scholarly journals Identifying carbohydrate-active enzymes of Cutaneotrichosporon oleaginosus using systems biology

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Tobias Fuchs ◽  
Felix Melcher ◽  
Zora Selina Rerop ◽  
Jan Lorenzen ◽  
Pariya Shaigani ◽  
...  
Keyword(s):  
Carbon Source ◽  
Molecular Mechanisms ◽  
Oleaginous Yeast ◽  
Consensus Sequence ◽  
Lipid Production ◽  
Carbon Sources ◽  
Cellular Growth ◽  
Biomass Waste ◽  
Systematic Analysis ◽  
Wide Range

Abstract Background 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. Results 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. Conclusions 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.

Partial purification and properties of pyruvate kinase and its regulatory role during lipid accumulation by the oleaginous yeast Rhodosporidium toruloides CBS 14

1985 ◽  
Vol 31 (5) ◽  
pp. 479-484 ◽  
Author(s):  
Christopher Thomas Evans ◽  
Colin Ratledge
Keyword(s):  
Pyruvate Kinase ◽  
Oleaginous Yeast ◽  
Lipid Production ◽  
Energy Charge ◽  
Rhodosporidium Toruloides ◽  
Regulatory Role ◽  
Relative Mass ◽  
Partial Purification ◽  
Wide Range ◽  
Fructose 1,6 Bisphosphate

Pyruvate kinase from the oleaginous yeast Rhodosporidium toruloides CBS 14 was partially purified and its properties investigated to determine its role during lipid production by this yeast. The enzyme (relative mass (Mr) = 190 000) showed a pH optimum of 8.0 and apparent Km values for K+, phosphoenolpyruvate (PEP), and ADP of 1.6 mM, 571 μM, and 120 μM, respectively. Enzyme activity was inhibited by citrate, isocitrate, ATP, GTP, and CTP and activated by fructose 1,6-bisphosphate, L-glutamate, and [Formula: see text] ions. Inhibition by citrate and ATP were both competitive with PEP with the Ki(citrate) = 340 μM and Ki(ATP) = 303 μM. The effect of ATP and cellular energy charge were critically dependent on the concentration of ADP present in the enzyme assay. Both L-glutamate and fructose 1,6-bisphosphate increased the affinity of the enzyme for both PEP and ADP and so were significant activators at nonsaturating substrate concentrations. [Formula: see text] ions increased the affinity of the enzyme for PEP, but not ADP. The modulation of pyruvate kinase activity by such a wide range of effectors is indicative of a major regulatory role in controlling the flux of carbon, through glycolysis, into lipid-synthesizing systems.

scholarly journals Production of galactitol from galactose by the oleaginous yeast Rhodosporidium toruloides IFO0880

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Sujit Sadashiv Jagtap ◽  
Ashwini Ashok Bedekar ◽  
Jing-Jing Liu ◽  
Yong-Su Jin ◽  
Christopher V. Rao
Keyword(s):  
Oleaginous Yeast ◽  
Lipid Production ◽  
Value Added ◽  
Building Blocks ◽  
Rhodosporidium Toruloides ◽  
Sugar Alcohols ◽  
Galactose Metabolism ◽  
Alcohol Production ◽  
Wide Range ◽  
Multiple Value

