Structure of a Complete ATP Synthase Dimer Reveals the Molecular Basis of Inner Mitochondrial Membrane Morphology.

We determined the structure of a complete, dimeric F1Fo-ATP synthase from yeast Yarrowia lipolytica mitochondria by a combination of cryo-EM and X-ray crystallography. The final structure resolves 58 of the 60 dimer subunits. Horizontal helices of subunit a in Fo wrap around the c-ring rotor, and a total of six vertical helices assigned to subunits a, b, f, i, and 8 span the membrane. Subunit 8 (A6L in human) is an evolutionary derivative of the bacterial b subunit. On the lumenal membrane surface, subunit f establishes direct contact between the two monomers. Comparison with a cryo-EM map of the F1Fo monomer identifies subunits e and g at the lateral dimer interface. They do not form dimer contacts but enable dimer formation by inducing a strong membrane curvature of ∼100°. Our structure explains the structural basis of cristae formation in mitochondria, a landmark signature of eukaryotic cell morphology.

A conserved arginine residue is critical for stabilizing the N2 FeS cluster in mitochondrial complex I.

Respiratory complex I (NADH:ubiquinone oxidoreductase), the first enzyme of the electron-transport chain, captures the free energy released by NADH oxidation and ubiquinone reduction to translocate protons across an energy-transducing membrane and drive ATP synthesis during oxidative phosphorylation. The cofactor that transfers the electrons directly to ubiquinone is an iron-sulfur cluster (N2) located in the NDUFS2/NUCM subunit. A nearby arginine residue (R121), which forms part of the second coordination sphere of the N2 cluster, is known to be posttranslationally dimethylated but its functional and structural significance are not known. Here, we show that mutations of this arginine residue (R121M/K) abolish the quinone-reductase activity, concomitant with disappearance of the N2 signature from the electron paramagnetic resonance (EPR) spectrum. Analysis of the cryo-EM structure of NDUFS2-R121M complex I at 3.7 Å resolution identified the absence of the cubane N2 cluster as the cause of the dysfunction, within an otherwise intact enzyme. The mutation further induced localized disorder in nearby elements of the quinone-binding site, consistent with the close connections between the cluster and substrate-binding regions. Our results demonstrate that R121 is required for the formation and/or stability of the N2 cluster and highlight the importance of structural analyses for mechanistic interpretation of biochemical and spectroscopic data on complex I variants.

Altering the fatty acid profile of Yarrowia lipolytica to mimic cocoa butter by genetic engineering of desaturases.

BACKGROUND: Demand for Cocoa butter is steadily increasing, but the supply of cocoa beans is naturally limited and under threat from global warming. One route to meeting the future demand for cocoa butter equivalent (CBE) could be to utilize microbial cell factories such as the oleaginous yeast Yarrowia lipolytica. RESULTS: The main goal was to achieve triacyl-glycerol (TAG) storage lipids in Y. lipolytica mimicking cocoa butter. This was accomplished by replacing the native Δ9 fatty acid desaturase (Ole1p) with homologs from other species and changing the expression of both Ole1p and the Δ12 fatty acid desaturase (Fad2p). We thereby abolished the palmitoleic acid and reduced the linoleic acid content in TAG, while the oleic acid content was reduced to approximately 40 percent of the total fatty acids. The proportion of fatty acids in TAG changed dramatically over time during growth, and the fatty acid composition of TAG, free fatty acids and phospholipids was found to be very different. CONCLUSIONS: We show that the fatty acid profile in the TAG of Y. lipolytica can be altered to mimic cocoa butter. We also demonstrate that a wide range of fatty acid profiles can be achieved while maintaining good growth and high lipid accumulation, which, together with the ability of Y. lipolytica to utilize a wide variety of carbon sources, opens up the path toward sustainable production of CBE and other food oils.

Increased Accumulation of Squalene in Engineered Yarrowia lipolytica through Deletion of PEX10 and URE2.

