Activating and Elucidating Metabolism of Complex Sugars in Yarrowia lipolytica

ABSTRACTThe oleaginous yeastYarrowia lipolyticais an industrially important host for production of organic acids, oleochemicals, lipids, and proteins with broad biotechnological applications. Albeit known for decades, the unique native metabolism ofY. lipolyticafor using complex fermentable sugars, which are abundant in lignocellulosic biomass, is poorly understood. In this study, we activated and elucidated the native sugar metabolism inY. lipolyticafor cell growth on xylose and cellobiose as well as their mixtures with glucose through comprehensive metabolic and transcriptomic analyses. We identified 7 putative glucose-specific transporters, 16 putative xylose-specific transporters, and 4 putative cellobiose-specific transporters that are transcriptionally upregulated for growth on respective single sugars.Y. lipolyticais capable of using xylose as a carbon source, but xylose dehydrogenase is the key bottleneck of xylose assimilation and is transcriptionally repressed by glucose.Y. lipolyticahas a set of 5 extracellular and 6 intracellular β-glucosidases and is capable of assimilating cellobiose via extra- and intracellular mechanisms, the latter being dominant for growth on cellobiose as a sole carbon source. Strikingly,Y. lipolyticaexhibited enhanced sugar utilization for growth in mixed sugars, with strong carbon catabolite activation for growth on the mixture of xylose and cellobiose and with mild carbon catabolite repression of glucose on xylose and cellobiose. The results of this study shed light on fundamental understanding of the complex native sugar metabolism ofY. lipolyticaand will help guide inverse metabolic engineering ofY. lipolyticafor enhanced conversion of biomass-derived fermentable sugars to chemicals and fuels.

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Snf1 Is a Regulator of Lipid Accumulation in Yarrowia lipolytica

Applied and Environmental Microbiology ◽

10.1128/aem.02079-13 ◽

2013 ◽

Vol 79 (23) ◽

pp. 7360-7370 ◽

Cited By ~ 54

Author(s):

John Seip ◽

Raymond Jackson ◽

Hongxian He ◽

Quinn Zhu ◽

Seung-Pyo Hong

Keyword(s):

Yarrowia Lipolytica ◽

Lipid Accumulation ◽

Oleaginous Yeast ◽

De Novo ◽

Lipid Synthesis ◽

Omega 3 ◽

Wild Type ◽

Content Type ◽

Reverse Transcription Pcr ◽

Dry Cell Weight

ABSTRACTIn the oleaginous yeastYarrowia lipolytica,de novolipid synthesis and accumulation are induced under conditions of nitrogen limitation (or a high carbon-to-nitrogen ratio). The regulatory pathway responsible for this induction has not been identified. Here we report that the SNF1 pathway plays a key role in the transition from the growth phase to the oleaginous phase inY. lipolytica. Strains with aY. lipolyticasnf1(Ylsnf1) deletion accumulated fatty acids constitutively at levels up to 2.6-fold higher than those of the wild type. When introduced into aY. lipolyticastrain engineered to produce omega-3 eicosapentaenoic acid (EPA),Ylsnf1deletion led to a 52% increase in EPA titers (7.6% of dry cell weight) over the control. Other components of theY. lipolyticaSNF1 pathway were also identified, and their function in limiting fatty acid accumulation is suggested by gene deletion analyses. Deletion of the gene encoding YlSnf4, YlGal83, or YlSak1 significantly increased lipid accumulation in both growth and oleaginous phases compared to the wild type. Furthermore, microarray and quantitative reverse transcription-PCR (qRT-PCR) analyses of theYlsnf1mutant identified significantly differentially expressed genes duringde novolipid synthesis and accumulation inY. lipolytica. Gene ontology analysis found that these genes were highly enriched with genes involved in lipid metabolism. This work presents a new role for Snf1/AMP-activated protein kinase (AMPK) pathways in lipid accumulation in this oleaginous yeast.