Abstract Background Sugar alcohols are commonly used as low-calorie sweeteners and can serve as potential building blocks for bio-based chemicals. Previous work has shown that the oleaginous yeast Rhodosporidium toruloides IFO0880 can natively produce arabitol from xylose at relatively high titers, suggesting that it may be a useful host for sugar alcohol production. In this work, we explored whether R. toruloides can produce additional sugar alcohols. Results Rhodosporidium toruloides is able to produce galactitol from galactose. During growth in nitrogen-rich medium, R. toruloides produced 3.2 ± 0.6 g/L, and 8.4 ± 0.8 g/L galactitol from 20 to 40 g/L galactose, respectively. In addition, R. toruloides was able to produce galactitol from galactose at reduced titers during growth in nitrogen-poor medium, which also induces lipid production. These results suggest that R. toruloides can potentially be used for the co-production of lipids and galactitol from galactose. We further characterized the mechanism for galactitol production, including identifying and biochemically characterizing the critical aldose reductase. Intracellular metabolite analysis was also performed to further understand galactose metabolism. Conclusions Rhodosporidium toruloides has traditionally been used for the production of lipids and lipid-based chemicals. Our work demonstrates that R. toruloides can also produce galactitol, which can be used to produce polymers with applications in medicine and as a precursor for anti-cancer drugs. Collectively, our results further establish that R. toruloides can produce multiple value-added chemicals from a wide range of sugars.

scholarly journals Rhodotorula toruloides Single Cell Oil Production Using Eucalyptus urograndis Hemicellulose Hydrolysate as a Carbon Source

Energies ◽  
2020 ◽  
Vol 13 (4) ◽  
pp. 795 ◽  
Author(s):  
Helberth Júnnior Santos Lopes ◽  
Nemailla Bonturi ◽  
Everson Alves Miranda
Keyword(s):  
Carbon Source ◽  
Lipid Production ◽  
Carbon Sources ◽  
Oil Production ◽  
Research Area ◽  
Microbial Oil ◽  
Hemicellulose Hydrolysate ◽  
Initial Ph ◽  
Different Temperatures ◽  
Active Research

Microbial oil is a potential substitute for vegetable oils in the biodiesel industry. Efforts to obtain cheap carbon sources for the cultivation of lipid-producing microorganisms comprise an active research area. This work aimed to extract the hemicellulose fraction from Eucalyptus uograndis and to use its hydrolysate as a carbon source for Rhodotorula toruloides (an oleaginous yeast) cultivation for microbial oil production. Hemicellulose hydrothermal extractions were performed at different temperatures, times, and ratios of solid to liquid (S/L). Temperature and time showed a stronger effect on the solubilization of hemicellulose. Hemicellulose extraction at 155 °C, 195 min, and an S/L ratio of 1/2 resulted in a hydrolysate with a xylose content of 37.0 g/l. R. toruloides cultivation in this hydrolysate showed that initial pH had a strong influence on cell growth. At an initial pH of 6.2, cells grew to 6.0 g/l of biomass with a lipid content of 50%. Therefore, we believe that E. urograndis hemicellulose hydrolysate could be a potential substrate for R. toruloides for lipid production based on the biorefinery concept.

scholarly journals A Comparative Review on Different Trophic Modes and Usable Carbon Sources for Microalgae Cultivation, Approaching to Optimize Lipid Production in Trade Scale

2020 ◽  
pp. 16-26
Author(s):  
Zahra Lari ◽  
Fatemeh Khosravitabar
Keyword(s):  
Carbon Source ◽  
Large Scale ◽  
Lipid Production ◽  
Carbon Sources ◽  
Point Of View ◽  
Microalgae Cultivation ◽  
Lipid Yield ◽  
Comparative Review

Microalgae are considered as an outstanding feedstock to produce high value lipid products like biodiesel and biomedicine. Reaching commercial maturity in this field is possible in the case of maximizing lipid yield and minimizing prime costs. In order to clarify the best features of carbon source (for microalgae cultivation) to reach optimum efficiency of biomass and lipid production, this paper reviews the merits and demerits of different trophic modes as well as type and concentration of carbon source. Furthermore carbon supplementation for large scale microalgae cultivation and lipid production is discussed as an economical point of view.

