Genomic organization of the yeast Yarrowia lipolytica

We produced electrophoretic karyotypes of the reference strain E150 and of seven other isolates from different geographical origins to study the genomic organization of the dimorphic yeast Yarrowia lipolytica. These karyotypes differed in the number and size of the chromosomal bands. The karyotype of the reference stain E150 consisted of five bands of between 2.6 and 4.9 Mb in size. This strain contained at least five rDNA clusters, from 190 to 620 kb in size, which were scattered over most of the chromosomes. The assignment of 43 markers, including rRNA genes and three centromeres, to the E150 bands defined five linkage groups. Hybridization to the karyotypes of other isolates with pools of markers of each linkage group showed that linkage groups I, II, IV and V were conserved in the strains tested whereas group III was not and was split between at least two chromosomes in most strains. Use of a meganuclease I-SceI site targeted to one locus of E150 linkage group III showed that two chromosomes actually comigrated in band III of this strain. Our results are compatible with six chromosomes defining the haploid complement of strains of Y. lipolytica and that, despite an unprecedented chromosome length polymorphism, the overall structure of the genome is conserved in different isolates. Received: 27 March 1997; in revised form: 8 July 1997 / Accepted: 9 July 1997     

Peroxisome biogenesis in the yeast Yarrowia lipolytica

Extensive perexisome proliferation during growth on oleic acid, combined with the availability of excellent genetic tools, makes the dimorphic yeast, Yarrowia lipolytica, a powerful model system to study the molecular mechanisms involved in peroxisome biogenesis. A combined genetic, biochemical, and morphological approach has revealed that the endoplasmic reticulum (ER) plays an essential role in the assembly of functional peroxisomes in this yeast. The trafficking of some membrane proteins to the peroxisomes occurs via the ER, results in their glyco-sylation in the ER lumen, does not involve transit through the Golgi, and requires the products of the SEC238, SRP54, PEX1, and PEX6 genes. The authors' data suggest a model for protein import into peroxisomes via two subpopulations of ER-derived vesicles that are distinct from secretory vesicles. A kinetic analysis of the trafficking of peroxisomal proteins in vivo has demonstrated that membrane and matrix proteins are initially targeted to multiple vesicular precursors that represent intermediates in the assembly pathway of peroxisomes. The authors have also recently identified a novel cytosolic chaperone, Pex20p, that assists in the oligomerization of thiolase in the cytosol and promotes its targeting to the peroxisome. These data provide the first evidence that a chaperone-assisted folding and oligomerization of thiolase in the cytosol is required for the import of this protein into the peroxisomal matrix.     

解脂耶氏酵母表达系统研究进展

解脂耶氏酵母（Yarrowia lipolytica）是非常规酵母中具代表性的一种,它底物广泛,尤其能利用有机酸（柠檬酸、异柠檬酸）,蛋白类（蛋白酶、脂肪酸、酯酶、磷酸酶、α-甘露糖苷酶、RNase）。烷烃类廉价物质作为底物分泌大量的代谢产物,自上世纪40年代被发现以来,越来越受到研究者的重视,并于上世纪90年代被开发成为一种新的酵母表达系统,用于42种异源蛋白的高效表达。综述了解脂耶氏酵母表达系统及其特点,有利于研究者从转录和翻译的水平研究异源蛋白在此菌中的表达分泌路径以及寻找到调控型启动子。     

Analysis of Yarrowia lipolytica extracellular lipase Lip2p glycosylation

Wild-type (WT) Yarrowia lipolytica strain secretes a major extracellular lipase Lip2p which is glycosylated. In silico sequence analysis reveals the presence of two potential N-glycosylation sites (N113IS and N134NT). Strains expressing glycosylation mutant forms were constructed. Esterase activities for the different forms were measured with three substrates: p-nitrophenol butyrate (p-NPB), tributyrin and triolein. Sodium dodecyl sulfate polacrylamide gel electrophoresis analysis of supernatant indicated that the suppression of the two sites of N-glycosylation did not affect secretion. S115V or N134Q mutations led to lipase with similar specific activity compared with WT lipase while a T136V mutation reduced specific activity toward p-NPB and tributyrin. Electrospray ionization MS of the WT entire protein led to an average mass of 36 950 Da, higher than the mass deduced from the amino acid sequence (33 385 Da) and to the observation of at least two different mannose structures: Man(8)GlcNAc(2) and Man(9)GlcNAc(2). LC-tandem MS analysis of the WT Lip2p after trypsin and endoproteinase Asp-N treatments led to high coverage (87%) of protein sequence but the peptides containing N113 and N134 were not identified. We confirmed that the presence of N-glycosylation occurred at both N113 and N134 by MS of digested proteins obtained after enzymatic deglycosylation or from mutant forms.     

