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.

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 Valorization of Brewers’ Spent Grain for the Production of Lipids by Oleaginous Yeast

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3052 ◽  
Author(s):  
Alok Patel ◽  
Fabio Mikes ◽  
Saskja Bühler ◽  
Leonidas Matsakas
Keyword(s):  
Lipid Content ◽  
Oleaginous Yeast ◽  
Lipid Production ◽  
Enzymatic Saccharification ◽  
Dry Weight ◽  
Lipid Concentration ◽  
Microwave Assisted ◽  
Spent Grain ◽  
Synthetic Media ◽  
By Products

Brewers’ spent grain (BSG) accounts for 85% of the total amount of by-products generated by the brewing industries. BSG is a lignocellulosic biomass that is rich in proteins, lipids, minerals, and vitamins. In the present study, BSG was subjected to pretreatment by two different methods (microwave assisted alkaline pretreatment and organosolv) and was evaluated for the liberation of glucose and xylose during enzymatic saccharification trials. The highest amount of glucose (46.45 ± 1.43 g/L) and xylose (25.15 ± 1.36 g/L) were observed after enzymatic saccharification of the organosolv pretreated BSG. The glucose and xylose yield for the microwave assisted alkaline pretreated BSG were 34.86 ± 1.27 g/L and 16.54 ± 2.1 g/L, respectively. The hydrolysates from the organosolv pretreated BSG were used as substrate for the cultivation of the oleaginous yeast Rhodosporidium toruloides, aiming to produce microbial lipids. The yeast synthesized as high as 18.44 ± 0.96 g/L of cell dry weight and 10.41 ± 0.34 g/L lipids (lipid content of 56.45 ± 0.76%) when cultivated on BSG hydrolysate with a C/N ratio of 500. The cell dry weight, total lipid concentration and lipid content were higher compared to the results obtained when grown on synthetic media containing glucose, xylose or mixture of glucose and xylose. To the best of our knowledge, this is the first report using hydrolysates of organosolv pretreated BSG for the growth and lipid production of oleaginous yeast in literature. The lipid profile of this oleaginous yeast showed similar fatty acid contents to vegetable oils, which can result in good biodiesel properties of the produced biodiesel.

scholarly journals Mutant Variants of the Substrate-Binding Protein DppA fromEscherichia coliEnhance Growth on Nonstandard γ-Glutamyl Amide-Containing Peptides

2018 ◽  
Vol 84 (13) ◽  
Author(s):  
Tilmann Kuenzl ◽  
Xiaochun Li-Blatter ◽  
Puneet Srivastava ◽  
Piet Herdewijn ◽  
Timothy Sharpe ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Protein ◽  
Substrate Binding ◽  
Building Blocks ◽  
Side Chains ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Wide Range ◽  
Amino Acid Side Chains ◽  
Substrate Binding Protein

ABSTRACTThe import of nonnatural molecules is a recurring problem in fundamental and applied aspects of microbiology. The dipeptide permease (Dpp) ofEscherichia coliis an ABC-type multicomponent transporter system located in the cytoplasmic membrane, which is capable of transporting a wide range of di- and tripeptides with structurally and chemically diverse amino acid side chains into the cell. Given this low degree of specificity, Dpp was previously used as an entry gate to deliver natural and nonnatural cargo molecules into the cell by attaching them to amino acid side chains of peptides, in particular, the γ-carboxyl group of glutamate residues. However, the binding affinity of the substrate-binding protein dipeptide permease A (DppA), which is responsible for the initial binding of peptides in the periplasmic space, is significantly higher for peptides consisting of standard amino acids than for peptides containing side-chain modifications. Here, we used adaptive laboratory evolution to identify strains that utilize dipeptides containing γ-substituted glutamate residues more efficiently and linked this phenotype to different mutations in DppA.In vitrocharacterization of these mutants by thermal denaturation midpoint shift assays and isothermal titration calorimetry revealed significantly higher binding affinities of these variants toward peptides containing γ-glutamyl amides, presumably resulting in improved uptake and therefore faster growth in media supplemented with these nonstandard peptides.IMPORTANCEFundamental and synthetic biology frequently suffer from insufficient delivery of unnatural building blocks or substrates for metabolic pathways into bacterial cells. The use of peptide-based transport vectors represents an established strategy to enable the uptake of such molecules as a cargo. We expand the scope of peptide-based uptake and characterize in detail the obtained DppA mutant variants. Furthermore, we highlight the potential of adaptive laboratory evolution to identify beneficial insertion mutations that are unlikely to be identified with existing directed evolution strategies.