Squalene is a triterpenoid serving as an ingredient of various products in the food, cosmetic, pharmaceutical industries. The oleaginous yeast Yarrowia lipolytica offers enormous potential as a microbial chassis for the production of terpenoids, such as carotenoid, limonene, linalool, and farnesene, as the yeast provides ample storage space for hydrophobic products. Here, we present a metabolic design that allows the enhanced accumulation of squalene in Y. lipolytica. First, we improved squalene accumulation in Y. lipolytica by overexpressing the genes (ERG and HMG) coding for the mevalonate pathway enzymes. Second, we increased the production of lipid where squalene is accumulated by overexpressing DGA1 (encoding diacylglycerol acyltransferase) and deleting PEX10 (for peroxisomal membrane E3 ubiquitin ligase). Third, we deleted URE2 (coding for a transcriptional regulator in charge of nitrogen catabolite repression [NCR]) to induce lipid accumulation regardless of the carbon-to-nitrogen ratio in culture media. The resulting engineered Y. lipolytica exhibited a 115-fold higher squalene content (22.0 mg/g dry cell weight) than the parental strain. These results suggest that the biological function of Ure2p in Y. lipolytica is similar to that in Saccharomyces cerevisiae, and its deletion can be utilized to enhance the production of hydrophobic target products in oleaginous yeast strains. IMPORTANCE This study demonstrated a novel strategy for increasing squalene production in Y. lipolytica. URE2, a bifunctional protein that is involved in both nitrogen catabolite repression and oxidative stress response, was identified and demonstrated correlation to squalene production. The data suggest that double deletion of PEX10 and URE2 can serve as a positive synergistic effect to help yeast cells in boosting squalene production. This discovery can be combined with other strategies to engineer cell factories to efficiently produce terpenoid in the future.

Disrupting a phospholipase A2 gene increasing lipid accumulation in the oleaginous yeast Yarrowia lipolytica.

AIMS: Phospholipase A2 (PLA2 ) is a diverse superfamily that hydrolyzes fatty acyl ester bonds at the sn-2 position of phospholipids. The correlation between phospholipid metabolism and the anabolism of neutral lipids remains unclear in yeasts. This study aims to explore the effects of PLA2 on lipid accumulation in the oleaginous yeast Yarrowia lipolytica. METHODS AND RESULTS: This study identified an actively expressed phospholipase A2 gene (PLA2-3, YAIL0_E16060g) in Y. lipolytica by quantitative PCR analysis. The gene PLA2-3 was disrupted in the strain po1gΔKu70 by homologous recombination and in the strain po1g-G3 by a CRISPR-Cas9 system, which caused an increase in stress sensitivity while the cell growth was not altered under fermentative conditions. Lipid production was performed in both flasks and bioreactors. The results showed that the lipid titre and lipid content were improved over 25% and 8-30%, respectively, in PLA2-3 disrupted strains compared to the controls. CONCLUSIONS: Disruption of the phospholipase PLA2-3 gene could effectively improve lipid production in Y. lipolytica. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presented a strategy on improving the lipid production of oleaginous yeasts and a similar strategy might be used in other oleaginous microbes.

Redox-coupled quinone dynamics in the respiratory complex I.

Complex I couples the free energy released from quinone (Q) reduction to pump protons across the biological membrane in the respiratory chains of mitochondria and many bacteria. The Q reduction site is separated by a large distance from the proton-pumping membrane domain. To address the molecular mechanism of this long-range proton-electron coupling, we perform here full atomistic molecular dynamics simulations, free energy calculations, and continuum electrostatics calculations on complex I from Thermus thermophilus We show that the dynamics of Q is redox-state-dependent, and that quinol, QH2, moves out of its reduction site and into a site in the Q tunnel that is occupied by a Q analog in a crystal structure of Yarrowia lipolytica We also identify a second Q-binding site near the opening of the Q tunnel in the membrane domain, where the Q headgroup forms strong interactions with a cluster of aromatic and charged residues, while the Q tail resides in the lipid membrane. We estimate the effective diffusion coefficient of Q in the tunnel, and in turn the characteristic time for Q to reach the active site and for QH2 to escape to the membrane. Our simulations show that Q moves along the Q tunnel in a redox-state-dependent manner, with distinct binding sites formed by conserved residue clusters. The motion of Q to these binding sites is proposed to be coupled to the proton-pumping machinery in complex I.

