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.     

Change in transport activities of vacuoles of the yeast Yarrowia lipolytica during its growth on glucose

Vacuoles were isolated from Yarrowia lipolytica yeast cells taken at various growth phases under carbon or nitrogen limitation. Vacuoles from the cells at the logarithmic growth phase showed a high activity of vacuolar H⁺-ATPase (0·9–1·1 U/mg protein) and efficiently generated chemical proton gradient and membrane potential across the tonoplast. Ca²⁺- and citrate transport were found to be maximal at this growth phase. At growth retardation and then in the stationary phase all the parameters studied decreased irrespective of the method of growth limitation. The citrate-transporting activity of vacuoles completely disappeared at growth retardation, also irrespective of the limitation method and irrespective of whether yeast cells overproduced citrate in the culture medium. The citrate-transporting system of Y. lipolytica vacuolar membrane is concluded not to be involved in citrate efflux and this efflux is probably performed by the plasmalemma transport system.     

The technological characteristics of Debaryomyces hansenii and Yarrowia lipolytica and their potential as starter cultures for production of Danablu

Six strains of Debaryomyces hansenii var. hansenii and Yarrowia lipolytica, respectively, originating from blue mould cheeses were examined for their potential use as starter cultures for the production of Danablu in laboratory studies. D. hansenii showed strong growth and assimilation of lactose, galactose, lactate and five out of six strains assimilated citric acid under the environmental conditions prevailing in Danablu during maturation at 10°C. Y. lipolytica was more sensitive to NaCl and did not assimilate lactose and galactose. Both yeasts hydrolysed tributyrin with the highest activity observed for Y. lipolytica. D. hansenii showed little if any release of free fatty acids from butterfat at 10°C. Y. lipolytica was strongly lipolytic. The strains of D. hansenii were not able to hydrolyse casein at 10°C whereas 4 of the 6 strains of Y. lipolytica degraded all components of the casein. Strain-specific interactions, in cheese agar resulting in inhibition of mycelial growth and sporulation of P. roqueforti was observed, especially for Y. lipolytica.     

An efficient method for production of kynurenic acid by Yarrowia lipolytica

Kynurenic acid (KYNA) is a compound derived from the tryptophan catabolic pathway. Antioxidant and neuroprotective properties have been confirmed for KYNA, which makes it an interesting and important metabolite of biomedical significance. In the present study, the yeast Yarrowia lipolytica was tested for KYNA biosynthesis. The results showed that Y. lipolytica strain S12 is able to produce KYNA in high concentrations (up to 21.38 μg/ml in culture broth and 494.16 μg/g cell dry weight in biomass) in optimized conditions in a medium supplemented with tryptophan. Different conditions of culture growth, including the source of carbon, its concentration and pH value of the medium, as well as the influence of an inhibitor or precursor of KYNA synthesis, were analysed. The obtained data confirmed the presence of KYNA metabolic pathway in the investigated yeast. To our best knowledge, this is the first study that reports KYNA production in the yeast Y. lipolytica in submerged fermentation.     

XVI. Yeast sequencing reports. A new essential gene GCR4 located in the upstream region of GCR1

A new essential gene of Saccharomyces cerevisiae was found upstream of GCR1. Its cloning and sequencing predict a 280 amino acid protein (32 577 Da). The predicted protein is fairly hydrophobic, and a search of the database did not identify any homologous proteins. A LEU2 disruption at codon 104 was lethal, but disruption at codon 221 showed a temperature-sensitive conditional growth phenotype. Abnormalities were observed in some glycolytic enzyme levels. The sequence has been submitted to GenBank-EMBL-DDBJ under Accession Number D29645.     

Yeast exo-β-glucanases can be used as efficient and readily detectable reporter genes in Saccharomyces cerevisiae

Yeast exo-1,3-β-glucanases are secretable proteins whose function is basically trophic and may also be involved in cell wall glucan hydrolytic processes. Since fluorescein di(β-D -glucopyranoside) is a fluorogenic substrate detectable and quantifiable by flow cytometry, it was used for testing the ability of the EXG1 gene product of Saccharomyces cerevisiae and its homologous gene in Candida albicans to function as reporter genes. These open reading frames were coupled to different promoters in multicopy plasmids, and exoglucanase activity quantified at flow cytometry. Exoglucanases were found to be useful tools for the study of promoter regions in S. cerevisiae. This technique has the advantage over other reporter gene systems—such as β-galactosidase fusions—that it does not require permeabilization of yeast cells and therefore it allows the recovery of viable cells—by sorting—after flow cytometry analysis.     

I. Yeast sequencing reports. Isolation and characterization of the LEU2 gene of Hansenula polymorpha

A DNA fragment carrying the LEU2 gene of methylotrophic yeast Hansenula polymorpha was isolated by complementation of the leuB mutation of Escherichia coli. The nucleotide sequence of the isolated DNA fragment contains an open reading frame of 363 codons, coding for a protein 80% identical to the LEU2 gene product of Saccharomyces cerevisiae. Further downstream, there is a partial reading frame with no obvious similarity to known proteins. The LEU2 gene of H. polymorpha cannot complement the leu2 mutation of S. cerevisiae. The sequence has been entered in the EMBL data library under the Accession Number U00889.     

