Molecular characterization of Yarrowia lipolytica and Candida zeylanoides isolated from poultry

Yeast isolates from raw and processed poultry products were characterized using PCR amplification of the internally transcribed spacer (ITS) 5.8S ribosomal DNA region (ITS-PCR), restriction analysis of amplified products, randomly amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE). ITS-PCR resulted in single fragments of 350 and 650 bp, respectively, from eight strains of Yarrowia lipolytica and seven strains of Candida zeylanoides. Digestion of amplicons with HinfI and HaeIII produced two fragments of 200 and 150 bp from Y. lipolytica and three fragments of 350, 150, and 100 bp from C. zeylanoides, respectively. Although these fragments showed species-specific patterns and confirmed species identification, characterization did not enable intraspecies typing. Contour-clamped heterogeneous electric field PFGE separated chromosomal DNA of Y. lipolytica into three to five bands, most larger than 2 Mbp, whereas six to eight bands in the range of 750 to 2,200 bp were obtained from C. zeylanoides. Karyotypes of both yeasts showed different polymorphic patterns among strains. RAPD analysis, using enterobacterial repetitive intergenic sequences as primers, discriminated between strains within the same species. Cluster analysis of patterns formed groups that correlated with the source of isolation. For ITS-PCR, extraction of DNA by boiling yeast cells was successfully used.     

Yarrowia lipolytica possesses two plasma membrane alkali metal cation/H+ antiporters with different functions in cell physiology

The family of Nha antiporters mediating the efflux of alkali metal cations in exchange for protons across the plasma membrane is conserved in all yeast species. Yarrowia lipolytica is a dimorphic yeast, phylogenetically very distant from the model yeast Saccharomyces cerevisiae. A search in its sequenced genome revealed two genes (designated as YlNHA1 and YlNHA2) with homology to the S. cerevisiae NHA1 gene, which encodes a plasma membrane alkali metal cation/H+ antiporter. Upon heterologous expression of both YlNHA genes in S. cerevisiae, we showed that Y. lipolytica antiporters differ not only in length and sequence, but also in their affinity for individual substrates. While the YlNha1 protein mainly increased cell tolerance to potassium, YlNha2p displayed a remarkable transport capacity for sodium. Thus, Y. lipolytica is the first example of a yeast species with two plasma membrane alkali metal cation/H+ antiporters differing in their putative functions in cell physiology; cell detoxification vs. the maintenance of stable intracellular pH, potassium content and cell volume.     

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。     

New finding and optimal production of a novel extracellular alkaline lipase from Yarrowia lipolytica NRRL Y-2178

Lipases are industrially useful versatile enzymes that catalyze numerous different reactions including hydrolysis of triglycerides, transesterification, and chiral synthesis of esters under natural conditions. Although lipases from various sources have been widely used in industrial applications, such as in food, chemical, pharmaceutical, and detergent industries, there are still substantial current interests in developing new microbial lipases, specifically those functioning in abnormal conditions. We screened 17 lipase-producing yeast strains, which were prescreened for substrate specificity of lipase from more than 500 yeast strains from the Agricultural Research Service Culture Collection (Peoria, IL, U.S.A.), and selected Yarrowia lipolytica NRRL Y-2178 as a best lipase producer. This report presents new finding and optimal production of a novel extracellular alkaline lipase from Y. lipolytica NRRL Y-2178. Optimal c ulture conditions f orlipase production by Y. lipolytica NRRL Y-2178 were 72 h incubation time, 27.5 degrees C, pH 9.0. Glycerol and glucose were efficiently used as the most efficient carbon sources, and a combination of yeast extract and peptone was a good nitrogen source for lipase production by Y. lipolytica NRRL Y-2178. These results suggested that Y. lipolytica NRRL Y-2178 showsgood industrial potential as a new alkaline lipase producer.     

Randomly amplified polymorphic DNA (RAPD) PCR for the identification of yeasts isolated from dairy products

In the present work randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) with primers M13 and RF2 was applied to the identification at species level of yeast strains isolated from cheeses. RAPD-PCR analysis of the type strains of different yeast species gave distinctive band profiles that allowed a clear differentiation of all the considered species. Forty-two of the 48 dairy associated yeasts were clearly assigned to the species Saccharomyces cerevisiae, Kluyveromyces marxianus (anamorph Candida kefyr), Kluyveromyces lactis (anamorph Candida sphaerica), Debaryomyces hansenii (anamorph Candida famata), Yarrowia lipolytica and Torulaspora delbrueckii (anamorph Candida colliculosa). The method, which is rapid and easy to perform, could be a useful tool for the identification of yeasts present in dairy products.     

