Expression of yeast homolog of the mammal BCRP gene coding for riboflavin efflux protein activates vitamin B2 production in the flavinogenic yeast Candida famata

Candida famata is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B₂) in response to iron limitation. Overproduced riboflavin accumulates in the cultural medium rather than in the cells suggesting existence of the special mechanisms involved in riboflavin excretion. The corresponding protein and gene have not been identified in yeasts. At the same time, the corresponding gene BCRP has been identified in mammal mammary glands. Several homologs of the mammal BCRP gene encoding putative riboflavin efflux protein (excretase) were identified in Debaryomyces hansenii. The closest homolog was expressed under the control of D. hansenii TEF1 promoter in the riboflavin overproducing strain of C. famata. Resulted transformants overexpressed the corresponding gene and produced 1.4- to 1.8-fold more riboflavin as compared with the parental strain. They also were characterized by overexpression of RIB1 and RIB6 genes of riboflavin synthesis and exhibited elevated specific activity of GTP-cyclohydrolase II. Membrane localization of the riboflavin excretase was confirmed by fluorescent microscopy.     

Candida famata (Candida flareri)

Candida famata (Candida flareri) belongs to the group of so‐called ‘flavinogenic yeasts’, capable of riboflavin oversynthesis under condition of iron starvation. Some strains of C. famata belong to the most flavinogenic organisms known and were used for industrial production of riboflavin for a long time in the USA. C. famata is characterized by high salt tolerance, growing at NaCl concentrations of up to 2.5 m . Development of basic tools for the metabolic engineering of C. famata, such as a transformation system, selective markers, insertional mutagenesis, a reporter system and others, are described. The developed tools were used for cloning and identification of structural and regulatory genes of riboflavin synthesis. The construction of improved yeast strains producing riboflavin, FMN and FAD, based on the industrial riboflavin‐producing strain dep8 and its non‐reverting analogue AF4, is also described. Copyright © 2012 John Wiley & Sons, Ltd.     

A novel kanamycin/G418 resistance marker for direct selection of transformants in Escherichia coli and different yeast species

We have developed a set of cloning vectors possessing a modified Tn903 kanamycin resistance gene that enables the selection of both kanamycin‐resistant transformants in Escherichia coli and G418‐resistant transformants in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha and Pichia pastoris. Expression of this gene in yeast is controlled by the H. polymorpha glyceraldehyde‐3‐phosphate dehydrogenase promoter, while expression in E. coli is governed by an upstream E. coli lacZ promoter. Applicability of the vectors for gene disruption in H. polymorpha and S. cerevisiae was demonstrated by inactivation of the HpMAL1 and URA3 genes, respectively. One of the vectors possesses a H. polymorpha ARS allowing plasmid maintenance in an episomal state. The small size of the vectors (2–2.5 kb) makes them convenient for routine DNA cloning. In addition, we report a novel approach for construction of gene disruption cassettes. Copyright © 2009 John Wiley & Sons, Ltd.     

Flocculation of the yeast Candida famata (Debaryomyces hansenii): An essential role for peptone

Aggregation of Candida famata (Debaryomyces hansenii) is consistent with being a form of lectin-mediated yeast flocculation. Flocculation of C. famata is unusual in that it requires the presence of peptone, either in the growth medium or added later to harvested cells in buffer. Flocculation after peptone addition was rapid, being largely complete within 10 min. Heat-killed cells also flocculated, arguing for direct participation of peptone in the flocculation binding mechanism. Flocculent C. famata cells progressively lost the ability to flocculate when washed with EDTA. Flocculation was fully restored by peptone addition; calcium addition was without effect. C. famata cells were able to agglutinate erythrocytes in the presence or absence of peptone. Pronase E-treated yeast lost both the ability to haemagglutinate and self-flocculate. Haemagglutination was not diminished by progressive EDTA washing, suggesting that surface lectins remained present and active on the yeast cell walls. Non-flocculating C. famata cells mutually flocculated with non-flocculent Schizosaccharomyces pombe cells, shown to have surface-exposed galactose residues. Mutual flocculation was lost following treatment of C. famata with Pronase E. It was concluded that the cell wall of C. famata contains lectins enabling haemagglutination and mutual flocculation but lacks carbohydrate receptors for these lectins. This yeast self-flocculates only via bridging multi-valent carbohydrates; these being present in peptone.     

Vector YFRp1 allows transformant selection in Saccharomyces cerevisiae via resistance to formaldehyde

Formaldehyde (FA), a chemical with low toxic potential, is used as sole selective agent for transformation in the yeast Saccharomyces cerevisiae. Neither stable auxotrophic markers in recipient cells nor defined synthetic media are needed when multicopy vector YFRp1, containing the yeast SFA gene, is employed for yeast transformation. The SFA gene gives stability to the vector and its yeast (and other) passenger genes when transformants are propagated in complex media supplemented with 3–5 mM-FA. Use of inexpensive FA and non-synthetic, undefined media will lower the cost of yeast transformant propagation considerably and thus make feasible large-volume industrial application of transformants containing YFRp1 derivatives.     