Cloning and characterization of the peroxisomal acyl CoA oxidase ACO3 gene from the alkane-utilizing yeast Yarrowia lipolytica

The ACO3 gene, which encodes one of the acyl-CoA oxidase isoenzymes, was isolated from the alkane-utilizing yeast Yarrowia lipolytica as a 10 kb genomic fragment. It was sequenced and found to encode a 701-amino acid protein very similar to other ACOs, 67·5% identical to Y. lipolytica Aco1p and about 40% identical to S. cerevisiae Pox1p. Haploid strains with a disrupted allele were able to grow on fatty acids. The levels of acyl-CoA oxidase activity in the ACO3 deleted strain, in an ACO1 deleted strain and in the wild-type strain, suggested that ACO3 encodes a short chain acyl-CoA oxidase isoenzyme. This narrow substrate spectrum was confirmed by expression of Aco3p in E. coli. © 1998 John Wiley & Sons, Ltd.     

Characterization of phosphatidic acid phosphatase activity in the oleaginous yeast Yarrowia lipolytica and its role in lipid biosynthesis

Phosphatidic acid phosphatase (PAP) catalyses the committed step of triacylglycerol (TAG) biosynthesis and thus regulates the amounts of TAG produced by the cell. TAG is the target of biotechnological processes developed for the production of food lipids or biofuels. These processes are using oleaginous microorganisms like the yeast Yarrowia lipolytica as the TAG producers. Thus manipulating key enzymatic activities like PAP in Y. lipolytica could drive lipid biosynthesis towards TAG production and increase TAG yields. In this study, PAP activity in Y. lipolytica was characterized in detail and its role in lipid biosynthesis was addressed. PAP activity increased 2.5‐fold with the addition of Mg²⁺ (1 mm ) in the assay mixture, which means that most of the PAP activity was due to Mg²⁺‐dependent PAP enzymes (e.g. Pah1, App1). In contrast, N‐ethylmaleimide (NEM) potently inhibited PAP activity, indicating the presence of NEM‐sensitive PAP enzymes (e.g. App1, Lpp1). Localization studies revealed that the majority of PAP activity resides in the membrane fraction, while the cytosolic fraction harbours only a small amount of activity. PAP activity was regulated in a growth‐dependent manner, being induced at the early exponential phase and declining thereafter. PAP activity did not correlate with TAG synthesis, which increased as cells progressed from the exponential phase to the early stationary phase. In stationary phase, TAG was mobilized with the concomitant synthesis of sterols and sterol esters. These results provide the first insights into the role of PAP in lipid biosynthesis by Y. lipolytica. Copyright © 2016 John Wiley & Sons, Ltd.     

Comparison of expression systems in the yeasts Saccharomyces cerevisiae,Hansenula polymorpha,Klyveromyces lactis,Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica

We have compared expression systems based on autonomously replicating vectors in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha and Yarrowia lipolytica in order to identify a more suitable host organism for use in the expression cloning method (Dalbøge and Heldt-Hansen, 1994) in which S. cerevisiae has traditionally been used. The capacity of the expression systems to secrete active forms of six fungal genes encoding the enzymes galactanase, lipase, polygalacturonase, xylanase and two cellulases was examined, as well as glycosylation pattern, plasmid stability and transformation frequency. All of the examined alternative hosts were able to secrete more active enzyme than S. cerevisiae but the relative expression capacity of the individual hosts varied significantly in a gene-dependent manner. One of the most attractive of the alternative host organisms, Y. lipolytica, yielded an increase which ranged from 4·5 times to more than two orders of magnitude. As the initially employed Y. lipolytica XPR2 promoter is unfit in the context of expression cloning, two novel promoter sequences for highly expressed genes present in only one copy on the genome were isolated. Based on sequence homology, the genes were identified as TEF, encoding translation elongation factor-1α and RPS7, encoding ribosomal protein S7. Using the heterologous cellulase II (celII) and xylanase I (xylI) as reporter genes, the effect of the new promoters was measured in qualitative and quantitative assays. Based on the present tests of the new promoters, Y. lipolytica appears as a highly attractive alternative to S. cerevisiae as a host organism for expression cloning. GenBank Accession Numbers: TEF gene promoter sequence: AF054508; RPS7 gene promoter sequence: AF054509. © 1998 John Wiley & Sons, Ltd.     