From yeast genetics to biotechnology

Roots of classical yeast genetics go back to the early work of Lindegreen in the 1930s, who studied thallism, sporulation and inheritance of wine yeast strains belonging to S. cerevisiae. Consequent mutation and hybridization of heterothallic S. cerevisae strains resulted in the discovery of life cycle and mating type system, as well as construction of the genetic map. Elaboration of induced mutation and controlled hybridization of yeast strains opened up new possibilities for the genetic analysis of technologically important properties and for the production of improved industrial strains, but a big drawback was the widely different genetic properties of laboratory and industrial yeast strains. Genetic analysis and mapping of industrial strains were generally hindered because of homothallism, poor sporulation and/or low spore viability of brewing and wine yeast strains [1, 2]. In spite of this, there are a few examples of the application of sexual hybridization in the study of genetic control of important technological properties, e.g. sugar utilization, flocculation and flavor production in brewing yeast strains [3] or in the improvement of ethanol producing S. cerevisiae strains [4]. Rare mating and application of karyogamy deficient (kar-) mutants also proved useful in strain improvement [5]. Importance of yeasts in biotechnology is enormous. This includes food and beverage fermentation processes where a wide range of yeast species are playing role, but S. cerevisiae is undoubtedly the most important species among them. New biotechnology is aiming to improve these technologies, but besides this, a completely new area of yeast utilization has been emerged, especially in the pharmaceutical and medical areas. Without decreasing the importance of S. cerevisiae, numerous other yeast species, e.g. Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica have gained increasing potentialities in the modern fermentation biotechnology [6]. Developments in yeast genetics, biochemistry, physiology and process engineering provided bases of rapid development in modern biotechnology, but elaboration of the recombinant DNA technique is far the most important milestone in this field. Other molecular genetic techniques, as molecular genotyping of yeast strains proved also very beneficial in yeast fermentation technologies, because dynamics of both the natural and inoculated yeast biota could be followed by these versatile DNA-based techniques.     

黑曲霉中内切菊粉酶基因及其全合成基因在解脂耶氏酵母中表达的比较

利用酵母密码子偏爱性将黑曲霉(Aspergillus niger)中的内切菊粉酶(Endoinu linase)基因通过基因全合成的方式合成为酵母密码子偏爱性的内切菊粉酶基因。然后将原始和全合成的内切菊粉酶基因克隆到解脂耶氏酵母表达载体PINA1296上,得重组解脂耶氏酵母表达载体pHBM2020、pHBM2021,将两种质粒分别转化解脂耶氏酵母(Yarrowia lipolytica)CLIB725,筛选得到重组解脂耶氏酵母CLIB725(pHBM2020)、CLIB725(pHBM2021),将两种重组酵母摇瓶培养,经SDS-PAGE、测酶活检测表明两种基因在解脂耶氏酵母中都有表达,全合成菊粉酶比原始菊粉酶酶活要高。     

<Emphasis Type="Italic">Metschnikowia viticola</Emphasis> sp. nov., a new yeast species from grape

Two yeast strains, producing needle-shaped ascospores under suitable conditions, were isolated from grapes grown in Hungary. Based on these two strains, Metschnikowia viticola (type strain NCAIM Y.01705, CBS 9950, JCM 12561) is proposed as a new yeast species. Considering its phenotypic features, the restriction fragment patterns of 18S rDNA and the sequence of the D1/D2 domain of 26S rDNA, the proposed new species is closely related to Candida kofuensis.     

Production of biotin by yeasts

A quantitative screening procedure for biotin and biotin vitamer production was conducted on 129 yeast strains able to grow in a biotin-free medium. Production of biotin and biotin vitamers varied considerably from strain to strain even within a species. The best producers of biotin were strains of Sporobolomyces roseus and Rhodotorula rubra whilst strains of Rhodotorula rubra and Yarrowia lipolytica produced the largest amounts of vitamers.     