Waste soybean frying oil for the production, extraction, and characterization of cell-wall-associated lipases from Yarrowia lipolytica.

The lipolytic yeast Yarrowia lipolytica produces cell-wall-associated lipases, namely Lip7p and Lip8p, that could have interesting properties as catalyst either in free (released lipase fraction-RLF) or cell-associated (cell-bound lipase fraction-CBLF) forms. Herein, a mixture of waste soybean frying oil, yeast extract and bactopeptone was found to favor the enzyme production. Best parameters for lipase activation and release from the cell wall by means of acoustic wave treatment were defined as: 26 W/cm2 for 1 min for CBLF and 52 W/cm2 for 2 min for RLF. Optimal pH and temperature values for lipase activity together with storage conditions were similar for both the free enzyme and cell-associated one: pH 7.0; T = 37 °C; and > 70% residual activity for 60 days at 4, - 4 °C and for 15 days at 30 °C.

The N-Acetylglucosamine Kinase from Yarrowia lipolytica Is a Moonlighting Protein.

In Yarrowia lipolytica, expression of the genes encoding the enzymes of the N-acetylglucosamine (NAGA) utilization pathway (NAG genes) becomes independent of the presence of NAGA in a Ylnag5 mutant lacking NAGA kinase. We addressed the question of whether the altered transcription was due to a lack of kinase activity or to a moonlighting role of this protein. Glucosamine-6-phosphate deaminase (Nag1) activity was measured as a reporter of NAG genes expression. The NGT1 gene encoding the NAGA transporter was deleted, creating a Ylnag5 ngt1 strain. In glucose cultures of this strain, Nag1 activity was similar to that of the Ylnag5 strain, ruling out the possibility that NAGA derived from cell wall turnover could trigger the derepression. Heterologous NAGA kinases were expressed in a Ylnag5 strain. Among them, the protein from Arabidopsis thaliana did not restore kinase activity but lowered Nag1 activity 4-fold with respect to a control. Expression in the Ylnag5 strain of YlNag5 variants F320S or D214V with low kinase activity caused a repression similar to that of the wild-type protein. Together, these results indicate that YlNag5 behaves as a moonlighting protein. An RNA-seq analysis revealed that the Ylnag5 mutation had a limited transcriptomic effect besides derepression of the NAG genes.

The Role of Hexokinase and Hexose Transporters in Preferential Use of Glucose over Fructose and Downstream Metabolic Pathways in the Yeast Yarrowia lipolytica.

The development of efficient bioprocesses requires inexpensive and renewable substrates. Molasses, a by-product of the sugar industry, contains mostly sucrose, a disaccharide composed of glucose and fructose, both easily absorbed by microorganisms. Yarrowia lipolytica, a platform for the production of various chemicals, can be engineered for sucrose utilization by heterologous invertase expression, yet the problem of preferential use of glucose over fructose remains, as fructose consumption begins only after glucose depletion what significantly extends the bioprocesses. We investigated the role of hexose transporters and hexokinase (native and fructophilic) in this preference. Analysis of growth profiles and kinetics of monosaccharide utilization has proven that the glucose preference in Y. lipolytica depends primarily on the affinity of native hexokinase for glucose. Interestingly, combined overexpression of either hexokinase with hexose transporters significantly accelerated citric acid biosynthesis and enhanced pentose phosphate pathway leading to secretion of polyols (31.5 g/L vs. no polyols in the control strain). So far, polyol biosynthesis was efficient in glycerol-containing media. Moreover, overexpression of fructophilic hexokinase in combination with hexose transporters not only shortened this process to 48 h (84 h for the medium with glycerol) but also allowed to obtain 23% more polyols (40 g/L) compared to the glycerol medium (32.5 g/L).