Development of the yeast flora of whole-crop maize during ensiling and during subsequent aerobiosis

The fresh maize plant was inhabited by non-fermenting basidiomycetous yeast species, viz. Candida ingeniosa, Cryptococcus laurentii, Sporobolomyces roseus, Sporidiobolus salmonicolor and Rhodotorula rubra. These species had vanished after 2 days of anaerobiosis in a laboratory-scale silo. After this time, ascomycetous species tolerant to acetic acid were isolated. Successively Candida holmii, C. milleri, C. krusei, C. lambica, C. famata, Geotrichum candidum and Hansenula anomala were encountered. Except for C. famata and G. candidum these yeasts vigorously ferment glucose. Total yeast counts were 10⁷ g^?1 during the first 2 weeks and gradually decreased to about 10⁵ g^?1 after 4 months. This shows that anaerobic silage provides an adverse environment for yeasts. Exposure of maize silage to air resulted in complete consumption of lactic and acetic acids within 5 days. This was due to growth of C. hoimii, C. lambica and C. milled. The physiologically similar yeast species C. holmii and C. milled were both abundant from the early stage of the fermentation and during aerobiosis. However, C. milled predominated in the first 2 weeks of anaerobiosis. The less frequently occurring yeast species C. famata, G. candidum and H. anomala assimilated lactic acid and ethanol as well as minor products of bacterial sugar fermentation such as acetoin and butan-2,3-diol but not diacetyl.     

Direct selection of Pichia pastoris expression strains using new G418 resistance vectors

The methylotrophic yeast, Pichia pastoris, is widely used as a host organism for the expression of heterologous proteins. Currently, the Zeocin and blasticidin resistance genes are the only dominant selectable markers that can be used for primary selection of transformants. In this report we describe new expression vectors that can be used to select directly for P. pastoris transformants using G418 resistance conferred by a modified Tn903kan(r) gene. Compared to other dominant markers, this system is more economical and offers a higher transformation efficiency, due to the small sizes of the cloning vectors, pKAN B and pKANalpha B (GenBank Accession Nos EU285585 and EU285586, respectively). Additionally, multicopy transformants can be generated using these new vectors.     

Isolation and sequence of the gene encoding ornithine decarboxylase,SPE1, from Candida albicans by complementation of a spe1Δ ctrain of Saccharomyces cerevisiae

The gene encoding ornithine decarboxylase, SPE1, from the pathogenic yeast Candida albicans has been isolated by complementation of an ornithine decarboxylase-negative (spe1Δ) strain of Saccharomyces cerevisiae. Four transformants, three of which contain plasmids with the SPE1 gene, were isolated by selection on polyamine-free medium. The C. albicans ornithine decarboxylase (ODC) showed high homology with other eukaryotic ODCs at both the amino acid and nucleic acid levels. The GenBank accession number for this gene is U85005. © 1997 John Wiley & Sons, Ltd.     

Efficient selection of hybrids by protoplast fusion using drug resistance markers and reporter genes in Saccharomyces cerevisiae

We have developed a selection system for hybrids by protoplast fusion using dominant selective drug resistance markers, Tn601(903) against geneticin and AUR1-C against aureobasidin A, and reporter genes, ADH1p-PHO5-ADH1t and CLN2p-CYC1-lacZ, in Saccharomyces cerevisiae. To examine the effectiveness of this system, plasmids with each marker and reporter gene were introduced into auxotrophic sake yeasts. From the resulting transformants, eight colonies were screened by protoplast fusion in combination with the drug resistance markers and the reporter genes. Among them, seven strains were judged as hybrids between parental strains by analysis of growth on a minimal medium. This selection system was applied to wine yeasts having no genetic markers. Six strains were regarded as hybrids between parental strains by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the MET2 gene and by karyotype analysis using a contour-clamped homogeneous electric field (CHEF). We propose that the plotoplast fusion using dominant selective geneticin- and aureobasidin A-resistance markers and reporter genes is useful for the selection of hybrids from wine yeasts, which are homothallic and have low sporulation ability.     

A disruption-replacement approach for the targeted integration of foreign genes in Hansenula polymorpha

A system has been developed which allows the selection of integrative transformants with replacement of the Hansenula polymorpha methanol oxidase gene (MOX) with expression cassettes carrying heterologous gene under the control of the MOX promoter. The system is convenient for comparison of the expression levels of different constructs integrated into the same locus of the H. polymorpha genome. This system was used to compare the secretion levels of human urinary plasminogen activator, the secretion of which was directed by different signal sequences.     

