Full recovery of the NADH:ubiquinone activity of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica by the addition of phospholipids

NADH:ubiquinone oxidoreductase (complex I) is the largest multiprotein complex of the mitochondrial respiratory chain. His-tagged complex I purified from the strictly aerobic yeast Yarrowia lipolytica exhibited electron transfer rates from NADH to n-decylubiquinone of less than 2% when compared to turnover numbers calculated for native mitochondrial membranes from this organism. Reactivation was observed upon addition of asolectin, purified phospholipids and different phospholipid mixtures. Maximal activities of 6-7 μmol NADH min⁻¹ mg⁻¹ were observed following incubation with a mixture of 76% phosphatidylcholine, 19% phosphatidylethanolamine and 5% cardiolipin. For full reactivation, 400-500 phospholipid molecules per complex I were needed. This demonstrated that the inactivation of complex I from Y. lipolytica by general delipidation could be fully reversed simply by returning the phospholipids that had been removed during the purification procedure. Thus, our homogeneous and highly pure complex I preparation had retained its full catalytic potential and no specific, functionally essential component had been lost. As the purified enzyme was also found to contain only substoichiometric amounts of ubiquinone-9 (0.2-0.4 mol/mol), a functional requirement of this endogeneous ubiquinone could also be excluded.     

Toxicity of fatty acid hydroperoxides towards Yarrowia lipolytica: Implication of their membrane fluidizing action

Linoleic acid hydroperoxide (HPOD), substrate of hydroperoxide lyase, an enzyme of the lipoxygenase pathway, can be transformed into many aromatic compounds, the so-called “green notes”. The presence of linoleic acid hydroperoxide in the culture medium of Yarrowia lipolytica, the yeast expressing the cloned hydroperoxide lyase of green bell pepper, undoubtedly exerted an inhibition on the growth and a toxic effect with 90% of yeast cells died after 120 min of exposition in 100 mM HPOD solution. The increase in cell membrane fluidity evaluated by measuring fluorescence generalized polarization with the increasing concentration of HPOD in the medium confirmed the fluidizing action of HPOD on yeast membrane. In addition, we determined by infrared spectroscopy measurement that this compound rapidly diffused into model phospholipids [1, 2-Dimyristoyl-D54-sn-Glycero-3-Phosphocholine (DMPC-D54)] bilayer, modifying their general physical state and their phase transition. In the presence of various concentrations of HPOD, the phase transition of DMPC-D54 occurred with an increase of both the corresponding wave number shift and the temperature range but the phase transition temperature was not modified. These results show that the toxic effects of HPOD on the yeast Yarrowia lipolytica may be initially linked to a strong interaction of this compound with the cell membrane phospholipids and components.     

Yarrowia lipolytica: A model and a tool to understand the mechanisms implicated in lipid accumulation

The oleaginous yeast Yarrowia lipolytica is known to inhabit various lipid-containing environments. One of the most striking features in this yeast is the presence of several multigene families involved in the metabolic pathways of hydrophobic substrate utilization. The complexity and the multiplicity of these genes give Y. lipolytica a wide capability range towards hydrophobic substrate (HS) utilization and storage. The combination of the increasing knowledge of this yeast's metabolism and the development of more efficient genetic tools is offering new perspectives in using Y. lipolytica as a model organism to study the mechanisms involved in lipid metabolism associated to fat uptake, storage, deposition, mobilization and regulation. Nutrient status and culture conditions seem to play a major role in obesity.     

Importance of the methyl-citrate cycle on glycerol metabolism in the yeast Yarrowia lipolytica

