Cloning of the mating-type gene MATA of the yeast Yarrowia lipolytica

Summary The mating type gene MA TA of the dimorphic yeast Yarrowia lipolytica was cloned. The strategy used was based on the presumed function of this gene in the induction of sporulation. A diploid strain homozygous for the mating type B was transformed with an integrative gene bank from an A wild-type strain. A sporulating transformant was isolated, which contained a plasmid with an 11.6 kb insert. This sequence was rescued from the chromosomal DNA of the transformant and deletion mapping was performed to localize the MAT insert. The MAT gene conferred both sporulating and non-mating phenotypes on a B/B diploid. A LEU2 sequence targeted to this locus segregated like a mating type-linked gene. The A strain did not contain silent copies of the MAT gene.     

Modeling hexanal production in oxido-reducing conditions by the yeast Yarrowia lipolytica

Hexanal produced by cells of a recombinant Yarrowia lipolytica yeast expressing the hydroperoxide lyase (HPL) from green bell pepper fruit was studied under oxido-reducing conditions using the reducing dithiotreitol and oxidizing potassium ferricyanide compounds. The combined effect of pH, linoleic acid 13-hydroperoxides concentration, temperature and oxido-reducing molecules on the hexanal production was studied. Significant positive effects for the hexanal production were found using high concentrations of hydroperoxides (100 mM, 30 g/L). Adding reducing molecules enhanced significantly hexanal production while the oxidizing molecules had an inhibitory effect. Combined effects of 13-hydroperoxides and dithiotreitol were optimised by a central composite design and a model was proposed. Finally, 6 mM (600 mg/L) of hexanal was obtained when 119 mM of 13-hydroperoxides (37 g/L) and 50 mM of dithiotreitol were introduced directly in the biocatalytic medium of the yeast Y. lipolytica.     

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.     

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...     

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...     

Induction of a non-specific permeability transition in mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, possessing a respiratory chain with the usual three points of energy conservation. High-amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating induction of the mitochondrial permeability transition due to opening of a pore (mPTP). Mitochondria from Y. lipolytica, lacking a natural mitochondrial Ca²⁺ uptake pathway, and from D. magnusii, harboring a high-capacitive, regulated mitochondrial Ca²⁺ transport system (Bazhenova et al. J Biol Chem 273:4372–4377, 1998a; Bazhenova et al. Biochim Biophys Acta 1371:96–100, 1998b; Deryabina and Zvyagilskaya Biochemistry (Moscow) 65:1352–1356, 2000; Deryabina et al. J Biol Chem 276:47801–47806, 2001) were very resistant to Ca²⁺ overload. However, exposure of yeast mitochondria to 50–100 μM Ca²⁺ in the presence of the Ca²⁺ ionophore ETH129 induced collapse of the membrane potential, possibly due to activation of the fatty acid-dependent Ca²⁺/nH⁺-antiporter, with no classical mPTP induction. The absence of response in yeast mitochondria was not simply due to structural limitations, since large-amplitude swelling occurred in the presence of alamethicin, a hydrophobic, helical peptide, forming voltage-sensitive ion channels in lipid membranes. Ca²⁺- ETH129-induced activation of the Ca²⁺/H⁺-antiport system was inhibited and prevented by bovine serum albumin, and partially by inorganic phosphate and ATP. We subjected yeast mitochondria to other conditions known to induce the permeability transition in animal mitochondria, i.e., Ca²⁺ overload (in the presence of ETH129) combined with palmitic acid (Mironova et al. J Bioenerg Biomembr 33:319–331, 2001; Sultan and Sokolove Arch Biochem Biophys 386:37–51, 2001), SH-reagents, carboxyatractyloside (an inhibitor of the ADP/ATP translocator), depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high phosphate concentrations. None of the above-mentioned substances or conditions induced a mPTP-like pore. It is thus evident that the permeability transition in yeast mitochondria is not coupled with Ca²⁺ uptake and is differently regulated compared to the mPTP of animal mitochondria.     