Expression Cassettes for Formaldehyde and Fluoroacetate Resistance,Two Dominant Markers in Saccharomyces cerevisiae

We employed two genes in constructing yeast expression cassettes for dominant, selectable markers. The Saccharomyces cerevisiae gene SFA1, encoding formaldehyde dehydrogenase, was placed under the control of the GPD1 promoter and CYC1 terminator. The Moraxella sp. strain B gene dehH1, encoding fluoroacetate dehalogenase, was placed under the control of both the GPD1 and CYC1 promoters. With these constructs it was possible to select directly for yeast strains resistant to either formaldehyde or fluoroacetate. Both selective agents are completely metabolized and inexpensive, making them very useful in the pursuit of yeast gene functions and for industrial applications. An additional advantage of the formaldehyde dehydrogenase marker is that it is an S. cerevisiae gene, thus allowing ‘all yeast’ constructs. © 1997 John Wiley & Sons, Ltd.     

Overexpression of the genes of glycerol catabolism and glycerol facilitator improves glycerol conversion to ethanol in the methylotrophic thermotolerant yeast Ogataea polymorpha

Production of fuel ethanol is one of the possible ways to utilize crude glycerol, substantial amounts of which are produced by biodiesel industry. Earlier, we have described construction of the recombinant strains of methylotrophic thermotolerant yeast Ogataea polymorpha with simultaneous overexpression of the genes PDC1 and ADH1, which produced increased amounts of ethanol from glycerol. In this work, we have further improved these strains by overexpression of genes involved either in oxidative (through dihydroxyacetone) or phosphorylative (through glycerol-3-phosphate) pathway of glycerol catabolism, as well as heterologous gene coding for glycerol transporter FPS1 from Komagataella phaffii (formerly, Pichia pastoris). Obtained recombinant strains produced up to 10.7 g/L of ethanol (with ethanol productivity 30 mg/g of biomass/hr and yield 132 mg/g of consumed glycerol) from pure glycerol and up to 3.55 g/L of ethanol (with ethanol productivity 11.6 mg/g of biomass/hr and yield 72.3 mg/g of consumed glycerol) from crude glycerol as a carbon source, which is approximately 15 times more relative to that of the O. polymorpha wild-type strain and 2.2 more relative to the earlier constructed strain.     

Tryptophan accumulation in Saccharomyces cerevisiae under the influence of an artificial yeast TRP gene cluster

Plasmid pME559, carrying all five yeast TRP genes, was constructed. This plasmid is a yeast/Escherichia coli shuttle vector based on pBR322 and 2 μm-DNA sequences derived from plasmid pJDB207. We studied in yeast (i) the stability of the plasmid under selective and non-selective conditions, (ii) expression of all five TRP genes and (iii) tryptophan accumulation in yeast transformants. These studies were conducted in comparison with an earlier construction, pME554, which differs from plasmid pME559 in the expression of the TRP1 gene and which carries the TRP2 wild type instead of the TRP2^fbr mutant allele. For stable maintenance of the plasmids in yeast a selection was necessary. Plasmid pME559 displayed normal expression of all TRP genes, and enzyme levels on average 23-fold higher than in the wild type strain were found. In comparison, the maximal tryptophan flux observed in such a plasmid-carrying strain was about ten-fold higher than the maximal flux capacity in the wild type strain.     

Cloning and characterization of the Hansenula polymorpha homologue of the Saccharomyces cerevisiae PMR1 gene

A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Hansenula polymorpha. The partial DNA fragment of the H. polymorpha homologue was initially obtained by a polymerase chain reaction and used to isolate the entire gene which encodes a protein of 918 amino acids. The putative gene product contains all ten of the conserved regions observed in P-type ATPases. The cloned gene product exhibits 60·3% amino acid identity to the S. cerevisiae PMR1 gene product and complemented the growth defect of a S. cerevisiae pmr1 null mutant in the EGTA-containing medium. The results demonstrate that the H. polymorpha gene encodes the functional homologue of the S. cerevisiae PMR1 gene product, a P-type Ca²⁺-ATPase. The DNA sequence of the H. polymorpha homologue has been submitted to GenBank with the Accession Number U92083. © 1998 John Wiley & Sons, Ltd.     

d-Xylose consumption by nonrecombinant Saccharomyces cerevisiae: A review

Xylose is the second most abundant sugar in nature. Its efficient fermentation has been considered as a critical factor for a feasible conversion of renewable biomass resources into biofuels and other chemicals. The yeast Saccharomyces cerevisiae is of exceptional industrial importance due to its excellent capability to ferment sugars. However, although S. cerevisiae is able to ferment xylulose, it is considered unable to metabolize xylose, and thus, a lot of research has been directed to engineer this yeast with heterologous genes to allow xylose consumption and fermentation. The analysis of the natural genetic diversity of this yeast has also revealed some nonrecombinant S. cerevisiae strains that consume or even grow (modestly) on xylose. The genome of this yeast has all the genes required for xylose transport and metabolism through the xylose reductase, xylitol dehydrogenase, and xylulokinase pathway, but there seems to be problems in their kinetic properties and/or required expression. Self-cloning industrial S. cerevisiae strains overexpressing some of the endogenous genes have shown interesting results, and new strategies and approaches designed to improve these S. cerevisiae strains for ethanol production from xylose will also be presented in this review.     