Glutamate dehydrogenases in the oleaginous yeast Yarrowia lipolytica.

Glutamate dehydrogenases (GDHs) are fundamental to cellular nitrogen and energy balance. Yet little is known about these enzymes in the oleaginous yeast Yarrowia lipolytica. The YALI0F17820g and YALI0E09603g genes, encoding potential GDH enzymes in this organism, were examined. Heterologous expression in gdh-null Saccharomyces cerevisiae and examination of Y. lipolytica strains carrying gene deletions demonstrate that YALI0F17820g (ylGDH1) encodes a NADP-dependent GDH whereas YALI0E09603g (ylGDH2) encodes a NAD-dependent GDH enzyme. The activity encoded by these two genes accounts for all measurable GDH activity in Y. lipolytica. Levels of the two enzyme activities are comparable during logarithmic growth on rich medium, but the NADP-ylGDH1p enzyme activity is most highly expressed in stationary and nitrogen starved cells by threefold to 12-fold. Replacement of ammonia with glutamate causes a decrease in NADP-ylGdh1p activity, whereas NAD-ylGdh2p activity is increased. When glutamate is both carbon and nitrogen sources, the activity of NAD-ylGDH2p becomes dominant up to 18-fold compared with that of NADP-ylGDH1p. Gene deletion followed by growth on different carbon and nitrogen sources shows that NADP-ylGdh1p is required for efficient nitrogen assimilation whereas NAD-ylGdh2p plays a role in nitrogen and carbon utilization from glutamate. Overexpression experiments demonstrate that ylGDH1 and ylGDH2 are not interchangeable. These studies provide a vital basis for future consideration of how these enzymes function to facilitate energy and nitrogen homeostasis in Y. lipolytica.

High-yield expression of extracellular lipase from Yarrowia lipolytica and its interactions with lipopeptide biosurfactants: A biophysical approach.

The unconventional yeast Yarrowia lipolytica is known as a producer of extracellular lipases. Here we overexpressed extracellular lipase (YlLip2) in yeast strain Y. lipolytica AJD ΔXΔA-Lip2 harboring the overexpression cassette of the YALI0A20350 gene under the strong hybrid promoter UAS1B16-TEF. To maintain a high level of YlLip2 production, two extracellular proteases of Y. lipolytica, AEPp and AXPp, were deleted. The purified recombinant YlLip2 presented optimal catalytic activities at 37 °C and pH 8.0. The effect of two lipopeptide biosurfactants, i.e., amphisin produced by Pseudomonas fluorescens DSS73 and viscosinamide secreted by P. fluorescens DR54, on the conformation and activity of YlLip2 was evaluated using spectral methods, surface tension, and the enzyme activity assay. YlLip2 demonstrated high tolerance of the tested biosurfactants and had greater activity retention after incubation with both biosurfactants. Finally, we observed that intrinsic fluorescence intensity of YlLip2 decreased significantly with increasing lipopeptides concentration ranging from 2.5 to 60 µM. Our results showed that both biosurfactants improve enzymatic activity of YlLip2 and might suggest better interaction of the substrate with the active site. These favorable characteristics make YlLip2 a prospective additive in the pharmaceutical, food, cosmetic, and detergent industries.

The influence of transketolase on lipid biosynthesis in the yeast Yarrowia lipolytica.