scholarly journals Harnessing the Power of Mutagenesis and Adaptive Laboratory Evolution for High Lipid Production by Oleaginous Microalgae and Yeasts

2020 ◽  
Vol 12 (12) ◽  
pp. 5125
Author(s):  
Neha Arora ◽  
Hong-Wei Yen ◽  
George P. Philippidis
Keyword(s):  
Genetic Engineering ◽  
Lipid Production ◽  
Random Mutagenesis ◽  
Strain Improvement ◽  
Production Costs ◽  
Screening Methods ◽  
Scale Production ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Oleaginous Microalgae

Oleaginous microalgae and yeasts represent promising candidates for large-scale production of lipids, which can be utilized for production of drop-in biofuels, nutraceuticals, pigments, and cosmetics. However, low lipid productivity and costly downstream processing continue to hamper the commercial deployment of oleaginous microorganisms. Strain improvement can play an essential role in the development of such industrial microorganisms by increasing lipid production and hence reducing production costs. The main means of strain improvement are random mutagenesis, adaptive laboratory evolution (ALE), and rational genetic engineering. Among these, random mutagenesis and ALE are straight forward, low-cost, and do not require thorough knowledge of the microorganism’s genetic composition. This paper reviews available mutagenesis and ALE techniques and screening methods to effectively select for oleaginous microalgae and yeasts with enhanced lipid yield and understand the alterations caused to metabolic pathways, which could subsequently serve as the basis for further targeted genetic engineering.

scholarly journals Optimization of carbon source efficiency for lipid production with the oleaginous yeast Saitozyma podzolica DSM 27192 applying automated continuous feeding

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Olga Gorte ◽  
Michaela Kugel ◽  
Katrin Ochsenreither
Keyword(s):  
Carbon Source ◽  
Process Optimization ◽  
Oleaginous Yeast ◽  
Lipid Production ◽  
Lipid Productivity ◽  
Sugar Uptake ◽  
Plant Oils ◽  
Microbial Lipids ◽  
Batch Phase ◽  
Xylonic Acid

Abstract Background Biotechnologically produced microbial lipids are of interest as potential alternatives for crude and plant oils. Their lipid profile is similar to plant oils and can therefore be a substitute for the production of biofuels, additives for food and cosmetics industry as well as building blocks for oleochemicals. Commercial microbial lipids production, however, is still not profitable and research on process optimization and cost reduction is required. This study reports on the process optimization using glucose or xylose with the unconventional oleaginous yeast Saitozyma podzolica DSM 27192 aiming to reduce the applied carbon source amount without sacrificing lipid productivity. Results By optimizing the process parameters temperature and pH, lipid productivity was enhanced by 40%. Thereupon, by establishing a two-phase strategy with an initial batch phase and a subsequent fed-batch phase for lipid production in which a constant sugar concentration of about 10 g/L was maintained, resulted in saving of ~ 41% of total glucose and ~ 26% of total xylose. By performing the automated continuous sugar feed the total sugar uptake was improved to ~ 91% for glucose and ~ 92% for xylose and thus, prevented waste of unused carbon source in the cultivation medium. In addition, reduced glucose cultivation resulted in to 28% higher cell growth and 19% increase of lipid titer. By using xylose, the by-product xylonic acid was identified for the first time as by-product of S. podzolica. Conclusions These findings provide a broad view of different cultivation process strategies with subsequent comparison and evaluation for lipid production with S. podzolica. Additionally, new biotechnological characteristics of this yeast were highlighted regarding the ability to produce valuable organic acids from sustainable and renewable sugars.

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.

scholarly journals Engineering improved ethylene production: Leveraging systems Biology and adaptive laboratory evolution

2021 ◽  
Author(s):  
Sophie Vaud ◽  
Nicole Pearcy ◽  
Marko Hanževački ◽  
Alexander M.W. Van Hagen ◽  
Salah Abdelrazig ◽  
...  
Keyword(s):  
Systems Biology ◽  
Ethylene Production ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution
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