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Understanding and Eliminating the Detrimental Effect of Thiamine Deficiency on the Oleaginous Yeast Yarrowia lipolytica

Applied and Environmental Microbiology ◽

10.1128/aem.02299-19 ◽

2019 ◽

Vol 86 (3) ◽

Cited By ~ 1

Author(s):

Caleb Walker ◽

Seunghyun Ryu ◽

Richard J. Giannone ◽

Sergio Garcia ◽

Cong T. Trinh

Keyword(s):

Energy Metabolism ◽

Yarrowia Lipolytica ◽

Thiamine Deficiency ◽

Oleaginous Yeast ◽

De Novo ◽

Lipid Production ◽

Cellular Metabolism ◽

Living Cells ◽

Lipid Biosynthesis ◽

Content Type

ABSTRACT Thiamine is a vitamin that functions as a cofactor for key enzymes in carbon and energy metabolism in all living cells. While most plants, fungi, and bacteria can synthesize thiamine de novo, the oleaginous yeast Yarrowia lipolytica cannot. In this study, we used proteomics together with physiological characterization to elucidate key metabolic processes influenced and regulated by thiamine availability and to identify the genetic basis of thiamine auxotrophy in Y. lipolytica. Specifically, we found that thiamine depletion results in decreased protein abundance for the lipid biosynthesis pathway and energy metabolism (i.e., ATP synthase), leading to the negligible growth and poor sugar assimilation observed in our study. Using comparative genomics, we identified the missing 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase (THI13) gene for the de novo thiamine biosynthesis in Y. lipolytica and discovered an exceptional promoter, P3, that exhibits strong activation and tight repression by low and high thiamine concentrations, respectively. Capitalizing on the strength of our thiamine-regulated promoter (P3) to express the missing gene from Saccharomyces cerevisiae (scTHI13), we engineered a thiamine-prototrophic Y. lipolytica strain. By comparing this engineered strain to the wild-type strain, we revealed the tight relationship between thiamine availability and lipid biosynthesis and demonstrated enhanced lipid production with thiamine supplementation in the engineered thiamine-prototrophic Y. lipolytica strain. IMPORTANCE Thiamine plays a crucial role as an essential cofactor for enzymes involved in carbon and energy metabolism in all living cells. Thiamine deficiency has detrimental consequences for cellular health. Yarrowia lipolytica, a nonconventional oleaginous yeast with broad biotechnological applications, is a native thiamine auxotroph whose affected cellular metabolism is not well understood. Therefore, Y. lipolytica is an ideal eukaryotic host for the study of thiamine metabolism, especially because mammalian cells are also thiamine auxotrophic and thiamine deficiency is implicated in several human diseases. This study elucidates the fundamental effects of thiamine deficiency on cellular metabolism in Y. lipolytica and identifies genes and novel thiamine-regulated elements that eliminate thiamine auxotrophy in Y. lipolytica. Furthermore, the discovery of thiamine-regulated elements enables the development of thiamine biosensors with useful applications in synthetic biology and metabolic engineering.

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Exploring Proteomes of Robust Yarrowia lipolytica Isolates Cultivated in Biomass Hydrolysate Reveals Key Processes Impacting Mixed Sugar Utilization, Lipid Accumulation, and Degradation

mSystems ◽

10.1128/msystems.00443-21 ◽

2021 ◽

Author(s):

Caleb Walker ◽

Bruce Dien ◽

Richard J. Giannone ◽

Patricia Slininger ◽

Stephanie R. Thompson ◽

...

Keyword(s):

Lignocellulosic Biomass ◽

Yarrowia Lipolytica ◽

Lipid Accumulation ◽

Oleaginous Yeast ◽

Neutral Lipids ◽

Lipid Degradation ◽

Content Type ◽

Sugar Utilization ◽

Biomass Hydrolysates ◽

Mixed Sugar

Yarrowia lipolytica is an important industrial oleaginous yeast due to its robust phenotypes for effective conversion of inhibitory lignocellulosic biomass hydrolysates into neutral lipids. While lipid accumulation has been well characterized in this organism, its interconnected lipid degradation phenotype is poorly understood during fermentation of biomass hydrolysates.

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Engineering the oleaginous yeast Yarrowia lipolytica to produce limonene from waste cooking oil

Biotechnology for Biofuels ◽

10.1186/s13068-019-1580-y ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 12

Author(s):

Yaru Pang ◽

Yakun Zhao ◽

Shenglong Li ◽

Yu Zhao ◽

Jian Li ◽

...