BACKGROUND: During the pentose phosphate pathway (PPP), two important components, NADPH and pentoses, are provided to the cell. Previously it was shown that this metabolic pathway is a source of reducing agent for lipid synthesis from glucose in the yeast Yarrowia lipolytica. Y. lipolytica is an attractive microbial host since it is able to convert untypical feedstocks, such as glycerol, into oils, which subsequently can be transesterified to biodiesel. However, the lipogenesis process is a complex phenomenon, and it still remains unknown which genes from the PPP are involved in lipid synthesis. RESULTS: To address this problem we overexpressed five genes from this metabolic pathway: transaldolase (TAL1, YALI0F15587g), transketolase (TKL1, YALI0E06479g), ribulose-phosphate 3-epimerase (RPE1, YALI0C11880g) and two dehydrogenases, NADP+-dependent glucose-6-phosphate dehydrogenase (ZWF1, YALI0E22649g) and NADP+-dependent 6-phosphogluconate dehydrogenase (GND1, YALI0B15598g), simultaneously with diacylglycerol acyltransferase (DGA1, YALI0E32769g) and verified each resulting strain's ability to synthesize fatty acid growing on both glycerol and glucose as a carbon source. Our results showed that co-expression of DGA1 and TKL1 results in higher SCO synthesis, increasing lipid content by 40% over the control strain (DGA1 overexpression). CONCLUSIONS: Simultaneous overexpression of DGA1 and TKL1 genes results in a higher lipid titer independently from the fermentation conditions, such as carbon source, pH and YE supplementation.

Subcellular engineering of lipase dependent pathways directed towards lipid related organelles for highly effectively compartmentalized biosynthesis of triacylglycerol derived products in Yarrowia lipolytica.

As an alternative to in vitro lipase dependent biotransformation and to traditional assembly of pathways in cytoplasm, the present study focused on targeting lipase dependent pathways to a subcellular compartment lipid body (LB), in combination with compartmentalization of associated pathways in other lipid relevant organelles including endoplasmic reticulum (ER) and peroxisome for efficient in vivo biosynthesis of fatty acid methyl esters (FAMEs) and hydrocarbons, in the context of improving Yarrowia lipolytica lipid pool. Through knock in and knock out of key genes involved in triacylglycerols (TAGs) biosynthesis and degradation, the TAGs content was increased to 51.5%, from 7.2% in parent strain. Targeting lipase dependent pathway to LB gave a 10-fold higher FAMEs titer (1028.0 mg/L) compared to cytosolic pathway (102.8 mg/L). Furthermore, simultaneously targeting lipase dependent pathway to LB, ER and peroxisome gave rise to the highest FAMEs titer (1644.8 mg/L). The subcellular compartment engineering strategy was extended to other lipase dependent pathways for fatty alkene and alkane biosynthesis, which resulted in a 14-fold titer enhancement compared to traditional cytosolic pathways. We developed yeast subcellular cell factories by directing lipase dependent pathways towards the TAGs storage organelle LB for efficient biosynthesis of TAG derived chemicals for the first time. The successful exploration of targeting metabolic pathways towards LB centered organelles is expected to promote subcellular compartment engineering for other lipid derived product biosynthesis.

Boosting the biosynthesis of betulinic acid and related triterpenoids in Yarrowia lipolytica via multimodular metabolic engineering.

