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

2019 ◽  
Vol 86 (3) ◽  
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.

scholarly journals Understanding and eliminating the detrimental effect of endogenous thiamine auxotrophy on metabolism of the oleaginous yeast Yarrowia lipolytica

2019 ◽  
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 ◽  
Missing Gene

ABSTRACTThiamine is an essential vitamin that functions as a cofactor for key enzymes in carbon and energy metabolism for 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 understand key metabolic processes influenced and regulated by thiamine availability and identified the genetic basis of thiamine auxotrophy in Y. lipolytica. Specifically, we found thiamine depletion results in decreased protein abundance of the lipid biosynthesis pathways and energy metabolism (i.e., ATP synthase), attributing to the negligible growth and poor sugar assimilation observed in our study. Using comparative genomics, we identified the missing gene scTHI13, encoding the 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase for the de novo thiamine synthesis in Y. lipolytica, and discovered an exceptional promoter, P3, that exhibits strong activation or tight repression by low and high thiamine concentrations, respectively. Capitalizing on the strength of our thiamine-regulated promoter (P3) to express the missing gene, we engineered the first thiamine-prototrophic Y. lipolytica reported to date. By comparing this engineered strain to the wildtype, we unveiled the tight relationship linking thiamine availability to lipid biosynthesis and demonstrated enhanced lipid production with thiamine supplementation in the engineered thiamine-prototrophic Y. lipolytica.IMPORTANCEThiamine plays a crucial role as an essential cofactor for enzymes in carbon and energy metabolism of all living cells. Thiamine deficiency has detrimental consequences on cellular health. Yarrowia lipolytica, a non-conventional oleaginous yeast with broad biotechnological applications, is a native thiamine auxotroph, whose effects on cellular metabolism are not well understood. Therefore, Y. lipolytica is an ideal eukaryotic host to study thiamine metabolism, especially as mammalian cells are also thiamine-auxotrophic and thiamine deficiency is implicated in several human diseases. This study elucidates the fundamentals of thiamine deficiency on cellular metabolism of Y. lipolytica and identifies genes and novel thiamine-regulated elements that eliminate thiamine auxotrophy in Y. lipolytica. Furthermore, discovery of thiamine-regulated elements enables development of thiamine biosensors with useful applications in synthetic biology and metabolic engineering.

scholarly journals Snf1 Is a Regulator of Lipid Accumulation in Yarrowia lipolytica

2013 ◽  
Vol 79 (23) ◽  
pp. 7360-7370 ◽  
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.

scholarly journals Genome Sequence of the Oleaginous Yeast Yarrowia lipolytica H222

2019 ◽  
Vol 8 (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.

scholarly journals Two Phytoplasmas Elicit Different Responses in the Insect VectorEuscelidius variegatusKirschbaum

2018 ◽  
Vol 86 (5) ◽  
Author(s):  
Luciana Galetto ◽  
Simona Abbà ◽  
Marika Rossi ◽  
Marta Vallino ◽  
Massimo Pesando ◽  
...  
Keyword(s):  
Immune Response ◽  
Energy Metabolism ◽  
Pathogenic Bacteria ◽  
De Novo ◽  
Transcriptome Assembly ◽  
Content Type ◽  
Flavescence Dorée ◽  
Starting Point ◽  
Potential Pathogen ◽  
Insect Fitness

