Cytosine deaminase MX cassettes as positive/negative selectable markers in Saccharomyces cerevisiae

We describe positive/negative selectable cytosine deaminase MX cassettes for use in Saccharomyces cerevisiae. The basis of positive selection for cytosine deaminase (Fcy1) activity is that (a) fcy1 strains are unable to grow on medium containing cytosine as a sole nitrogen source and (b) fcy1 ura3 strains are unable to grow on medium containing cytosine as the sole pyrimidine source. Conversely, as 5-fluorocytosine (5FC) is toxic to cytosine deaminase-producing cells, fcy1 strains are resistant to 5FC. FCY1MX and FCA1MX cassettes, containing open reading frames (ORFs) of S. cerevisiae FCY1 and Candida albicans FCA1, respectively, were constructed and used to disrupt targeted genes in S. cerevisiae fcy1 strains. In addition, new direct repeat cassettes, kanPR, FCA1PR, FCY1PR and CaURA3PR, were developed to allow efficient deletion of target genes in cells containing MX3 repeats. Finally, the FCY1- and FCA1MX3 or PR direct repeat cassettes can be readily recycled after 5FC counter-selection on both synthetic and rich media.     

Molecular cloning and biochemical characterization of a 3'(2'),5'-bisphosphate nucleotidase from Debaryomyces hansenii

The enzyme 3'(2'),5'-bisphosphate nucleotidase catalyses a reaction that converts 3'-phosphoadenosine-5'-phosphate (PAP) to adenosine-5'-phosphate (AMP) and inorganic phosphate (Pi). The enzyme from Saccharomyces cerevisiae is highly sensitive to sodium and lithium and is thus considered to be the in vivo target of salt toxicity in yeast. In S. cerevisiae, the HAL2 gene encodes this enzyme. We have cloned a homologous gene, DHAL2, from the halotolerant yeast Debaryomyces hansenii. DNA sequencing of this clone revealed a 1260 bp open reading frame (ORF) that putatively encoded a protein of 420 amino acid residues. S. cerevisiae transformed with DHAL2 gene displayed higher halotolerance. Biochemical studies showed that recombinant Dhal2p could efficiently utilize PAP (K(m)17 microM) and PAPS (K(m)48 microM) as substrate. Moreover, we present evidence that, in comparison to other homologues from yeast, Dhal2p displays significantly higher resistance towards lithium and sodium ions.     

Kluyveromyces lactis SSO1 and SEB1 genes are functional in Saccharomyces cerevisiae and enhance production of secreted proteins when overexpressed

The SEB1/SBH1 and the SSO genes encode components of the protein secretory machinery functioning at the opposite ends, ER translocation and exocytosis, respectively, of the secretory pathway in Saccharomyces cerevisiae. Overexpression of these genes can rescue temperature-sensitive (ts) growth defect of many sec mutants impaired in protein secretion. Furthermore, their overexpression in wild-type yeast enhances production of secreted proteins in S. cerevisiae, which suggests that they may be rate-limiting factors in this process. Here we report isolation of Kluyveromyces lactis homologues of these genes. KlSSO1 and KlSEB1 were isolated as clones capable of rescuing growth of ts sso2-1 and seb1Delta seb2Delta sem1Delta strains, respectively, at the restrictive temperature. The encoded Kluyveromyces proteins are up to 70% identical with the S. cerevisiae homologues at the amino acid level and can functionally replace them. Interestingly, KlSSO1 and KlSEB1 show similar enhancing effect on production of a secreted protein as the SSO and SEB1 genes of S. cerevisiae when overexpressed. In accordance with the high homology level of the secretory pathway proteins in different yeast species, the polyclonal antibodies raised against S. cerevisiae Seb1p, Sso2p and Sec4p can detect homologous proteins in cell lysates of K. lactis and Pichia pastoris, the latter also in Candida utilis. The GenBank Accession Nos are AF307983 (K. lactis SSO1) and AF318314 (K. lactis SEB1).     