A phosphatidylinositol/phosphatidylcholine transfer protein is required for differentiation of the dimorphic yeast Yarrowia lipolytica from the yeast to the mycelial form

The SEC14SC gene encodes the phosphatidylinositol/phosphatidylcholine transfer protein (PI/PC-TP) of Saccharomyces cerevisiae. The SEC14SC gene product (SEC14pSC) is associated with the Golgi complex as a peripheral membrane protein and plays an essential role in stimulating Golgi secretory function. We report the characterization of SEC14YL, the structural gene for the PI/PC-TP of the dimorphic yeast Yarrowia lipolytica. SEC14YL encodes a primary translation product (SEC14YL) that is predicted to be a 497-residue polypeptide of which the amino- terminal 300 residues are highly homologous to the entire SEC14pSC, and the carboxyl-terminal 197 residues define a dispensible domain that is not homologous to any known protein. In a manner analogous to the case for SEC14pSC, SEC14pYL localizes to punctate cytoplasmic structures in Y. lipolytica that likely represent Golgi bodies. However, SEC14pYL is neither required for the viability of Y. lipolytica nor is it required for secretory pathway function in this organism. This nonessentiality of SEC14pYL for growth and secretion is probably not the consequence of a second PI/PC-TP activity in Y. lipolytica as cell-free lysates prepared from delta sec14YL strains are devoid of measurable PI/PC-TP activity in vitro. Phenotypic analyses demonstrate that SEC14pYL dysfunction results in the inability of Y. lipolytica to undergo the characteristic dimorphic transition from the yeast to the mycelial form that typifies this species. Rather, delta sec14YL mutants form aberrant pseudomycelial structures as cells enter stationary growth phase. The collective data indicate a role for SEC14pYL in promoting the differentiation of Y. lipolytica cells from yeast to mycelia, and demonstrate that PI/PC-TP function is utilized in diverse ways by different organisms.     

Essential role of YlMPO1, a novel Yarrowia lipolytica homologue of Saccharomyces cerevisiae MNN4, in mannosylphosphorylation of N- and O-linked glycans

Mannosylphosphorylation of N- and O-glycans, which confers negative charges on the surfaces of cells, requires the functions of both MNN4 and MNN6 in Saccharomyces cerevisiae. To identify genes relevant to mannosylphosphorylation in the dimorphic yeast Yarrowia lipolytica, the molecular functions of five Y. lipolytica genes showing significant sequence homology with S. cerevisiae MNN4 and MNN6 were investigated. A set of mutant strains in which Y. lipolytica MNN4 and MNN6 homologues were deleted underwent glycan structure analysis. In contrast to S. cerevisiae MNN4 (ScMNN4), the Y. lipolytica MNN4 homologue, MPO1 (YlMPO1), encodes a protein that lacks the long KKKKEEEE repeat domain at its C terminus. Moreover, just a single disruption of YlMPO1 resulted in complete disappearance of the acidic sugar moiety in both the N- and O-linked glycan profiles. In contrast, even quadruple disruption of all ScMNN6 homologues, designated YlKTR1, YlKTR2, YlKTR3, and YlKTR4, resulted in no apparent reduction in acidic sugar moieties. These findings strongly indicate that YlMpo1p performs a significant role in mannosylphosphorylation in Y. lipolytica with no involvement of the Mnn6p homologues. Mutant strains harboring the YlMPO1 gene disruption may serve as useful platforms for engineering Y. lipolytica glycosylation pathways for humanized glycans without any yeast-specific acidic modifications.     

Characterization of three endophytic, indole-3-acetic acid-producing yeasts occurring in Populus trees

Three endophytic yeast, one isolated from stems of wild cottonwood (Populus trichocarpa), two from stems of hybrid poplar (P. trichocarpa × Populus deltoides), were characterized by analyzing three ribosomal genes, the small subunit (18S), internal transcribed spacer (ITS), and D1/D2 region of the large subunit (26S). Phenotypic characteristics of the yeast isolates were also obtained using a commercial yeast identification kit and used for assisting the species identification. The isolate from wild cottonwood was identified to be closest to species Rhodotorula graminis. The two isolates from hybrid poplar were identified to be species Rhodotorula mucilaginosa. In addition, the three yeast isolates were observed to be able to produce indole-3-acetic acid (IAA), a phytohormone which can promote plant growth, when incubated with l-tryptophan. To our knowledge, the yeast strains presented in this study were the first endophytic yeast strains isolated from species of Populus.     