BACKGROUND: Betulinic acid is a pentacyclic lupane-type triterpenoid and a potential antiviral and antitumor drug, but the amount of betulinic acid in plants is low and cannot meet the demand for this compound. Yarrowia lipolytica, as an oleaginous yeast, is a promising microbial cell factory for the production of highly hydrophobic compounds due to the ability of this organism to accumulate large amounts of lipids that can store hydrophobic products and supply sufficient precursors for terpene synthesis. However, engineering for the heterologous production of betulinic acid and related triterpenoids has not developed as systematically as that for the production of other terpenoids, thus the production of betulinic acid in microbes remains unsatisfactory. RESULTS: In this study, we applied a multimodular strategy to systematically improve the biosynthesis of betulinic acid and related triterpenoids in Y. lipolytica by engineering four functional modules, namely, the heterogenous CYP/CPR, MVA, acetyl-CoA generation, and redox cofactor supply modules. First, by screening 25 combinations of cytochrome P450 monooxygenases (CYPs) and NADPH-cytochrome P450 reductases (CPRs), each of which originated from 5 different sources, we selected two optimal betulinic acid-producing strains. Then, ERG1, ERG9, and HMG1 in the MVA module were overexpressed in the two strains, which dramatically increased betulinic acid production and resulted in a strain (YLJCC56) that exhibited the highest betulinic acid yield of 51.87 ± 2.77 mg/L. Then, we engineered the redox cofactor supply module by introducing NADPH- or NADH-generating enzymes and the acetyl-CoA generation module by directly overexpressing acetyl-CoA synthases or reinforcing the ß-oxidation pathway, which further increased the total triterpenoid yield (the sum of the betulin, betulinic acid, betulinic aldehyde yields). Finally, we engineered these modules in combination, and the total triterpenoid yield reached 204.89 ± 11.56 mg/L (composed of 65.44% betulin, 23.71% betulinic acid and 10.85% betulinic aldehyde) in shake flask cultures. CONCLUSIONS: Here, we systematically engineered Y. lipolytica and achieved, to the best of our knowledge, the highest betulinic acid and total triterpenoid yields reported in microbes. Our study provides a suitable reference for studies on heterologous exploitation of P450 enzymes and manipulation of triterpenoid production in Y. lipolytica.

Enantioselective synthesis of enantiopure chiral alcohols using carbonyl reductases screened from Yarrowia lipolytica.

AIMS: We aimed to explore Yarrowia lipolytica carbonyl reductases as effective biocatalysts and to develop efficient asymmetric reduction systems for chiral alcohol synthesis. METHODS AND RESULTS: Yarrowia lipolytica carbonyl reductase genes were obtained via homologous sequence amplification strategy. Two carbonyl reductases, YaCRI and YaCRII, were identified and characterized, and used to catalyse the conversion of 2-hydroxyacetophenone (2-HAP) to optically pure (S)-1-phenyl-1,2-ethanediol. Enzymatic assays revealed that YaCRI and YaCRII exhibited specific activities of 6·96 U mg-1 (99·8% e.e.) and 7·85 U mg-1 (99·9% e.e.), respectively, and showed moderate heat resistance at 40-50°C and acid tolerance at pH 5·0-6·0. An efficient whole-cell two-phase system was established using reductase-expressing recombinant Escherichia coli. The conversion of 2-HAP (20·0 g l-1 ) conversion with the solvent of dibutyl phthalate was approximately 70-fold higher than in water. Furthermore, the two recombinant E. coli displayed biocatalyst activity and enantioselectivity towards several different carbonyl compounds, and E. coli BL21 (DE3)/pET-28a-yacrII showed a broad substrate spectrum. CONCLUSIONS: A new whole-cell recombinant E. coli-based bioreduction system for enantiopure alcohol synthesis with high enantioselectivity at high substrate concentrations was developed. SIGNIFICANCE AND IMPACT OF THE STUDY: We proposed a promising approach for the efficient preparation of enantiopure chiral alcohols.

High-Level Production of a Thermostable Mutant of Yarrowia lipolytica Lipase 2 in Pichia pastoris.

As a promising biocatalyst, Yarrowia lipolytica lipase 2 (YlLip2) is limited in its industrial applications due to its low thermostability. In this study, a thermostable YlLip2 mutant was overexpressed in Pichia pastoris and its half-life time was over 30 min at 80 °C. To obtain a higher protein secretion level, the gene dosage of the mutated lip2 gene was optimized and the lipase activity was improved by about 89%. Then, the YlLip2 activity of the obtained strain further increased from 482 to 1465 U/mL via optimizing the shaking flask culture conditions. Subsequently, Hac1p and Vitreoscilla hemoglobin (VHb) were coexpressed with the YlLip2 mutant to reduce the endoplasmic reticulum stress and enhance the oxygen uptake efficiency in the recombinant strains, respectively. Furthermore, high-density fermentations were performed in a 3 L bioreactor and the production of the YlLip2 mutant reached 9080 U/mL. The results demonstrated that the expression level of the thermostable YlLip2 mutant was predominantly enhanced via the combination of these strategies in P. pastoris, which forms a consolidated basis for its large-scale production and future industrial applications.