scholarly journals Rhodococcus as Biofactories for Microbial Oil Production

Molecules ◽  
2021 ◽  
Vol 26 (16) ◽  
pp. 4871
Author(s):  
Héctor M. Alvarez ◽  
Martín A. Hernández ◽  
Mariana P. Lanfranconi ◽  
Roxana A. Silva ◽  
María S. Villalba
Keyword(s):  
Molecular Mechanisms ◽  
Carbon Sources ◽  
Oil Production ◽  
Dry Weight ◽  
Industrial Wastes ◽  
Natural Environments ◽  
Microbial Oil ◽  
Qualitative And Quantitative ◽  
Physiological Properties ◽  
Wide Range

Bacteria belonging to the Rhodococcus genus are frequent components of microbial communities in diverse natural environments. Some rhodococcal species exhibit the outstanding ability to produce significant amounts of triacylglycerols (TAG) (>20% of cellular dry weight) in the presence of an excess of the carbon source and limitation of the nitrogen source. For this reason, they can be considered as oleaginous microorganisms. As occurs as well in eukaryotic single-cell oil (SCO) producers, these bacteria possess specific physiological properties and molecular mechanisms that differentiate them from other microorganisms unable to synthesize TAG. In this review, we summarized several of the well-characterized molecular mechanisms that enable oleaginous rhodococci to produce significant amounts of SCO. Furthermore, we highlighted the ability of these microorganisms to degrade a wide range of carbon sources coupled to lipogenesis. The qualitative and quantitative oil production by rhodococci from diverse industrial wastes has also been included. Finally, we summarized the genetic and metabolic approaches applied to oleaginous rhodococci to improve SCO production. This review provides a comprehensive and integrating vision on the potential of oleaginous rhodococci to be considered as microbial biofactories for microbial oil production.

scholarly journals (p)ppGpp and Its Role in Bacterial Persistence: New Challenges

2020 ◽  
Vol 64 (10) ◽  
Author(s):  
Olga Pacios ◽  
Lucia Blasco ◽  
Inés Bleriot ◽  
Laura Fernandez-Garcia ◽  
Antón Ambroa ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Osmotic Shock ◽  
Carbon Sources ◽  
Stringent Response ◽  
Temperature Shift ◽  
Bacterial Persistence ◽  
Development Of Resistance ◽  
Wide Range ◽  
Antibiotic Failure

ABSTRACT Antibiotic failure not only is due to the development of resistance by pathogens but can also often be explained by persistence and tolerance. Persistence and tolerance can be included in the “persistent phenotype,” with high relevance for clinics. Two of the most important molecular mechanisms involved in tolerance and persistence are toxin-antitoxin (TA) modules and signaling via guanosine pentaphosphate/tetraphosphate [(p)ppGpp], also known as “magic spot.” (p)ppGpp is a very important stress alarmone which orchestrates the stringent response in bacteria; hence, (p)ppGpp is produced during amino acid or fatty acid starvation by proteins belonging to the RelA/SpoT homolog family (RSH). However, (p)ppGpp levels can also accumulate in response to a wide range of signals, including oxygen variation, pH downshift, osmotic shock, temperature shift, or even exposure to darkness. Furthermore, the stringent response is not only involved in responses to environmental stresses (starvation for carbon sources, fatty acids, and phosphates or heat shock), but it is also used in bacterial pathogenesis, host invasion, and antibiotic tolerance and persistence. Given the exhaustive and contradictory literature surrounding the role of (p)ppGpp in bacterial persistence, and with the aim of summarizing what is known so far about the magic spot in this bacterial stage, this review provides new insights into the link between the stringent response and persistence. Moreover, we review some of the innovative treatments that have (p)ppGpp as a target, which are in the spotlight of the scientific community as candidates for effective antipersistence agents.

scholarly journals Draft Genome Sequence of the Oleaginous Yeast Cryptococcus albidus var. albidus

2016 ◽  
Vol 4 (3) ◽  
Author(s):  
Shashwat Vajpeyi ◽  
Kartik Chandran
Keyword(s):  
Genome Sequence ◽  
Oleaginous Yeast ◽  
Lipid Production ◽  
Draft Genome ◽  
Carbon Sources ◽  
Lipid Synthesis ◽  
Draft Genome Sequence ◽  
Bioprocess Development ◽  
Cryptococcus Albidus ◽  
Organic Carbon Sources