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。     

Dominant mutations affecting expression of pH-regulated genes inYarrowia lipolytica

In the yeastYarrowia lipolytica the levels of the alkaline extracellular protease (AEP) and acid extracellular protease (AXP) are controlled by the pH of the growth medium. When the pH of growth medium is kept close to 4.0, levels of AXP are high and those of AEP are low, whereas at pH above 6.0 the opposite is true. Mutations which mimic the effects on the protease system of growth at alkaline pH have been identified in two genes,RPH1 andRPH2, inY. lipolytica. Detailed genetic studies showed that mutations in these two genes are dominant in heterozygous diploids, and that their effects are additive in haploid double mutants. These mutants show that pH regulates AEP expression independently from other metabolic signals. These mutants are not detectably affected in their growth rates, nor in internal pH homeostasis.     

工业微生物解脂耶氏酵母及其应用研究*

解脂耶氏酵母是一种可利用多种底物发酵生产多种产品的非常规酵母,环境适应性强、易培养、安全性高。因此,该物种作为一种新型的生物工程菌株引起了科学界的广泛关注。近年来,工业生物技术因绿色、循环、低碳等优势成为新兴工业技术,在国内外得到了快速发展。介绍了解脂耶氏酵母的特征及其代谢生产各类化合物的方法,并通过对工业生物技术与传统化学化工技术的比较分析,阐述了工业生物技术的特点、研究现状及应用前景。     

Production of 3-hydroxy-γ-decalactone, the precursor of two decenolides with flavouring properties, by the yeast Yarrowia lipolytica

3-Hydroxy-γ-decalactone is the precursor of dec-2 and dec-3-en-4-olides which are valuable aroma compounds not yet produced. To promote the accumulation of this lactone, the yeast Yarrowia lipolytica was placed in different environmental conditions aiming at altering β-oxidation fluxes. The concentration of substrate, pH, aeration and dissolved oxygen level were modified. We observed an important accumulation at low aeration (0.40 molar yields) and, to a lesser extent, at lower pH (0.15). As oxygen played a key-role, we evaluated its effect at fixed dissolved oxygen and at the pH which was the most favourable to the biotransformation (pH 4.5). At 5% and 30% dissolved oxygen, yields reached 0.50. β-Oxidation fluxes are very dependent on the presence of oxygen and conditions of accumulation of 3-hydroxy-γ-decalactone with very high yields were identified. These results are an important step in the production of the two decenolides. Moreover, they show the high dependence of β-oxidation fluxes on environmental conditions and relate these conditions to the accumulation of intermediates, results that are of interest to all the processes using yeast on lipids or alkanes.     

解脂耶氏酵母基于木质纤维素原料生产化学品研究进展*

解脂耶氏酵母具有遗传背景清晰、分子操作体系较为成熟、抗逆性强、底物谱广、有机酸和蛋白质分泌能力强等优点,在微生物发酵生产化学品领域极具应用潜力。木质纤维素是丰富的可再生生物质资源,以木质纤维素原料替代化石原料生产化学品对于缓解全球能源危机、保障粮食安全等意义重大。解脂耶氏酵母可以天然代谢木质纤维素水解产生的葡萄糖,但对其他水解产物(如木糖)的利用效率极低。综述解脂耶氏酵母利用木质纤维素原料的代谢途径及改造策略,以木质纤维素原料生产化学品为例,重点讨论该过程中的主要瓶颈问题及解决办法,为后续研究提供参考。     

工业微生物解脂耶氏酵母及其应用研究*

解脂耶氏酵母是一种可利用多种底物发酵生产多种产品的非常规酵母,环境适应性强、易培养、安全性高。因此,该物种作为一种新型的生物工程菌株引起了科学界的广泛关注。近年来,工业生物技术因绿色、循环、低碳等优势成为新兴工业技术,在国内外得到了快速发展。介绍了解脂耶氏酵母的特征及其代谢生产各类化合物的方法,并通过对工业生物技术与传统化学化工技术的比较分析,阐述了工业生物技术的特点、研究现状及应用前景。     

Cloning of the gene encoding the catalytic subunit of casein kinase II from the yeast Yarrowia lipolytica