High-level production of extracellular lipase by Yarrowia lipolytica mutants from methyl oleate

The yeast Yarrowia lipolytica degrades efficiently low-cost hydrophobic substrates for the production of various added-value products such as lipases. To obtain yeast strains producing high levels of extracellular lipase, Y. lipolytica DSM3286 was subjected to mutation using ethyl methanesulfonate (EMS) and ultraviolet (UV) light. Twenty mutants were selected out of 1600 mutants of Y. lipolytica treated with EMS and UV based on lipase production ability on selective medium. A new industrial medium containing methyl oleate was optimized for lipase production. In the 20 L bioreactor containing new industrial medium, one UV mutant (U6) produced 356 U/mL of lipase after 24h, which is about 10.5-fold higher than that produced by the wild type strain. The properties of the mutant lipase were the same as those of the wild type: molecular weight 38 kDa, optimum temperature 37°C and optimum pH 7. Furthermore, the nucleotide sequences of extracellular lipase gene (LIP2) in wild type and mutant strains were determined. Only two silent substitutions at 362 and 385 positions were observed in the ORF region of LIP2. Two single substitutions and two duplications of the T nucleotide were also detected in the promoter region. LIP2 sequence comparison of the Y. lipolytica DSM3286 and U6 strains shows good targets to effective DNA recombinant for extracellular lipase of Y. lipolytica.     

Radiation resistance ofSalmonella

Summary The ionizing radiation resistances of sixSalmonella species were examined. The experimental variables were the suspending medium, the presence or absence of air, and the temperature during the irradiation process.S. typhimurium ATCC 14028,S. enteritidis ATCC 9186,S. newport ATCC 6962,S. dublin ATCC 15480,S. anatum ATCC 9270, andS. arizonae ATCC 29933 were suspended in phosphate buffer (0.1 M, pH 7.0), brain heart infusion broth (BHI) or mechanically deboned chicken and exposed to gamma radiation from cesium-137 at 0.12 kGy per min. The radiation resistance of theSalmonella increased approximately two-fold when assayed in sterile mechanically deboned chicken rather than in buffer or BHI. The average radiation (0.30 to 1.20 kGy) D-value for all sixSalmonella strains was 0.56 kGy in mechanically deboned chicken.S. enteritidis was significantly more resistant to ionizing radiation than the other five strains ofSalmonella tested on mechanically deboned chicken. The temperature of irradiation but not the presence or absence of air significantly influenced the survival ofS. typhimurium andS. enteritidis in mechanically deboned chicken. Treatment of chicken meat with ionizing radiation would be an effective means for control ofSalmonella contamination.     

A comparison of clinical and food Saccharomyces cerevisiae isolates on the basis of potential virulence factors

Saccharomyces cerevisiae is the most widely used yeast in industrial/commercial food and beverage production and is even consumed as a nutritional supplement. Various cases of fungemia caused by this yeast species in severely debilitated traumatized or immune-deficient patients have been reported in recent years, suggesting that this species could be an opportunistic pathogen in such patients. To determine whether the industrial S. cerevisiae strains can be included in this virulent group of strains, we carried out a comparative study between clinical and industrial yeasts based on the various phenotypic traits associated with pathogenicity in two other yeast species (Candida albicans and Cryptococcus neoformans). The majority of the clinical isolates were found to secrete higher levels of protease and phospholipase, grow better at 42°C and show strong pseudohyphal growth relative to industrial yeasts. However three industrial yeast strains, one commercial wine strain, baker’s yeast and one commercial strain of S. cerevisiae (var. boulardii), were exceptions and based on their physiological traits these yeasts would appear to be related to clinical strains.     

Evolutionary relationships among pathogenic Candida species and relatives.

Small subunit rRNA sequences have been determined for 10 of the most clinically important pathogenic species of the yeast genus Candida (including Torulopsis [Candida] glabrata and Yarrowia [Candida] lipolytica) and for Hansenula polymorpha. Phylogenetic analyses of these sequences and those of Saccharomyces cerevisiae, Kluyveromyces marxianus var. lactis, and Aspergillus fumigatus indicate that Candida albicans, C. tropicalis, C. parapsilosis, and C. viswanathii form a subgroup within the genus. The remaining significant pathogen, T. glabrata, falls into a second, distinct subgroup and is specifically related to S. cerevisiae and more distantly related to C. kefyr (psuedotropicalis) and K. marxianus var. lactis. The 18S rRNA sequence of Y. lipolytica has evolved rapidly in relation to the other Candida sequences examined and appears to be only distantly related to them. As anticipated, species of several other genera appear to bear specific relationships to members of the genus Candida.