Cloning, sequencing and characterization of the alpha-aminoadipate reductase gene (LYS2) from Saccharomycopsis fibuligera

The gene putatively encoding alpha-aminoadipate reductase (AAR) was isolated successfully by degenerate PCR and chromosome walking, based on cassette PCR methods, from the dimorphous yeast Saccharomycopsis fibuligera PD70 and was named SfLYS2. Sequence analysis revealed that it contained a putative open reading frame (ORF) of 4161 bp and encoded a polypeptide of 1386 amino acids. The deduced translation product shared an identity of 53% and 51% to the Lys2p homologues of Candida albicans and Saccharomyces cerevisiae, respectively. An atypical TATA box and a GCN4-box element were found in the 5'-upstream region. Genomic Southern hybridization suggested the presence of a single locus of SfLYS2 in the S. fibuligera genome. Expression of the ORF of SfLYS2 in a lys2(-) strain of S. cerevisiae could functionally complement the lysine mutant of the S. cerevisiae strain. S. fibuligera could use lysine as the sole nitrogen source but its growth was inhibited on the alpha-aminoadipate (AA) medium. Approximately 90% of the mutants of S. cerevisiae resistant to AA are lysine auxotrophs; in contrast all the mutants of S. fibuligera resistant to AA recovered in this work were not lysine auxotrophs.     

The uracil permease of Schizosaccharomyces pombe: A representative of a family of 10 transmembrane helix transporter proteins of yeasts

The uracil permease gene of Schizosaccharomyces pombe was cloned and sequenced. The deduced protein sequence shares strong similarities with five open reading frames from Saccharomyces cerevisiae, namely the uracil permease encoded by the FUR4 gene, the allantoin permease encoded by DAL4, a putative uridine permease (YBL042C) and two unknown ORFs YOR071c and YLR237w. A topological model retaining ten transmembrane helices, based on predictions and on experimental data established for the uracil permease of S. cerevisiae by Galan and coworkers (1996), is discussed for the four closest proteins of this family of transporters. The sequence of the uracil permease gene of S. pombe has been deposited in the EMBL data bank under Accession Number X98696. © 1998 John Wiley & Sons, Ltd.     

Amino acid residues important for substrate specificity of the amino acid permeases Can1p and Gnp1p in Saccharomyces cerevisiae.

Deletion of the general amino acid permease gene GAP1 abolishes uptake of L-citrulline in Saccharomyces cerevisiae, resulting in the inability to grow on L-citrulline as sole nitrogen source. Selection for suppressor mutants that restored growth on L-citrulline led to isolation of 21 mutations in the arginine permease gene CAN1. One similar mutation was found in the glutamine-asparagine permease gene GNP1. L-[(14)C]citrulline uptake measurements confirmed that suppressor mutations in CAN1 conferred uptake of this amino acid, while none of the mutant permeases had lost the ability to transport L-[(14)C]arginine. Substrate specificity seemed to remain narrow in most cases, and broad substrate specificity was only observed in the cases where mutations affect two proline residues (P148 and P313) that are both conserved in the amino acid-polyamine-choline (APC) transporter superfamily. We found mutations affecting six predicted domains (helices III and X, and loops 1, 2, 6 and 7) of the permeases. Helix III and loop 7 are candidates for domains in direct contact with thetransported amino acid. Helix III was affected in both CAN1 (Y173H, Y173D) and GNP1 (W239C) mutants and has previously been found to be important for substrate preference in other members of the family. Furthermore, the mutations affecting loop 7 (residue T354, S355, Y356) are close to a glutamate side chain (E367) potentially interacting with the positively charged substrate, a notion supported by conservation of the side chain in permeases for cationic substrates.     

Kinetics of active sucrose transport in Saccharomyces cerevisiae

The kinetic analysis of active sucrose-H+ uptake by Saccharomyces cerevisiae revealed the presence of two transport systems with high and low affinity for sucrose. The MAL2T permease has a low affinity (K(m) = 120 +/-20 mM) for sucrose, while the alpha-glucoside transporter encoded by the AGT1 gene is a high affinity sucrose-H+ symporter (K(m) = 7.9+/-0.8 mM) that increases the specific growth rate of cells growing on sucrose.     

Function of Candida glabrata ABC transporter gene,PDH1

The rapid increase in azole resistance during treatment of patients infected with Candida glabrata may be due to increased azole efflux mediated by ABC transporters, as occurs with increased expression of PDR5 in Saccharomyces cerevisiae. Two known C. glabrata homologues of PDR5 influencing azole susceptibility are PDH1 (CgCDR2) and CgCDR1. Disruption of PDH1 in a cgcdr1::ura3 strain increased susceptibility to rhodamine 6G, cycloheximide and chloramphenicol, and also increased rhodamine 6G accumulation, all properties of pdr5 null mutants. Overexpression of PDH1 in S. cerevisiae complemented the pdr5 mutation by reversing susceptibility to rhodamine 6G, chloramphenicol and cycloheximide, as well as by decreasing rhodamine 6G intracellular concentration. Expression of PDH1 in a C. glabrata cgcdr1::ura3 pdh1Delta::ura3 mutant using a multicopy plasmid almost completely restored the wild-type phenotype, showing that PDH1 at higher levels of expression can replace CgCDR1. Because PDH1 and CgCDR1 have both been reported to have upstream sequences similar to the Pdr1p- and Pdr3p-binding elements of PDR5, we sought similarities in regulation between the three genes. Abundance of PDH1 and CgCDR1 mRNA in C. glabrata was increased by rhodamine 6G, cycloheximide and oligomycin, properties in common with PDR5. PDH1, CgCDR1 and PDR5 have striking similarities in function and regulation.     