The Yarrowia lipolytica PAH1 homologue contributes but is not required for triacylglycerol biosynthesis during growth on glucose

The PAH1-encoded phosphatidate phosphatase (PAP) catalyzes the Mg²⁺-dependent dephosphorylation of phosphatidate to produce diacylglycerol, which can be acylated to form triacylglycerol (TAG). In the model oleaginous yeast Yarrowia lipolytica, TAG is the major lipid produced, and its biosynthesis requires a continuous supply of diacylglycerol, which can be provided by the PAP reaction. However, the regulation of Pah1 has not been studied in detail in Y. lipolytica, and thus its contribution to the biosynthesis of TAG in this yeast is not well understood. In this work, we examined the regulation of the PAH1-mediated PAP activity and Pah1 abundance and localization in cells growing on glucose. We found that Pah1 abundance and localization were regulated in a growth-dependent manner, yet the loss of Pah1 did not have a major effect on PAP activity. We also examined the effects of the Y. lipolytica pah1Δ mutation on cell physiology and lipid biosynthesis. The lack of Pah1 in the pah1Δ mutant resulted in a moderate decrease in TAG levels and an increase in phospholipid levels. These results showed that Pah1 contributed to TAG biosynthesis in Y. lipolytica but also suggested the presence of other activities in the pah1Δ mutant that compensate for the loss of Pah1. Also, the levels of linoleic acid were elevated in pah1Δ cells with a concomitant decrease in the oleic acid levels suggesting that the pah1Δ mutation affected the biosynthesis of fatty acids.     

Yarrowia lipolytica SRP54 Homolog and Translocation of Kar2p

To investigate the role of Srp54p in protein translocation, the Yarrowia lipolytica SRP54 homolog was cloned. Sequencing revealed an open reading frame of 536 amino acids coding for a 57·2 kilodalton polypeptide with 55 to 57% sequence identity to Srp54ps of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and mouse. Like these Srp54ps, Y. lipolytica Srp54p has an N-terminal domain with a highly conserved GTP-binding site and a methionine-rich C-terminal domain. Differing results regarding the essentiality of SRP subunits were obtained. SRP54 is important but not essential for growth, but it was reconfirmed that at least one SRP RNA gene is essential. Cells with SRP54 deleted grow about six times more slowly than wild type; faster-growing colonies, still growing much slower than wild type, appeared quite frequently. In srp54Δ cells, no untranslocated alkaline extracellular protease precursor was detected. Therefore, to develop another reporter molecule the Y. lipolytica KAR2 homolog was cloned and Kar2p antibodies were produced. For Kar2p an untranslocated precursor was detected in srp54Δ but not in wild-type cells, suggesting that its translocation was defective in the srp54Δ cells. These results confirm an in vivo role for SRP in protein translocation in Y. lipolytica, suggest that SRP RNA or an SRP core-particle has functions not shared by Srp54p, and show that, as in S. cerevisiae and Sz. pombe, reporter molecules differ in their dependency on SRP for translocation. The SRP54 and KAR2 sequences have been deposited in GenBank under Accession Numbers U42418 and U63136.     

Induced expression of bacterial β-glucosidase activity in saccharomyces

The bglA gene which encodes a β-glucosidase from Bacillus polymyxa, has been expressed in Saccharomyces cerevisiae under control of the yeast CYC-GAL promoter. Strains have been constructed which carry the gene in different locations: in a multicopy plasmid, a single integration at the URA3 locus, or multiple integrations at the RDN1 locus. Integrative transformation at RDN1 yielded genetically stable clones with a high level of β-glucosidase activity. Coordinated overexpression of the GAL4 inducer protein further increased the level of enzyme activity, although eventually caused the lysis of the cultures. Diploid, triploid and tetraploid strains derived from the transformants with multiple integrations were constructed and expression of β-glucosidase activity in different conditions of growth was assayed. While per-cell activity increased with ploidy, specific activity was about the same in strains of equivalent genotype regardless of ploidy. Genetically stable and regulated expression in Saccharomyces of β-glucosidase activity is interesting for the development of strains able to ferment β-glycosidic sugars (i.e. cellobiose and lactose). From another point of view, the bglA product proved to be a convenient reporter enzyme to monitor heterologous gene expression.     

Effect of Dyes on the Growth of Food Yeasts

Organic dyes were incorporated in YM medium individually at various concentrations (1:1,000 to 1:1,000,000 dilutions) to test their effects on growth of 33 food-related yeasts (e.g. Saccharomyces spp. and Candida spp.). Of 101 dyes tested, many (e.g. Bismarck brown Y, Brilliant cresyl blue, Congo red, Crystal violet, Diphenyl red 4BS supra, Erionyl Red B, Jannus green B, Malachite green, Methyl green, Methyl red, Methyl violet 2B, Nylomine red C-G, Phenosafranin, Safranin Y, Telon yellow GRL) showed potential application for selective isolation and differentiation of specific food yeast species. Candida lipolytica can be effectively selected from other yeasts on a Crystal violet medium (1: 1,000) and Malachite green medium (1:250,000) by providing distinctive white coarsely folded colonies. Trypan blue, Atlantic sky blue FF, Erie brilliant violet B, Eriosin blue B, and Jenner's stain contributed their characteristic violet or blue to the colonies, which may help counting or differentiation on primary plating agar.