Keyword(s):

Carbon Source ◽

Yarrowia Lipolytica ◽

Oleaginous Yeast ◽

Waste Cooking Oil ◽

Biologically Active ◽

Citrus Limon ◽

Mentha Spicata ◽

Cooking Oil ◽

Limonene Synthase ◽

Pharmaceutical Industries

Abstract Background Limonene is an important biologically active natural product widely used in the food, cosmetic, nutraceutical and pharmaceutical industries. However, the low abundance of limonene in plants renders their isolation from plant sources non-economically viable. Therefore, engineering microbes into microbial factories for producing limonene is fast becoming an attractive alternative approach that can overcome the aforementioned bottleneck to meet the needs of industries and make limonene production more sustainable and environmentally friendly. Results In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was successfully engineered to produce both d-limonene and l-limonene by introducing the heterologous d-limonene synthase from Citrus limon and l-limonene synthase from Mentha spicata, respectively. However, only 0.124 mg/L d-limonene and 0.126 mg/L l-limonene were produced. To improve the limonene production by the engineered yeast Y. lipolytica strain, ten genes involved in the mevalonate-dependent isoprenoid pathway were overexpressed individually to investigate their effects on limonene titer. Hydroxymethylglutaryl-CoA reductase (HMGR) was found to be the key rate-limiting enzyme in the mevalonate (MVA) pathway for the improving limonene synthesis in Y. lipolytica. Through the overexpression of HMGR gene, the titers of d-limonene and l-limonene were increased to 0.256 mg/L and 0.316 mg/L, respectively. Subsequently, the fermentation conditions were optimized to maximize limonene production by the engineered Y. lipolytica strains from glucose, and the final titers of d-limonene and l-limonene were improved to 2.369 mg/L and 2.471 mg/L, respectively. Furthermore, fed-batch fermentation of the engineered strains Po1g KdHR and Po1g KlHR was used to enhance limonene production in shake flasks and the titers achieved for d-limonene and l-limonene were 11.705 mg/L (0.443 mg/g) and 11.088 mg/L (0.385 mg/g), respectively. Finally, the potential of using waste cooking oil as a carbon source for limonene biosynthesis from the engineered Y. lipolytica strains was investigated. We showed that d-limonene and l-limonene were successfully produced at the respective titers of 2.514 mg/L and 2.723 mg/L under the optimal cultivation condition, where 70% of waste cooking oil was added as the carbon source, representing a 20-fold increase in limonene titer compared to that before strain and fermentation optimization. Conclusions This study represents the first report on the development of a new and efficient process to convert waste cooking oil into d-limonene and l-limonene by exploiting metabolically engineered Y. lipolytica strains for fermentation. The results obtained in this study lay the foundation for more future applications of Y. lipolytica in converting waste cooking oil into various industrially valuable products.

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Genome Sequence of the Oleaginous Yeast Yarrowia lipolytica H222

Microbiology Resource Announcements ◽

10.1128/mra.01547-18 ◽

2019 ◽

Vol 8 (4) ◽

Cited By ~ 4

Author(s):

Hugo Devillers ◽

Cécile Neuvéglise

Keyword(s):

Yarrowia Lipolytica ◽

Genome Sequence ◽

Genome Assembly ◽

Oleaginous Yeast ◽

De Novo ◽

Chromosomal Rearrangements ◽

Content Type ◽

Yeast Yarrowia Lipolytica

Here, we report the genome sequence of the oleaginous yeast Yarrowia lipolytica H222. De novo genome assembly shows three main chromosomal rearrangements compared to that of strain E150/CLIB122.

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Aeration and Stirring in Yarrowia lipolytica Lipase Biosynthesis during Batch Cultures with Waste Fish Oil as a Carbon Source

Fermentation ◽

10.3390/fermentation7020088 ◽

2021 ◽

Vol 7 (2) ◽

pp. 88

Author(s):

Paulina Snopek ◽

Dorota Nowak ◽

Bartłomiej Zieniuk ◽

Agata Fabiszewska

Keyword(s):