One-step transformation of the dimorphic yeast Yarrowia lipolytica

An efficient one-step transformation method for the dimorphic yeast Yarrowia lipolytica is described. Using cells grown overnight on agar plates, the whole process is carried out within 1 h. The transformant clones could be recovered on selective plates as early as 36–48 h after plating. The efficiency was better than 10⁵ transformants/μg replicative plasmid DNA. Effects of cell density, dithiothreitol, heat shock, poly(ethylene glycol) 4000 concentration and the wetness of selective plates were investigated. Received: 17 February 1997 / Received revision: 4 April 1997 / Accepted: 19 April 1997     

Extensive Divergence Between Mating-Type Chromosomes of the Anther-Smut Fungus

Genomic regions that determine mating compatibility are subject to distinct evolutionary forces that can lead to a cessation of meiotic recombination and the accumulation of structural changes between members of the homologous chromosome pair. The relatively recent discovery of dimorphic mating-type chromosomes in fungi can aid the understanding of sex chromosome evolution that is common to dioecious plants and animals. For the anther-smut fungus, Microbotryum lychnidis-dioicae (= M. violaceum isolated from Silene latifolia), the extent of recombination cessation on the dimorphic mating-type chromosomes has been conflictingly reported. Comparison of restriction digest optical maps for the two mating-type chromosomes shows that divergence extends over 90% of the chromosome lengths, flanked at either end by two pseudoautosomal regions. Evidence to support the expansion of recombination cessation in stages from the mating-type locus toward the pseudoautosomal regions was not found, but evidence of such expansion could be obscured by ongoing processes that affect genome structure. This study encourages the comparison of forces that may drive large-scale recombination suppression in fungi and other eukaryotes characterized by dimorphic chromosome pairs associated with sexual life cycles.     

Extracellular RNase produced by Yarrowia lipolytica

Production of extracellular RNase(s) by Yarrowia lipolytica CX161-1B was examined in media between pHs 5 and 7. RNase production occurred during the exponential growth phase. High-molecular-weight nitrogen compounds supported the highest levels of RNase production. Several RNases were detected in the supernatant medium. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the RNases had estimated molecular weights of 45,000, 43,000, and 34,000. It was found that Y. lipolytica secretes only one RNase (the 45,000-molecular-weight RNase) and that the 43,000 and 34,000-molecular-weight RNases are degradation products of this RNase. The alkaline extracellular protease secreted by Y. lipolytica was shown to have a major role in the 45,000- to 43,000-molecular-weight conversion, and it was demonstrated that the 45,000-molecular-weight RNase could be purified from a mutant which does not produce the alkaline extracellular protease. Purification of the RNase from a wild-type strain resulted in purification of the 43,000-molecular-weight RNase. This RNase was a glycoprotein with a molecular weight of 44,000 as estimated by gel filtration, an isoelectric point of pH 4.8, and a pH optimum between 6.5 and 7.0.     

Engineering Yarrowia lipolytica for Campesterol Overproduction

Campesterol is an important precursor for many sterol drugs, e.g. progesterone and hydrocortisone. In order to produce campesterol in Yarrowia lipolytica, C-22 desaturase encoding gene ERG5 was disrupted and the heterologous 7-dehydrocholesterol reductase (DHCR7) encoding gene was constitutively expressed. The codon-optimized DHCR7 from Rallus norvegicus, Oryza saliva and Xenapus laevis were explored and the strain with the gene DHCR7 from X. laevis achieved the highest titer of campesterol due to D409 in substrate binding sites. In presence of glucose as the carbon source, higher biomass conversion yield and product yield were achieved in shake flask compared to that using glycerol and sunflower seed oil. Nevertheless, better cell growth rate was observed in medium with sunflower seed oil as the sole carbon source. Through high cell density fed-batch fermentation under carbon source restriction strategy, a titer of 453±24.7 mg/L campesterol was achieved with sunflower seed oil as the carbon source, which is the highest reported microbial titer known. Our study has greatly enhanced campesterol accumulation in Y. lipolytica, providing new insight into producing complex and desired molecules in microbes.     

Budding in the Dimorphic Fungus Phialophora dermatitidis

Ultrastructural comparisons of yeast and hyphal bud formation in Phialophora dermatitidis reveal that bud initiation is characterized by a blastic rupture of the outer portion of the yeast or hyphal wall and the emergence of a bud protuberance through the resulting opening. The wall of the emerging bud is continuous, with only an inner wall layer of the parental yeast or hypha. The outer, ruptured portion of the parental wall typically forms a collar around the constricted emergence region of the developing bud. The cytoplasm within the very young emerging bud invariably contains a small number of membrane-bound vesicles. The septum formed between the daughter bud and the parental yeast or hypha is a complete septum devoid of a septal pore, septal pore plug, or any associated Woronin bodies characteristic of simple septa of the moniliform or true hyphae. These observations suggest that yeast bud formation and lateral hyphal bud formation in the dimorphic fungus P. dermatitidis involve a growth process which occurs identically in both the yeast and mold phase of this human pathogenic organism.     