We report the complete draft genome sequence of Cryptococcus albidus var. albidus , an oleaginous yeast, which can utilize various organic carbon sources for lipid synthesis. Availability of this genome will help elucidate factors driving lipid accumulation in C. albidus and contribute toward bioprocess development and optimization for engineered lipid production.

scholarly journals Enhanced inhibitor tolerance and increased lipid productivity through adaptive laboratory evolution in the oleaginous yeast Metshnikowia pulcherrima

2020 ◽  
Author(s):  
Robert H. Hicks ◽  
Yuxin Sze ◽  
Christopher J. Chuck ◽  
Daniel A. Henk
Keyword(s):  
Formic Acid ◽  
Oleaginous Yeast ◽  
Lipid Production ◽  
Dry Weight ◽  
Lipid Productivity ◽  
Adaptive Laboratory Evolution ◽  
Stirred Tank Bioreactor ◽  
Inhibitor Tolerance ◽  
Laboratory Evolution ◽  
Wide Range

AbstractMicrobial lipid production from second generation feedstocks presents a sustainable route to future fuels, foods and bulk chemicals. The oleaginous yeast Metshnikowia pulcherrima has previously been investigated as a potential platform organism for lipid production due to its ability to be grown in non-sterile conditions and metabolising a wide range of oligo- and monosaccharide carbon sources within lignocellulosic hydrolysates. However, the generation of inhibitors from depolymerisation causes downstream bioprocessing complications, and despite M. pulcherrima’s comparative tolerance, their presence is deleterious to both biomass and lipid formation. Using either a single inhibitor (formic acid) or an inhibitor cocktail (formic acid, acetic acid, fufural and HMF), two strategies of adaptive laboratory evolution were performed to improve M. pulcherrima’s fermentation inhibitor tolerance. Using a sequential batch culturing approach, the resulting strains from both strategies had increased growth rates and reduced lag times under inhibiting conditions versus the progenitor. Interestingly, the lipid production of the inhibitor cocktail evolved strains markedly increased, with one strain producing 41% lipid by dry weight compared to 22% of the progenitor. The evolved species was cultured in a non-sterile 2L stirred tank bioreactor and accumulated lipid rapidly, yielding 6.1 g/L of lipid (35% cell dry weight) within 48 hours; a lipid productivity of 0.128 g L-1 h-1. Furthermore, the lipid profile was analogous to palm oil, consisting of 39% C16:0 and 56% C18:1 after 48 hours.

The Role of the ABL1/YAP1/P73 Axis in Prevention of DNA Damage-Mediated Apoptosis in Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 725-725
Author(s):  
Francesca Cottini ◽  
Teru Hideshima ◽  
Martin Sattler ◽  
Federico Caligaris-Cappio ◽  
Kenneth C. Anderson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Damage ◽  
Western Blot ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Caspase 3 ◽  
Nuclear Translocation ◽  
Cellular Growth ◽  
Wide Range ◽  
Low Expression