Structural insight into the substrate specificity of acyl-CoA oxidase1 from Yarrowia lipolytica for short-chain dicarboxylyl-CoAs.

Acyl-CoA oxidase (ACOX) plays an important role in fatty acid degradation. The enzyme catalyzes the first reaction in peroxisomal fatty acid ß-oxidation by reducing acyl-CoA to 2-trans-enoyl-CoA. The yeast Yarrowia lipolytica is able to utilize fatty acids, fats, and oil as carbon sources to produce valuable bioproducts. We determined the crystal structure of ACOX1 from Y. lipolytica (YlACOX1) at a resolution of 2.5 Å. YlACOX1 forms a homodimer, and the monomeric structure is composed of four domains, the Nα, Nß, Cα1, and Cα2. The FAD cofactor is bound at the dimerization interface between the Nß- and Cα1-domains. The substrate-binding tunnel formed by the interface between the Nα-, Nß-, and Cα1-domains is located proximal to FAD. Amino acid and structural comparisons of YlACOX1 with other ACOXs show that the substrate-binding pocket of YlACOX1 is much smaller than that of the medium- or long-chain ACOXs but is rather similar to that of the short-chain ACOXs. Moreover, the hydrophilicity of residues constituting the end region of the substrate-binding pocket in YlACOX1 is quite similar to those in the short-chain ACOXs but different from those of the medium- or long-chain ACOXs. These observations provide structural insights how YlACOX1 prefers short-chain dicarboxylyl-CoAs as a substrate.

Phosphatidate phosphatase activity is induced during lipogenesis in the oleaginous yeast Yarrowia lipolytica.

Phosphatidate (PA) phosphatase dephosphorylates the membrane phospholipid PA to diacylglycerol (DAG) that can be used for the synthesis of the storage lipid triacylglycerol (TAG). In Yarrowia lipolytica, TAG biosynthesis is induced during the lipogenic phase, which results in the accumulation of this lipid in cells. The accumulation of TAG during lipogenesis requires the supply of DAG, but the source of this DAG is not known in Y. lipolytica. In this study, the regulation of PA phosphatase during lipogenesis and its contribution to TAG biosynthesis was examined in Y. lipolytica. Lipogenesis was triggered by growing cells in high-glucose media, whereas control cultures were grown in low-glucose media. PA phosphatase activity increased in a time-dependent manner as high-glucose cells progressed in the lipogenic phase. In contrast, the activity decreased in low-glucose cells that did not accumulate lipids. An analysis of the subcellular localization of the PA phosphatase activity showed that the membrane-associated activity increased during lipogenesis. The significance of this increase can be explained by the fact that only the membrane-associated PA phosphatase activity is responsible for the production of DAG. Taken together, these results indicate that PA phosphatase is involved in TAG biosynthesis during lipogenesis in Y. lipolytica.

Understanding Functional Roles of Native Pentose-Specific Transporters for Activating Dormant Pentose Metabolism in Yarrowia lipolytica.

