on the glucose-containing media the opposite effect was observed. Trichosporon oleaginosus can accumulate above 50% of total lipids in dry cell mass during continuous and batch culture in the nitrogen-limited conditions. In the continuous culture relatively high productivity of total lipid accumulation (0.67 g dm−3 h−1) was observed on the glucose containing media. The type of substrate limitation has considerable impact on the fatty acid composition of yeast cell lipids. The predicted biodiesel properties (based on the fatty acids composition of lipids) were found to meet the CEN quality standards. CONCLUSION The results obtained show that T. oleaginosus has great potential for feasible biodiesel production, especially when sugars from industrial and agricultural waste are used as a substrate. © 2016 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Background: Oleaginous organisms are a promising, renewable source of single cell oil. Lipid accumulation is mainly induced by limitation of nutrients such as nitrogen, phosphorus or sulfur. The oleaginous yeast Trichosporon oleaginosus accumulates up to 70% w/w lipid under nitrogen stress, while cultivation in non-limiting media only yields 9% w/w lipid. Uncoupling growth from lipid accumulation is key for the industrial process applicability of oleaginous yeasts. This study evaluates the effects of rapamycin on TOR specific signaling pathways associated with lipogenesis in Trichosporon oleaginosus for the first time. Results: Supplementation of rapamycin to nutrient rich cultivation medium led to an increase in lipid yield of up to 38% g/L. This effect plateaued at 40 μM rapamycin. Interestingly, the fatty acid spectrum resembled that observed with cultivation under nitrogen limitation. Significant changes in growth characteristics included a 19% increase in maximum cell density and a 12% higher maximum growth rate. T. oleaginosus only has one Tor gene much like the oleaginous yeast Rhodosporidium toruloides. Consequently, we analyzed the effect of rapamycin on T. oleaginosus specific TORC signaling using bioinformatic methodologies. Conclusions: We confirm, that target of rapamycin complex 1 (TORC1) is involved in control of lipid production and cell proliferation in T. oleaginosus and present a homology based signaling network. Signaling of lipid induction by TORC1 and response to carbon depletion to this complex appear to be conserved, whereas response to nitrogen limitation and autophagy are not. This work serves as a basis for further investigation regarding the control and induction of lipid accumulation in oil yeasts. [ABSTRACT FROM AUTHOR]

Lipid production by Trichosporon oleaginosus was first studied in fed-batch operated stirred-tank bioreactors on a milliliter- and liter-scale making use of typical sugar monomers and a sugar mixture that may be derived from microalgae biomass hydrolysis after the extraction of lipids. 20.3 g L −1 lipids (58% of dry cell mass) were produced with T. oleaginosus in a defined medium at nitrogen starvation in the fed-batch process with a typical microalgae derived carbohydrate mixture (60% glucose, 20% mannose, 20% galactose). Real microalgae hydrolysate resulted in superior growth of T. oleaginosus but no enhanced lipid formation was possible due to nitrogen and phosphorus excess in the hydrolysate. Phosphate precipitation and the application of a continuously operated membrane bioreactor with total cell retention due to the low sugar concentrations (∼40 g L −1 ) in the microalgae hydrolysate resulted in the production of 30 g L −1 lipids (53% of dry cell mass) with T. oleaginosus at high space-time-yields of 0.33 g lipids L −1 h −1 . A high apparent lipid yield of 0.43 g lipids g −1 sugars consumed (130% of the theoretical maximum) was achieved with the microalgae hydrolysate most likely due to the additional conversion of other carbon sources (e.g. uronic acids, peptides) in the hydrolysate. [ABSTRACT FROM AUTHOR]

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.

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., Competing Interests: Conflict of Interest: All authors declare no conflicts of interest in this paper.