A novel approach to trigger lipid accumulation and/or citrate production in vivo through the inactivation of the 2-methyl-citrate dehydratase in Yarrowia lipolytica was developed. In nitrogen-limited cultures with biodiesel-derived glycerol utilized as substrate, the Δphd1 mutant (JMY1203) produced 57.7 g/L of total citrate, 1.6-fold more than the wild-type strain, with a concomitant glycerol to citrate yield of 0.91 g/g. Storage lipid in cells increased at the early growth stages, suggesting that inactivation of the 2-methyl-citrate dehydratase would mimic nitrogen limitation. Thus, a trial of JMY1203 strain was performed with glycerol under nitrogen-excess conditions. Compared with the equivalent nitrogen-limited culture, significant quantities of lipid (up to ∼31% w/w in dry weight, 1.6-fold higher than the nitrogen-limited experiment) were produced. Also, non-negligible quantities of citric acid (up to ∼26 g/L, though 0.57-fold lower than the nitrogen-limited experiment) were produced, despite remarkable nitrogen presence into the medium, indicating the construction of phenotype that constitutively accumulated lipid and secreted citrate in Y. lipolytica during growth on waste glycerol utilized as substrate.     

Isolation of bioactive peptides from tryptone that modulate lipase production in Yarrowia lipolytica

In this work the effect of several organic nitrogen sources on lipase production in Yarrowia lipolytica LgX64.81 overproducing mutant was studied. Among them, tryptone and peptone showed the most prominent stimulatory effect. Interestingly, only tryptic and peptic casein digest were found to highly induce lipase biosynthesis while lipase production was very limited in the presence of casein digest from papain and pronase-catalysed hydrolysis and absent in case of chymotryptic digest. It was also demonstrated that the stimulatory peptides should be present in the culture medium at specific proportions and molecular size to match the physiological requirement of Yarrowia lipolytica strain for lipase biosynthesis.     

Yarrowia lipolytica and pollutants: Interactions and applications

Yarrowia lipolytica is a dimorphic, non-pathogenic, ascomycetous yeast species with distinctive physiological features and biochemical characteristics that are significant in environment-related matters. Strains naturally present in soils, sea water, sediments and waste waters have inherent abilities to degrade hydrocarbons such as alkanes (short and medium chain) and aromatic compounds (biphenyl and dibenzofuran). With the application of slow release fertilizers, design of immobilization techniques and development of microbial consortia, scale-up studies and in situ applications have been possible. In general, hydrocarbon uptake in this yeast is mediated by attachment to large droplets (via hydrophobic cell surfaces) or is aided by surfactants and emulsifiers. Subsequently, the internalized hydrocarbons are degraded by relevant enzymes innately present in the yeast. Some wild-type or recombinant strains also detoxify nitroaromatic (2,4,6-trinitrotoluene), halogenated (chlorinated and brominated hydrocarbons) and organophosphate (methyl parathion) compounds. The yeast can tolerate some metals and detoxify them via different biomolecules. The biomass (unmodified, in combination with sludge, magnetically-modified and in the biofilm form) has been employed in the biosorption of hexavalent chromium ions from aqueous solutions. Yeast cells have also been applied in protocols related to nanoparticle synthesis. The treatment of oily and solid wastes with this yeast reduces chemical oxygen demand or value-added products (single cell oil, single cell protein, surfactants, organic acids and polyalcohols) are obtained. On account of all these features, the microorganism has established a place for itself and is of considerable value in environment-related applications.     

A yeast sterol carrier protein with fatty-acid and fatty-acyl-CoA binding activity

The 14-kDa sterol carrier protein 2 (SCP2) domain is present in Eukaria, Bacteria and Archaea, and has been implicated in the transport and metabolism of lipids. We report the cloning, expression, purification and physicochemical characterization of a SCP2 from the yeast Yarrowia lipolytica (YLSCP2). Analytical size-exclusion chromatography, circular dichroism and fluorescence spectra, indicate that recombinant YLSCP2 is a well-folded monomer. Thermal unfolding experiments show that SCP2 maximal stability is at pH 7.0-9.0. YLSCP2 binds cis-parinaric acid and palmitoyl-CoA with KD values of 81+/-40 nM and 73+/-33 nM, respectively, sustaining for the first time the binding of fatty acids and their CoA esters to a nonanimal SCP2. The role of yeast SCP2 and other lipid binding proteins in transport, storage and peroxisomal oxidation of fatty acids is discussed.     