Gene sam1 encoding adenosylmethionine synthetase: effects of its expression in the fission yeast Schizosaccharomyces pombe

By screening gene libraries of Schizosaccharomyces pombe with a DNA fragment encoding part of the Saccharomyces cerevisiae S-adenosylmethionine synthetase (SAMS), we isolated the fission yeast sam1 gene. Its sequence exhibits good homology to SAMSs of other organisms and reveals the motifs characteristic for SAMSs. SAMS activity and sam1 mRNA levels decrease when cells enter stationary phase. In haploid strains, gene sam1 is essential for growth; if weakly expressed, cells mate and sporulate at a reduced rate. Strains overexpressing sam1 exhibit methionine-sensitive growth. This methionine-induced growth inhibition is partially relieved by adenine. We assume that methionine reduces the level of one or several adenine nucleotides by a SAMS-mediated mechanism. Intracellular SAM levels increase drastically by exogenously added methionine. This increase predicts that mutants exhibiting methionine revertible phenotypes can be indicative for mutations in proteins exhibiting SAM-dependent functions. In agreement with this prediction, we show that mutant pmt2-5 has this phenotype and that gene pmt2 encodes a potential SAM-dependent enzyme.     

Subtelomeric sequence from the right arm of Schizosaccharomyces pombe chromosome I contains seven permease genes

The sequence has been determined of 80 888 bp of contiguous subtelomeric DNA, including the isp5 gene, from the right arm of chromosome I of Schizosaccharomyces pombe; 27 open reading frames (ORFs) longer than 100 codons are present, giving a density of one gene per 3.0 kb. Seven of the predicted proteins are members of the major facilitator superfamily (MFS) of transport proteins, including four amino acid permease homologues, bringing this family of amino acid permease sequences to 17 in Sz. pombe, and a phylogenetic analysis is presented. Also encoded is an allantoate permease homologue, a sulphate permease homologue and a probable urea active transporter. Predicted non-membrane proteins include a 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase), a class III aminotransferase, serine acetyltransferase, protein-L-isoaspartate O-methyltransferase, alpha-glucosidase, alpha-galactosidase, esterase/lipase, oxidoreductase of the short-chain dehydrogenase/reductase (SDR) family, aldehyde dehydrogenase, formamidase, amidase, flavohaemoprotein, a putative translation initiation inhibitor and a protein with similarity to a filamentous fungal conidiation-specific protein. The remaining six ORFs are likely to encode proteins, either because they have sequence similarity with hypothetical proteins or because they are known to be transcribed. Introns are scarce in the sequenced region: only three ORFs contain introns, with only one having multiple introns. The sequenced region also contains a single Tf1 transposon long terminal repeat (LTR). The sequence is derived from cosmid clones c869, c922 and c1039 and has been submitted to the EMBL database under entries SPAC869 (Accession No. AL132779), SPAC922 (AL133522) and SPAC1039 (AL133521).     

The allantoin and uracil permease gene sequences of Saccharomyces cerevisiae are nearly identical.

We have determined the structure of the allantoin permease (DAL4) gene of Saccharomyces cerevisiae. The gene putatively encodes a hydrophobic protein with a M(r) of 71,755. It possesses the alternating hydrophobic-hydrophilic regions similar to those found in many other integral membrane proteins. The most striking feature of the allantoin permease component encoded by DAL4 is its striking similarity to the uracil permease component encoded by FUR4. Although data available indicate that these proteins do not share any overlap of function, their predicted protein sequences are 68% identical, 81% similar, and their DNA sequences are 70% identical. The upstream regulatory region of DAL4 contains all of the characterized cis-acting elements previously reported for inducible allantoin pathway genes: six sequences homologous to UASNTR, the element responsible for nitrogen catabolite repression-sensitive activation of allantoin pathway gene expression, and two sequences homologous to the cis-acting element responsible for inducer-responsiveness of the allantoin pathway genes, UIS. The finding of these homologous sequences predicted to exist on the basis of DAL4's expression characteristics, supports and strengthens the suggestion that these elements mediate the functions we have previously ascribed to them.     