Pentoses, including xylose and arabinose, are the second most prevalent sugars in lignocellulosic biomass that can be harnessed for biological conversion. Although Yarrowia lipolytica has emerged as a promising industrial microorganism for production of high-value chemicals and biofuels, its native pentose metabolism is poorly understood. Our previous study demonstrated that Y. lipolytica (ATCC MYA-2613) has endogenous enzymes for d-xylose assimilation, but inefficient xylitol dehydrogenase causes Y. lipolytica to assimilate xylose poorly. In this study, we investigated the functional roles of native sugar-specific transporters for activating the dormant pentose metabolism in Y. lipolytica By screening a comprehensive set of 16 putative pentose-specific transporters, we identified two candidates, YALI0C04730p and YALI0B00396p, that enhanced xylose assimilation. The engineered mutants YlSR207 and YlSR223, overexpressing YALI0C04730p and YALI0B00396p, respectively, improved xylose assimilation approximately 23% and 50% in comparison to YlSR102, a parental engineered strain overexpressing solely the native xylitol dehydrogenase gene. Further, we activated and elucidated a widely unknown native l-arabinose assimilation pathway in Y. lipolytica through transcriptomic and metabolic analyses. We discovered that Y. lipolytica can coconsume xylose and arabinose, where arabinose utilization shares transporters and metabolic enzymes of some intermediate steps of the xylose assimilation pathway. Arabinose assimilation is synergistically enhanced in the presence of xylose, while xylose assimilation is competitively inhibited by arabinose. l-Arabitol dehydrogenase is the rate-limiting step responsible for poor arabinose utilization in Y. lipolytica Overall, this study sheds light on the cryptic pentose metabolism of Y. lipolytica and, further, helps guide strain engineering of Y. lipolytica for enhanced assimilation of pentose sugars.IMPORTANCE The oleaginous yeast Yarrowia lipolytica is a promising industrial-platform microorganism for production of high-value chemicals and fuels. For decades since its isolation, Y. lipolytica has been known to be incapable of assimilating pentose sugars, xylose and arabinose, that are dominantly present in lignocellulosic biomass. Through bioinformatic, transcriptomic, and enzymatic studies, we have uncovered the dormant pentose metabolism of Y. lipolytica Remarkably, unlike most yeast strains, which share the same transporters for importing hexose and pentose sugars, we discovered that Y. lipolytica possesses the native pentose-specific transporters. By overexpressing these transporters together with the rate-limiting d-xylitol and l-arabitol dehydrogenases, we activated the dormant pentose metabolism of Y. lipolytica Overall, this study provides a fundamental understanding of the dormant pentose metabolism of Y. lipolytica and guides future metabolic engineering of Y. lipolytica for enhanced conversion of pentose sugars to high-value chemicals and fuels.

Heteroexpression and biochemical characterization of a glucose-6-phosphate dehydrogenase from oleaginous yeast Yarrowia lipolytica.

Yarrowia lipolytica, a nonpathogenic, nonconventional, aerobic and dimorphic yeast, is considered an oleaginous microorganism due to its excellent ability to accumulate large amounts of lipids. Glucose-6-phosphate dehydrogenase (G6PD) is one of two key enzymes involved in the lipid accumulation in this fungi, which catalyzes the oxidative dehydrogenation of glucose-6-phosphate to 6-phosphoglucono-δ-lactone with the reduction of NADP+ to NADPH. In this study, the full-length gene of G6PD from Y. lipolytica (YlG6PD) was cloned without intron and heterogeneously expressed in E. coli. Then, YlG6PD was purified and biochemically characterized in details. Kinetic analysis showed that YlG6PD was completely dependent on NADP+ and its apparent Km for NADP+ was 33.3 µM. The optimal pH was 8.5 and the maximum activity was around 47.5 °C. Heat-inactivation profiles revealed that it remained 50% of maximal activity after incubation at 48 °C for 20 min YlG6PD activity was competitively inhibited by NADPH with a Ki value of 56.04 µM. Most of the metal ions have no effect on activity, but Zn2+ was a strong inhibitor. Furthermore, the determinants in the coenzyme specificity of YlG6PD were investigated. Kinetic analysis showed that the single mutant R52D completely lost the ability to utilize NADP+ as its coenzyme, suggesting that Arg-52 plays a decisive role in NADP+ binding in YlG6PD. The identification of Y. lipolytica G6PD may provide useful scientific information for metabolic engineering of this yeast as a model for bio-oil production.