Mutational analysis of Arabidopsis thaliana plant uncoupling mitochondrial protein

In this study, point mutations were introduced in plant uncoupling mitochondrial protein AtUCP1, a typical member of the plant uncoupling protein (UCP) gene subfamily, in amino acid residues Lys147, Arg155 and Tyr269, located inside the so-called UCP-signatures, and in two more residues, Cys28 and His83, specific for plant UCPs. The effects of amino acid replacements on AtUCP1 biochemical properties were examined using reconstituted proteoliposomes. Residue Arg155 appears to be crucial for AtUCP1 affinity to linoleic acid (LA) whereas His83 plays an important role in AtUCP1 transport activity. Residues Cys28, Lys147, and also Tyr269 are probably essential for correct protein function, as their substitutions affected either the AtUCP1 affinity to LA and its transport activity, or sensitivity to inhibitors (purine nucleotides). Interestingly, Cys28 substitution reduced ATP inhibitory effect on AtUCP1, while Tyr269Phe mutant exhibited 2.8-fold increase in sensitivity to ATP, in accordance with the reverse mutation Phe267Tyr of mammalian UCP1.     

Adaptive character of metabolism in Eothenomys miletus in Hengduan Mountains region during cold acclimation

Environmental factors play an important role in the seasonal adaptation of body mass and thermogenesis in small, wild mammals. The purpose of the present study was to test the hypothesis that ambient temperature was a cue to trigger the seasonal adjustments in body mass, energy intake, uncoupling protein 1 (UCP1) in brown adipose tissue (BAT), and other biochemical characteristics of Eothenomys miletus during 49 days of cold exposure. Our data demonstrated that cold acclimation induced a remarkable decrease in body mass, a significant increase in energy intake and metabolic rate, and high expression of UCP1 in BAT of E. miletus. Biochemical characteristics of BAT and liver respiration were also increased following cold acclimation. These data suggest that E. miletus reduced the body mass and increased energy intake and expenditure under cold acclimation. Increased expression of UCP1 was potentially involved in the regulation of energy metabolism and thermogenic capacity following cold acclimation.     

发酵产泡性能降低的解脂耶氏酵母菌株的获得及其评价

微生物发酵过程中泡沫的产生是发酵领域遇到的共性问题。在不影响发酵性能的前提下抑制菌株的产泡,对简化操作以及降低发酵成本具有较为重要的意义。解脂耶氏酵母(Yarrowia lipolytica,之前称为Candida lipolytica)是一种常用的合成生物学底盘,也是合成赤藓糖醇等功能糖醇的生产菌株。但在发酵合成赤藓糖醇的过程中会产生大量的泡沫,需要添加消泡剂以消除泡沫。【目的】本研究旨在开发一种产泡能力显著降低的解脂耶氏酵母新菌株,以减少赤藓糖醇发酵过程中消泡剂的添加。【方法】本研究利用解脂耶氏酵母中非同源靶向重组占支配地位的原理,采用一段外源DNA随机插入基因组的手段,随机突变基因组,改变菌株的发酵产泡性能,使突变株在发酵过程中不产泡或者降低其产泡的能力。【结果】通过筛选,获得一株在发酵过程中产泡性能显著降低的工程菌株,该菌株在保留高效合成赤藓糖醇性能的同时,显著降低了泡沫的产生。【结论】所获得的菌株对工业发酵合成赤藓糖醇具有较为重要的意义,也为控制其他微生物发酵过程中泡沫的生成提供了思路。     

Yarrowia lipolytica as a model for bio-oil production

The yeast Yarrowia lipolytica has developed very efficient mechanisms for breaking down and using hydrophobic substrates. It is considered an oleaginous yeast, based on its ability to accumulate large amounts of lipids. Completion of the sequencing of the Y. lipolytica genome and the existence of suitable tools for genetic manipulation have made it possible to use the metabolic function of this species for biotechnological applications. In this review, we describe the coordinated pathways of lipid metabolism, storage and mobilization in this yeast, focusing in particular on the roles and regulation of the various enzymes and organelles involved in these processes. The physiological responses of Y. lipolytica to hydrophobic substrates include surface-mediated and direct interfacial transport processes, the production of biosurfactants, hydrophobization of the cytoplasmic membrane and the formation of protrusions. We also discuss culture conditions, including the mode of culture control and the culture medium, as these conditions can be modified to enhance the accumulation of lipids with a specific composition and to identify links between various biological processes occurring in the cells of this yeast. Examples are presented demonstrating the potential use of Y. lipolytica in fatty-acid bioconversion, substrate valorization and single-cell oil production. Finally, this review also discusses recent progress in our understanding of the metabolic fate of hydrophobic compounds within the cell: their terminal oxidation, further degradation or accumulation in the form of intracellular lipid bodies.     