Putative xylose and arabinose reductases in Saccharomyces cerevisiae

Saccharomyces cerevisiae mutants, in which open reading frames (ORFs) displaying similarity to the aldo-keto reductase GRE3 gene have been deleted, were investigated regarding their ability to utilize xylose and arabinose. Reduced xylitol formation from D-xylose in gre3 mutants of S. cerevisiae suggests that Gre3p is the major D-xylose-reducing enzyme in S. cerevisiae. Cell extracts from the gre3 deletion mutant showed no detectable xylose reductase activity. Decreased arabitol formation from L-arabinose indicates that Gre3p, Ypr1p and the protein encoded by YJR096w are the major arabinose reducers in S. cerevisiae. The ypr1 deletion mutant showed the lowest specific L-arabinose reductase activity in cell extracts, 3.5 mU/mg protein compared with 7.4 mU/mg protein for the parental strain with no deletions, and the lowest rate of arabitol formation in vivo. In another set of S. cerevisiae strains, the same ORFs were overexpressed. Increased xylose and arabinose reductase activity was observed in cell extracts for S. cerevisiae overexpressing the GRE3, YPR1 and YJR096w genes. These results, in combination with those obtained with the deletion mutants, suggest that Gre3p, Ypr1p and the protein encoded by YJR096w are capable of xylose and arabinose reduction in S. cerevisiae. Both the D-xylose reductase and the L-arabinose reductase activities exclusively used NADPH as co-factor.     

递推式ARTP-UV复合诱变筛选高产β-葡萄糖苷酶菌株

为了提高产酶能力,采用常压室温等离子体(ARTP)与紫外(UV)复合诱变技术对酿酒酵母G13、G21菌株进行递推式复合诱变,经过一轮ARTP诱变两轮UV诱变,摇瓶培养筛选到两株高产β-葡萄糖苷酶的菌株Dea-G13D1Z2、Dma-G21D1Z2,经连续5代发酵生产试验,产酶水平可分别稳定在240.93和173.38 U/mL,是出发菌株G13、G21酶活的4.61倍和3.59倍。递推式ARTP-UV是一种有效的微生物突变育种的方法,可有效应用于微生物育种工作。     

Cloning of the Candida albicans nucleoside transporter by complementation of nucleoside transport-deficient Saccharomyces

The nucleoside permease gene (i.e. NUP) from Candida albicans was cloned by complementation of Saccharomyces cerevisiae deficient in nucleoside transport capability. The permease transported adenosine and guanosine and was sensitive to the mammalian nucleoside transport inhibitors: dipyridamole and NBMPR. It did not transport uridine, cytidine, adenine, guanine or uracil. The inability to transport uridine indicated that the NUP gene product was different from the Candida uridine permease, which also transported cytosine and adenosine. The NUP gene coded for a protein of 407 amino acids in size which was approximately the size of the human, Giardia and E. coli nucleoside permeases. It did not, however, exhibit any significant degree of homology with these transporters. The GenBank accession number for the Candida NUP gene is AF016246. © 1998 John Wiley & Sons, Ltd.     

A permease exhibiting a dual role for lysine and biotin uptake in Saccharomyces cerevisiae

Among Saccharomyces cerevisiae strains each defective in one of 11 amino acid permeases, a lysine permease disruptant (DK) exhibited about 2-fold reductions in maximum cell density and fermentation ability compared to the parent in a synthetic medium. These unusual properties of DK were found to result from the requirement of biotin for growth, in contrast to the parent whose growth was not dependent on external biotin. The rate of 14C-labeled biotin uptake and the intracellular free biotin content of DK were 2-2.5 fold lower than in the parent. We suggest that lysine permease in S. cerevisiae has the ability to transport both lysine and biotin.     

有机溶剂耐受性酵母细胞的脂肪酸组成分析

本文以出发菌株酿酒酵母(Saccharomyces cerevisiae)、筛选所得乙醇耐受菌株Y-c-8和丙酮耐受菌株B-g-5为研究对象,分析测定了三种不同菌株的脂肪酸组成,从而证实有机溶剂会引起酵母细胞脂肪酸成分发生改变,主要是合成更多长链不饱和脂肪酸以适应不良环境.本研究为有机溶剂对微生物细胞的毒性机制提供了理论支持,为有机介质中微生物全细胞催化的工业化应用提供了理论支持.