Preparation of dodecanol-tolerant strains of Yarrowia lipolytica

Dodecanol (1% v/v) and dodecanoic acid (1% w/v) inhibited growth of Yarrowia lipolytica in complex media supplemented with glucose but dodecanedioic acid (1% w/v) was not toxic. Dodecanol-tolerant strains were prepared from the wild type strain H222 as well as the acyl-CoA oxidase deleted (deltaPOX2, POX3, POX5) strain MTLY35. These strains grew in rich media containing up to 10% (v/v) dodecanol. Dodecanol-tolerant strains remained dodecanol tolerant after they had been cultured in rich media without dodecanol. No significant amount of dodecanedioic acid was accumulated by the dodecanol-tolerant strains when grown on glucose in the presence of dodecanol.     

Different effectors of dimorphism in Yarrowia lipolytica

Yarrowia lipolytica is an ascomycete with biotechnological potential. In common media, the fungus grows as a mixture of yeast-like and short mycelial cells. The environmental factors that affect dimorphism in the wild-type strain, W29, and its auxotrophic derivative, PO1a, were analyzed. In both strains, pH was the most important factor regulating the dimorphic transition. Mycelium formation was maximal at pH near neutrality and decreased as pH was lowered to become almost null at pH 3. Carbon and nitrogen sources, namely glucose and ammonium, were also important for mycelium formation; and their effect was antagonized by some alternative carbon and nitrogen sources. Citrate was an important positive effector of mycelium growth. Anaerobic stress induced formation of mycelial cells. The importance of the protein kinase A pathway was suggested by the inhibition of mycelium growth by cAMP. We propose that the interplay of these factors regulates the adaptation of the fungus, to better exploit its natural ecological niches.     

解脂亚罗酵母P12单倍体的制备

[背景]目前解脂亚罗酵母在实验研究和工业生产方面的应用越来越广泛,但相较于常规酵母而言,解脂亚罗酵母缺乏简便有效的遗传转化体系,致使其在基因表达调控方面存在较大困难.同时,酵母的染色体倍性也会对基因敲除效果产生影响,选择单倍体细胞作为功能基因改造的受体可以避免等位基因之间相互作用的影响,解决多倍体细胞基因敲除不完全的问...     

Factors Affecting the Morphogenetic Switch in Yarrowia lipolytica

Yarrowia lipolytica is a dimorphic yeast usually isolated from dairy products. Here we described methods for inducing in a homogeneous way a true yeast-hypha transition in liquid medium. As a first step, the cells must be synchronized in the G1 phase of the cell cycle by nitrogen starvation. Using either N-acetylglucosamine (GlcNAc) or serum as the only carbon sources, more than 90% of the cells form hypha after 4–6 h of incubation. Bovine albumin is also able to induce the yeast-hypha transition, although to a lesser extent. The addition of glucose to cultures growing with GlcNAc arrest the morphogenetic switch but not when added to cultures growing in the presence of serum. Serum also induces invasive growth in solid medium. Neither pH, nitrogen starvation, nor temperature play a relevant role in the morphogenetic switch. Our results suggest that, as occurs in Candida albicans, at least two morphogenetic signal pathways exist in Y. lipolytica. Received: 20 March 2001 / Accepted: 17 April 2001     

Biosynthesis of biotin vitamers by Yarrowia lipolytica

The hydrocarbon utilizing yeast Yarrowia lipolyyica NCYC 1421 produces biotin and its vitamers when grown on glucose in biotin-free media. Levels of production can be influenced by the medium composition. Growth in the presence of longchained fatty acids greatly increases biotin vitamer production. The biotin vitamers produced are normally dethiobiotin and 7-keto, 8-aminopelargonic acid. The addition of succinic acid at 0.5 g per litre causes the vitamer 7, 8-diaminopelargonic acid to be produced at high levels. The biotin antagonist α-dehydrobiotin inhibits the growth of Yarrowia lipolytica . Mutants can be readily isolated which show resistance to α-dehydrobiotin, but these do not produce greater amounts of biotin or its vitamers.     

Phosphatase activity during growth of Yarrowia lipolytica

Abstract The yeast Yarrowia lipolytica produces four patterns of phosphatase activity during growth in the presence or absence of inorganic phosphate in the medium. Activities had pH optima at 4.2, 5.8, about pH 6.5 and pH 9.0. The level of all four phosphatase activities depended on the presence of inorganic phosphate in the medium.     

Efficient selection of hygromycin-B-resistant Yarrowia lipolytica transformants

 The yeast Yarrowia lipolytica was shown to be sensitive to the aminoglycoside antibiotic hygromycin B. Spontaneous resistants appeared at a frequency of (2–5)×10^-7 in media containing 100 mg/l drug. In order to develop a new selective marker for the transformation of this yeast, we constructed new plasmids expressing the Escherichia coli hygromycin-resistance gene (hph) under the control of the promoter and terminator sequences of the strongly expressed XPR2 gene of Y. lipolytica. Direct selection of hygromycin-B-resistant transformants on complete medium was very efficient and resulted in transformation frequencies comparable to those observed with conventional auxotrophic markers. This new marker can be used for integrating single copies of plasmid and for gene disruption and provides a convenient tag for genetic studies. Received: 16 February 1996/Received revision: 12 April 1996/Accepted: 15 April 1996