Evolution of mitochondrial uncoupling proteins: novel invertebrate UCP homologues suggest early evolutionary divergence of the UCP family

Current hypothesis about the evolution of uncoupling proteins (UCPs) proposed by suggests that UCP4 is the earliest form of UCP ancestral to all other UCP orthologues. However, this hypothesis is difficult to reconcile with a narrow tissue distribution of UCP4 (which is a brain-specific isoform), suggesting highly specialized rather than anfcestral function for this protein. We searched for UCP2, UCP3, and UCP5 homologues in invertebrate genomes using amplification with degenerate primers designed against UCP2-specific conserved sequences and/or BLASTP search with stringent ad hoc criteria to distinguish between homologues and orthologues of different UCPs. Our study identified invertebrate UCP homologues similar to UCP2 and 3 (which we termed UCP6) and an invertebrate homologue of UCP5. Phylogenetic analysis indicates that there are at least three clades of UCPs in invertebrates, which are closely related to vertebrate UCP1-3, UCP4, and UCP5, respectively, and shows early evolutionary divergence of UCPs, which pre-dates the divergence of protostomes and deuterostomes. It also suggests that the newly identified UCP6 proteins from invertebrates are ancestral to the vertebrate UCP1, UCP2, and UCP3, and that divergence of these three vertebrate orthologues occurred late in evolution of the vertebrates. This study refutes the hypothesis of Hanak and Jezek (2001) that UCP4 is an ancestral form for all UCPs, and shows early evolutionary diversification of this protein family, which corresponds to their proposed functional diversity in regulation of proton leak, antioxidant defense and apoptosis.     

Biosynthesis of homoeriodictyol from eriodictyol by flavone 3′-O-methyltransferase from recombinant Yarrowia lioplytica: Heterologous expression,biochemical characterization,and optimal transformation

In this work, we attempted to synthesize homoeriodictyol by transferring one methyl group of S-adenosyl-l-methionine (SAM) to eriodictyol using flavone 3′-O-methyltransferase ROMT-9, which was produced by recombinant Yarrowia lipolytica. Specifically, the ROMT-9 gene from rice was synthesized and cloned into the multi-copy integrative vector pINA1297, and was further expressed in Y. lipolytica with a growth phase-dependent constitutive promoter hp4d. The highest ROMT-9 activity reached 5.53 U/L after 4 days of culture in shake flask. The optimal pH and temperature of the purified ROMT-9 were 8.0 and 37 °C, respectively. The purified enzyme was stable up to 40 °C, and retained more than 80% of its maximal activity between pH 6.5 and 9.0. The recombinant ROMT-9 did not require Mg²⁺ for catalysis, while was completely inhibited in the presence of 5 mM Zn²⁺, Cu²⁺, Ba²⁺, Al³⁺, or Ni²⁺. The purified ROMT-9 was used to synthesize homoeriodictyol, and the maximal transformation ratio reached 52.4% at 16 h under the following conditions: eriodictyol 0.2 g/L, ROMT-9 0.16 g/L, SAM 0.2 g/L, CH₃OH 6% (v/v), temperature 37 °C, and pH 8.0. This work provides an alternative strategy for efficient synthesis of homoeriodictyol and compared to the traditional plant extraction or chemical synthesis, the biotransformation approach generates less environmental pollution and has a great potential for the sustainable production of homoeriodictyol.     