Hygromycin‐resistance vectors for gene expression in Pichia pastoris

Pichia pastoris is a common host organism for heterologous protein expression and metabolic engineering. Zeocin‐, G418‐, nourseothricin‐ and blasticidin‐resistance genes are the only dominant selectable markers currently available for selecting P. pastoris transformants. We describe here new P. pastoris expression vectors that confer a hygromycin resistance base on the Klebsiella pneumoniae hph gene. To demonstrate the application of the vectors for intracellular and secreted protein expression, green fluorescent protein (GFP) and human serum albumin (HSA) were cloned into the vectors and transformed into P. pastoris cells. The resulting strains expressed GFP and HSA constitutively or inducibly. The hygromycin resistance marker was also suitable for post‐transformational vector amplication (PTVA) for obtaining strains with high plasmid copy numbers. A strain with multiple copies of the HSA expression cassette after PTVA had increased HSA expression compared with a strain with a single copy of the plasmid. To demonstrate compatibility of the new vectors with other vectors bearing antibiotic‐resistance genes, P. pastoris was transformed with the Saccharomyces cerevisiae genes GSH1, GSH2 or SAM2 on plasmids containing genes for resistance to Zeocin, G418 or hygromycin. The resulting strain produced glutathione and S‐adenosyl‐l ‐methionine at levels approximately twice those of the parent strain. The new hygromycin‐resistance vectors allow greater flexibility and potential applications in recombinant protein production and other research using P. pastoris. Copyright © 2014 John Wiley & Sons, Ltd.     

Isolation and expression of the gene encoding mitochondrial ADP/ATP carrier (AAC) from the pathogenic yeast Candida parapsilosis

A gene homologous to Saccharomyces cerevisiae AAC genes coding for mitochondrial ADP/ATP carriers has been cloned from the pathogenic yeast Candida parapsilosis. A probe obtained by PCR amplification from C. parapsilosis DNA, using primers derived from the conserved transmembrane region of yeast ADP/ATP carriers, was used for screening of the C. parapsilosis genomic library. The cloned gene was sequenced and found to encode a polypeptide of 303 amino acids that shows homology with other yeast and fungal mitochondrial ADP/ATP carriers. The gene was designated CpAAC1 and was able to complement the growth phenotypes of S. cerevisiae double deletion mutant (Δaac2; Δaac3). The expression of the CpAAC1 gene was reduced under semi‐anaerobic conditions and it was affected at normal aerobic conditions by the nature of carbon sources used for growth. Hybridization experiments indicate that C. parapsilosis possesses a single gene encoding a mitochondrial ADP/ATP carrier. The GenBank Accession No. for the C. parapsilosis CpAAC1 gene is AF085429. Copyright © 1999 John Wiley & Sons, Ltd.     

A CONVENIENT DOMINANT SELECTION MARKER FOR GENE TRANSFER IN INDUSTRIAL STRAINS OF SACCHAROMYCES YEAST: SMRI ENCODED RESISTANCE TO THE HERBICIDE SULFOMETURON METHYL

The SMR1-410 gene of S. cerevisiae, encoding resistance to the herbicide sulfometuron methyl (SM), was used as a dominant selection marker in yeast replicating and yeast integrating vectors for the transformation of wild type strains of baking, brewing (ale and lager), distilling, wine and sake Saccharomyces yeasts. Transformation of lithium treated cells by a YEp vector resulted in transformation frequencies ranging from 200 to 8,000 transformants per 10 ug of DNA. Utilizing a yeast integrating vector with SMR1–410 as the only yeast DNA sequences, it was demonstrated that a single copy of SMR1–410 is sufficient to confer stably inherited SM resistance. Thus the SMR1–410 sequence has the unique ability to act as a selectable marker and to also provide a site for chromosomal integration. Since transformants were resistant to levels at least seven fold higher than wild type strains the resistance phenotype was clearly expressed and easily scored in all industrial strains tested. Unlike other selection markers derived from mammalian or bacterial cells, SMR1–410 is derived from S. cerevisiae. Thus industrial utilization of this marker as a means of genetically improving food and beverage strains of Saccharomyces yeasts by recombinant DNA technology is enhanced, as government regulatory agencies are likely to view it in a more favourable light.