Abstract Abstract 725 Background: Genome integrity plays a crucial role in the development of normal plasma cells to eliminate aberrant ones. Multiple myeloma (MM) is a plasma cell malignancy characterized by complex heterogeneous cytogenetic abnormalities. MM cells show constitutive DNA Damage Response (DDR) and activate compensatory mechanisms to prevent DNA-damage mediated apoptosis. Here we define the molecular mechanisms of these protective effects. Methods: A panel of 15 MM cell lines was used. Blood and BM samples from healthy volunteers and MM patients were obtained after informed consent and subjected to Ficoll-Paque density sedimentation to get mononuclear cells (MNCs). Patient MM cells were isolated from BM MNCs by CD138-positive selection. Lentiviral delivery system was used for expression and knock-down of YAP1 in KMS-18, KMS-20, MM.1S and UTMC-2 MM cell lines. The biologic impact of YAP1 phenotype was evaluated using cell growth, viability and apoptosis assays. Results: We confirmed that a wide range of MM patient cells and MM cell lines have markers of constitutive DDR, including phosphorylation of H2A.X, ATM, ATR, Chk2 and Chk1, assessed by western blot analysis and immunofluorescence. However, these MM cells do not show basal level of apoptosis. Specifically, cleaved forms of caspase 3 and PARP are lacking in non-treated cells, and the absence of co-expression of cleaved caspase 3 with phospho-H2A.X by immunofluorescence confirms that phospho-H2A.X positive cells are viable cells. Since DDR is present in both p53-wild-type (wt) and p53-mutated cell lines, we examined whether ABL1/YAP1/p73 axis represents an alternative and crucial pathway to avoid DNA-damage mediated apoptosis. Indeed, ABL1 is up-regulated and predominantly localizes in the nucleus, a potential apoptotic stimulus, in MM cells, as assessed by western blot and immunofluorescence. To define if ABL1 nuclear localization was triggered by DDR, we treated MM cells with a specific ATM inhibitor (Ku55933) and a DNA damaging agent, doxorubicin. Inhibition of DDR in both p53 wt cell lines (MM.1S and H929) and p53 mutated cell lines (UTMC-2, JJN-3 and KMS-20) causes ABL1 cytoplasmic retention, while doxorubicin increases ABL1 nuclear translocation. Co-treatment with doxorubicin and ABL1 inhibitor STI-571 rescues MM cells from doxorubicin-mediated cell death. In particular, apoptotic cells decrease from 47.2% to 21.5% in U266, from 55.3% to 12.4% in MM.1S, and from 57.9% to 19.1% in UTMC-2 cells in response to combination treatment. To delineate the molecular mechanisms whereby MM cells repress ABL1 pro-apoptotic function, we focused on YAP1, a downstream target of the Hippo pathway involved in ABL1 cascade. We explored a large dataset of aCGH data on MM patients and discovered that YAP1 genomic locus (chr. 11q22) is deleted, associated with BIRC2 and BIRC3 in 11% of patients. This genetic abnormality was also found in KMS-18 and KMS-20 cell lines. Although YAP1 expression was normal in peripheral blood MNCs (PBMCs), its expression was decreased in the majority of patient MM cells and MM cell lines regardless of the presence of focal deletion. Importantly, low expression of YAP1 is associated with poor prognosis of MM patients. To further delineate the biologic significance of YAP1 in MM cells, we re-expressed pLENTI4-YAP1-EGFP in MM cell lines with either YAP1 deletion (KMS-18, KMS-20) or YAP1 low expression (MM.1S). We also silenced YAP1 using two different specific shRNAs in UTMC-2 MM cell line. As expected, YAP1 re-expression reduces cellular growth and increases apoptosis in all cell lines tested (25.7%, 37.1% and 32.3% apoptotic KMS-20, KMS-18 and MM.1S cells, respectively), condition that was further enhanced by doxorubicin treatment. Previous studies have shown that P73 is expressed in MM even though at low levels and we here show that they inversely correlate with YAP1 protein expression. Importantly, YAP1 re-expression increases p73 stability and promotes transcription of p73-target genes including BAX, PUMA and p21. In contrast, UTMC-2-YAP1−/− cells show improved survival with lower levels of basal apoptosis and higher resistance to treatment with bortezomib or doxorubicin. Conclusion: YAP1 mediates a strong apoptotic signal for MM cells. Thus, activation and/or overexpression of YAP1 represent a novel therapeutic strategy to improve outcome of patients with MM. Disclosures: Anderson: Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder and Scientific Founder, Scientific Founder and Scientific Founder Other.

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