Casein kinase II from the yeast Yarrowia lipolytica is a heterotetramer of the form αα′β₂. We report on the cloning and sequencing of a partial cDNA and of the complete genomic DNA coding for the catalytic α subunit of the casein kinase II from this yeast species. The sequence of the gene coding for this enzyme has been analyzed. No intron was found in the gene, which is present in a single copy. The deduced amino acid sequence of the gene shows high similarity with those of α subunit described in other species, although, uniquely, Y. lipolytica CKIIα lacks cysteines. We find that the α subunit sequence of Y. lipolytica CKII is shown greater homology with the corresponding protein from S. pombe than with that from S. cerevisiae. We have analyzed CKIIα expression and CKIIα activity. We show that expression of this enzyme is regulated. The catalytic subunit is translated from a single mRNA, and the enzyme is present at a very low level in Y. lipolytica, as in other yeasts. Received: 20 December1997 / Accepted: 19 June 1997     

解脂耶氏酵母中囊泡蛋白YlSec15的鉴定及功能研究

解脂耶氏酵母(Yarrowia lipolytica)进行出芽繁殖时,决定未来分裂平面的出芽位点不是随机选取的,而是选择在前一次细胞分裂位置的对侧出芽,即进行双极出芽。目前对解脂耶氏酵母双极出芽的分子调控机制并不清楚。通过观察蛋白定位及过量表达的方法研究了解脂耶氏酵母中囊泡蛋白YlSec15的功能。结果表明:YlSec15在细胞中有明显的极性定位,在细胞的小芽内以及大中芽的芽颈处富集,过量表达YlSec15抑制了菌丝的形成并使得部分细胞的出芽位点选择方式由双极出芽转变为随机出芽,而引起这一变化的原因可能是由于过量的YlSec15在细胞中不能进行正常的极性定位。此外,YlSec15可能是通过YlRas2介导的信号通路参与调控细胞的菌丝形成及双极出芽。这一发现丰富了解脂耶氏酵母中双极出芽的分子调控机制,也证明了极性生长与囊泡运输之间是相互影响的。     

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.     

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.     

The Chlamydomonas chloroplast clpP gene contains translated large insertion sequences and is essential for cell growth

Sequence determination of the chloroplast clpP gene from two distantly related Chlamydomonas species (C. reinhardtii and C. eugametos) revealed the presence of translated large insertion sequences (IS1 and IS2) that divide the clpP gene into two or three sequence domains (SDs) and are not found in homologous genes in other organisms. These insertion sequences do not resemble RNA introns, and are not spliced out at the mRNA level. Instead, each insertion sequence forms a continuous open reading frame with its upstream and downstream sequence domains. IS1 specifies a potential polypeptide sequence of 286 and 318 amino acid residues in C. reinhardtii and C. eugametos, respectively. IS2 encodes a 456 amino acid polypeptide and is present only in C. eugametos. The two Chlamydomonas IS1 sequences show substantial similarity; however, there is no significant sequence similarity either between IS1 and IS2 or between these insertion sequences and any other known protein coding sequences. The C. reinhardtii clpP gene was further shown to be essential for cell growth, as demonstrated through targeted gene disruption by particle gun-mediated chloroplast transformation. Only heteroplasmic transformants could be obtained, even under mixotrophic growth conditions. The heteroplasmic transformants were stable only under selection pressure for the disrupted clpP, rapidly segregated into wild-type cells when the selection pressure was removed, and grew significantly more slowly than wildtype cells under phototrophic conditions.     

解脂耶氏酵母细胞表面展示乳糖水解酶高效水解乳糖

乳糖是婴幼儿获取能量的重要碳源之一,但乳糖需要在乳糖酶的作用下水解成半乳糖与葡萄糖后才能被吸收。缺少乳糖分解酶的婴幼儿在摄入含乳糖的食品后,未被消化的乳糖会直接进入大肠,刺激大肠蠕动加快,造成一系列不适应症状即乳糖不耐症,我国属于乳糖不耐症高发国家。因此,解决乳糖的体外水解问题对减轻该症状有重要的意义。研究通过将β-半乳糖苷酶(也称为乳糖水解酶)表面展示在食品安全微生物解脂耶氏酵母(Yarrowia lipolytica)细胞表面,通过培养获得该酵母,然后直接利用酵母细胞来水解乳糖生成半乳糖与葡萄糖。采用该工程酵母细胞(HCY10),能在24小时内完全水解50g/L的乳糖,生成半乳糖与葡萄糖。该方法具有高效、简便的优点,能为乳糖的高效水解提供一条新的途径。