Carbon Source ◽

Fish Oil ◽

Yarrowia Lipolytica ◽

Lipolytic Activity ◽

Culture Conditions ◽

Agitation Speed ◽

Oxygen Deficit ◽

Shear Stresses ◽

Process Selection ◽

Batch Cultures

Yarrowia lipolytica is one of the most studied non-conventional forms of yeast, exhibiting a high secretory capacity and producing many industrially important and valuable metabolites. The yeast conceals a great biotechnological potential to synthesize organic acids, sweeteners, microbial oil, or fragrances. The vast majority of bioprocesses are carried out in bioreactors, where suitable culture conditions are provided. In the current study, the effect of agitation speed (200–600 rpm) and air flow rate (0.0375–2.0 dm3/(dm3 × min)) on the biomass yield and lipase activity of Y. lipolytica KKP 379 is analyzed in a growth medium containing waste fish oil. The increase of aeration intensity limited the period of oxygen deficit in the medium. Simultaneously, an increase in lipolytic activity was observed from 2.09 U/cm3 to 14.21 U/cm3; however, an excessive agitation speed likely caused oxidative or shear stresses, and a reduction in lipolytic activity was observed. Moreover, it is confirmed that the synthesis of lipases is related to oxygen consumption, pH, and the yeast growth phase, and appropriate process selection may provide two advantages, namely, the maximum use of the waste carbon source and the production of lipolytic enzymes that are valuable in many industries.

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Simultaneous lipid biosynthesis and recovery for oleaginous yeast Yarrowia lipolytica

Biotechnology for Biofuels ◽

10.1186/s13068-019-1576-7 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 3

Author(s):

Pratik Prashant Pawar ◽

Annamma Anil Odaneth ◽

Rajeshkumar Natwarlal Vadgama ◽

Arvind Mallinath Lali

Keyword(s):

Yarrowia Lipolytica ◽

Oil Recovery ◽

Oleaginous Yeast ◽

Fermentation Broth ◽

Continuous Production ◽

Oil Production ◽

Biofuel Production ◽

Oil Yield ◽

Microbial Oil

Abstract Background Recent trends in bioprocessing have underlined the significance of lignocellulosic biomass conversions for biofuel production. These conversions demand at least 90% energy upgradation of cellulosic sugars to generate renewable drop-in biofuel precursors (Heff/C ~ 2). Chemical methods fail to achieve this without substantial loss of carbon; whereas, oleaginous biological systems propose a greener upgradation route by producing oil from sugars with 30% theoretical yields. However, these oleaginous systems cannot compete with the commercial volumes of vegetable oils in terms of overall oil yields and productivities. One of the significant challenges in the commercial exploitation of these microbial oils lies in the inefficient recovery of the produced oil. This issue has been addressed using highly selective oil capturing agents (OCA), which allow a concomitant microbial oil production and in situ oil recovery process. Results Adsorbent-based oil capturing agents were employed for simultaneous in situ oil recovery in the fermentative production broths. Yarrowia lipolytica, a model oleaginous yeast, was milked incessantly for oil production over 380 h in a media comprising of glucose as a sole carbon and nutrient source. This was achieved by continuous online capture of extracellular oil from the aqueous media and also the cell surface, by fluidizing the fermentation broth over an adsorbent bed of oil capturing agents (OCA). A consistent oil yield of 0.33 g per g of glucose consumed, corresponding to theoretical oil yield over glucose, was achieved using this approach. While the incorporation of the OCA increased the oil content up to 89% with complete substrate consumptions, it also caused an overall process integration. Conclusion The nondisruptive oil capture mediated by an OCA helped in accomplishing a trade-off between microbial oil production and its recovery. This strategy helped in realizing theoretically efficient sugar-to-oil bioconversions in a continuous production process. The process, therefore, endorses a sustainable production of molecular drop-in equivalents through oleaginous yeasts, representing as an absolute microbial oil factory.

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The effect of Medicare Part D on prescription drug composition and demand

Journal of Economic Studies ◽

10.1108/jes-08-2013-0109 ◽

2015 ◽

Vol 42 (2) ◽

pp. 170-185 ◽

Cited By ~ 4

Author(s):

David Zimmer

Keyword(s):