ABSTRACTPhytoplasmas are plant-pathogenic bacteria transmitted by hemipteran insects. The leafhopperEuscelidius variegatusis a natural vector of chrysanthemum yellows phytoplasma (CYp) and a laboratory vector of flavescence dorée phytoplasma (FDp). The two phytoplasmas induce different effects on this species: CYp slightly improves whereas FDp negatively affects insect fitness. To investigate the molecular bases of these different responses, transcriptome sequencing (RNA-seq) analysis ofE. variegatusinfected with either CYp or FDp was performed. The sequencing provided the firstde novotranscriptome assembly for a phytoplasma vector and a starting point for further analyses on differentially regulated genes, mainly related to immune system and energy metabolism. Insect phenoloxidase activity, immunocompetence, and body pigmentation were measured to investigate the immune response, while respiration and movement rates were quantified to confirm the effects on energy metabolism. The activation of the insect immune response upon infection with FDp, which is not naturally transmitted byE. variegatus, confirmed that this bacterium is mostly perceived as a potential pathogen. Conversely, the acquisition of CYp, which is naturally transmitted byE. variegatus, seems to increase the insect fitness by inducing a prompt response to stress. This long-term relationship is likely to improve survival and dispersal of the infected insect, thus enhancing the opportunity of phytoplasma transmission.

scholarly journals Activating and Elucidating Metabolism of Complex Sugars in Yarrowia lipolytica

2015 ◽  
Vol 82 (4) ◽  
pp. 1334-1345 ◽  
Author(s):  
Seunghyun Ryu ◽  
Julie Hipp ◽  
Cong T. Trinh
Keyword(s):  
Carbon Source ◽  
Yarrowia Lipolytica ◽  
Oleaginous Yeast ◽  
Sugar Metabolism ◽  
Fermentable Sugars ◽  
Content Type ◽  
Inverse Metabolic Engineering ◽  
Fundamental Understanding ◽  
Important Host ◽  
Shed Light

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.

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

mSystems ◽  
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.

scholarly journals Impact of Activation of the Neotrehalosadiamine/Kanosamine Biosynthetic Pathway on the Metabolism of Bacillus subtilis

2021 ◽  
Vol 203 (9) ◽  
Author(s):  
Natsumi Saito ◽  
Huong Minh Nguyen ◽  
Takashi Inaoka
Keyword(s):  
Bacillus Subtilis ◽  
Energy Metabolism ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Tca Cycle ◽  
Pentose Phosphate ◽  
Growth Defect ◽  
Metabolome Analysis ◽  
Content Type ◽  
Tca Cycle Intermediates

ABSTRACT The pentose phosphate (PP) pathway is one of the major sources of cellular NADPH. A Bacillus subtilis zwf mutant that lacks glucose-6-phosphate dehydrogenase (the enzyme that catalyzes the first step of the PP pathway) showed inoculum-dose-dependent growth. This growth defect was suppressed by glcP disruption, which causes the upregulation of the autoinducer neotrehalosadiamine (NTD)/kanosamine biosynthetic pathway. A metabolome analysis showed that the stimulation of NTD/kanosamine biosynthesis caused significant accumulation of tricarboxylic acid (TCA) cycle intermediates and NADPH. Because the major malic enzyme YtsJ concomitantly generates NADPH through malate-to-pyruvate conversion, de novo NTD/kanosamine biosynthesis can result in an increase in the intracellular NADPH pool via the accumulation of malate. In fact, a zwf mutant grew in malate-supplemented medium. Artificial induction of glcP in the zwf mutant caused a reduction in the intracellular NADPH pool. Moreover, the correlation between the expression level of the NTD/kanosamine biosynthesis operon ntdABC and the intracellular NADPH pool was confirmed. Our results suggest that NTD/kanosamine has the potential to modulate carbon energy metabolism through an autoinduction mechanism. IMPORTANCE Autoinducers enable bacteria to sense cell density and to coordinate collective behavior. NTD/kanosamine is an autoinducer produced by B. subtilis and several close relatives, although its physiological function remains unknown. The most important finding of this study was the significance of de novo NTD/kanosamine biosynthesis in the modulation of the central carbon metabolism in B. subtilis. We showed that NTD/kanosamine biosynthesis caused an increase in the NADPH pool via the accumulation of TCA cycle intermediates. These results suggest a possible role for NTD/kanosamine in carbon energy metabolism. As Bacillus species are widely used for the industrial production of various useful enzymes and compounds, the NTD/kanosamine biosynthetic pathway might be utilized to control metabolic pathways in these industrial strains.

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