代谢工程改造解脂耶氏酵母合成羧酸的研究进展

解脂耶氏酵母是一种具有独特生理代谢特征的非常规酵母.它具有可以利用多种廉价碳源、低pH值耐受性好、分泌能力强等优点,因此非常适合用于各种工业产品的微生物发酵.目前,解脂耶氏酵母已被证实具有高效生产多种(同源或异源)有机羧酸的能力.本文对近年来利用代谢工程及合成生物学技术改造解脂耶氏酵母生产羧酸的实例进行了总结,并重点介...     

解脂耶氏酵母表达调控工具的开发及天然产物合成的研究进展

解脂耶氏酵母是一种重要的产油酵母,由于其能利用多种疏水性底物,具有良好的耐酸、耐盐等胁迫耐受性,具有高通量的三羧酸循环,可提供充足的乙酰辅酶A前体等特点,被认为是生产萜类、聚酮类和黄酮类等天然产物的理想宿主,在代谢工程领域有着广泛的应用。近年来,越来越多的基因编辑、表达和调控工具被逐渐开发,这促进了解脂耶氏酵母合成各种天然产物的研究。文中综述了近年来解脂耶氏酵母中基因表达和天然产物合成方面的研究进展,并探讨了在该酵母中异源合成天然产物所面临的挑战和可能的解决方案。     

蔗糖异构酶PalI在解脂耶氏酵母中的表面展示

【背景】蔗糖异构酶(PalI)生物转化蔗糖是目前生产异麦芽酮糖的主要方法,但在生产过程中存在的蔗糖异构酶转化蔗糖副产物比例较高、游离酶需要分离纯化等问题限制了异麦芽酮糖工业生产的应用。【目的】构建蔗糖异构酶PalI在解脂耶氏酵母(Yarrowia lipolytica) Po1g中的表面展示菌株,以降低蔗糖异构酶转化蔗糖的副产物比例及其纯化成本。【方法】为获得具有生产PalI能力的Y.lipolytica Po1g表面展示菌株,通过重叠延伸PCR将克雷伯氏菌(Klebsiella singaporensis)LX3的PalI的编码基因PalI与全基因合成的来自Y.lipolytica细胞壁的锚定蛋白Pir1融合,转入Y.lipolytica Po1g中构建表面展示菌株。利用3,5-二硝基水杨酸(3,5-dinitrosalicylic acid,DNS)比色定糖法测定表面展示的PalI酶活力并对其酶学性质进行探究,通过高效液相色谱法分析其转化蔗糖的产物。【结果】构建了蔗糖异构酶表面展示菌株Pir1-PalI/Po1g,经DNS法测得展示在Y. lipolytica Po1g表面的Pal...     

脂肪酸对中华哲水蚤摄食两种海洋微藻的指示作用

在室内以饥饿培养为对照,以海洋原甲藻(Prorocentrum micans)和中肋骨条藻(Skeletonema costatum)培养中华哲水蚤(Calanus sinicus),研究了各脂肪酸标记对中华哲水蚤摄食不同饵料的指示作用。结果显示,海洋原甲藻中18 ∶ 4ω3、22 ∶ 6ω3含量较高,中肋骨条藻中16 ∶ 1ω7、20 ∶ 5ω3的含量较高。二者分别表现出典型的甲藻门和硅藻门的脂肪酸组成特征。中华哲水蚤的脂肪酸组成有两个特点:(1)20 ∶ 5ω3和22 ∶ 6ω3的含量均较高;(2)其体内表征桡足类浮游植物食性、由桡足类自身合成的20 ∶ 1和22 ∶ 1脂肪酸占有相当的比例。虽然中华哲水蚤对不同脂肪酸的吸收和转化效率不同,但以脂肪酸作为标记还是成功的指示了中华哲水蚤对微藻的摄食。在饥饿培养中,首先消耗的是那些浮游动物自身不能合成的多不饱和脂肪酸,而结构脂肪酸都表现出了较高的保守性。结合各脂肪酸标记变化趋势和Pearson相关性分析的结果认为,18 ∶ 4ω3、18 ∶ 4ω3/16 ∶ 1ω7、∑18/∑16能较好的指示中华哲水蚤对海洋原甲藻的摄食,仅16 ∶ 1ω7/18 ∶ 4ω3能指示中华哲水蚤对中肋骨条藻的摄食。