Prescription Drug ◽

Medicare Part D ◽

Medicare Part ◽

Part D ◽

Content Type ◽

The Us ◽

Upper Respiratory ◽

Nationally Representative ◽

The Impact ◽

Shed Light

Purpose – The US Medicare Modernization Act of 2003 introduced optional prescription drug coverage, beginning in 2006, widely known as Medicare Part D. This paper uses up-to-date nationally representative survey data to investigate the impact of Part D not only on drug spending and consumption, but also on the composition of drug consumption. The paper aims to discuss these issues. Design/methodology/approach – Specifically, the paper investigates whether Part D impacted the number of therapeutic classes for which drugs were prescribed, and also whether Part D lead to increased usage of drugs for specific medical conditions that typically receive drug-intensive therapies. Findings – In addition to confirming findings from previous studies, this paper shows that Part D increased the number of therapeutic classes to which seniors receive drugs by approximately four classes. Part D also lead to increased usage of drugs used to treat upper respiratory disease, hypertension, and diabetes. Originality/value – While mostly concurring with previous studies on the spending impacts of Part D, this paper is the first to shed light on other impacts of Part D, specifically with respect to its impact on therapeutic classes for which drugs are prescribed.

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Stable isotope and chemical inhibition analyses suggested the existence of a non-mevalonate-like pathway in the yeast Yarrowia lipolytica

Scientific Reports ◽

10.1038/s41598-021-85170-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sivamoke Dissook ◽

Tomohisa Kuzuyama ◽

Yuri Nishimoto ◽

Shigeru Kitani ◽

Sastia Putri ◽

...

Keyword(s):

Yarrowia Lipolytica ◽

Oleaginous Yeast ◽

Isoprenoid Biosynthesis ◽

Mep Pathway ◽

Authentic Standard ◽

Chemical Inhibition ◽

Chromatographic Peaks ◽

Methyl Erythritol Phosphate ◽

Limiting Condition ◽

Yeast Yarrowia Lipolytica

AbstractMethyl erythritol phosphate (MEP) is the metabolite found in the MEP pathway for isoprenoid biosynthesis, which is known to be utilized by plants, algae, and bacteria. In this study, an unprecedented observation was found in the oleaginous yeast Yarrowia lipolytica, in which one of the chromatographic peaks was annotated as MEP when cultivated in the nitrogen limiting condition. This finding raised an interesting hypothesis of whether Y. lipolytica utilizes the MEP pathway for isoprenoid biosynthesis or not, because there is no report of yeast harboring the MEP pathway. Three independent approaches were used to investigate the existence of the MEP pathway in Y. lipolytica; the spiking of the authentic standard, the MEP pathway inhibitor, and the 13C labeling incorporation analysis. The study suggested that the mevalonate and MEP pathways co-exist in Y. lipolytica and the nitrogen limiting condition triggers the utilization of the MEP pathway in Y. lipolytica.

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Identification of the ATPase Subunit of the Primary Maltose Transporter in the Hyperthermophilic Anaerobe Thermotoga maritima

Applied and Environmental Microbiology ◽

10.1128/aem.00930-17 ◽

2017 ◽

Vol 83 (18) ◽

Cited By ~ 4

Author(s):

Raghuveer Singh ◽

Derrick White ◽

Paul Blum

Keyword(s):

Thermotoga Maritima ◽

Mutant Cell ◽

Sugar Metabolism ◽

Genetic System ◽

Gene Replacement ◽

Atpase Subunit ◽

Content Type ◽

Fermentative Hydrogen Production ◽

Atpase Subunits ◽

Fermentative Hydrogen

ABSTRACT Thermotoga maritima is a hyperthermophilic anaerobic bacterium that produces molecular hydrogen (H2) by fermentation. It catabolizes a broad range of carbohydrates through the action of diverse ABC transporters. However, in T. maritima and related species, highly similar genes with ambiguous annotation obscure a precise understanding of genome function. In T. maritima, three putative malK genes, all annotated as ATPase subunits, exhibited high identity to each other. To distinguish between these genes, malK disruption mutants were constructed by gene replacement, and the resulting mutant cell lines were characterized. Only a disruption of malK3 produced a defect in maltose catabolism. To verify that the mutant phenotype arose specifically from malK3 inactivation, the malK3 mutation was repaired by recombination, and maltose catabolism was restored. This study demonstrates the importance of a maltose ABC-type transporter and its relationship to sugar metabolism in T. maritima. IMPORTANCE The application and further development of a genetic system was used here to investigate gene paralogs in the hyperthermophile Thermotoga maritima. The occurrence of three ABC transporter ATPase subunits all annotated as malK was evaluated using a combination of genetic and bioinformatic approaches. The results clarify the role of only one malK gene in maltose catabolism in a nonmodel organism noted for fermentative hydrogen production.

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