The CUP1 promoter of Saccharomyces cerevisiae is inducible by copper in Pichia pastoris

We report the construction of a Pichia pastoris integrating vector which contains the inducible CUP1 promoter from Saccharomyces cerevisiae. We show that the promoter is indeed inducible by copper when used in P. pastoris and that the level of induction is dependent on the amount of copper in the medium.     

KlROM2 encodes an essential GEF homologue in Kluyveromyces lactis

Cellular integrity in yeasts is ensured by a rigid cell wall whose synthesis is controlled by a MAP kinase signal transduction cascade. In Saccharomyces cerevisiae upstream regulatory components of this MAP kinase pathway involve a single protein kinase C, which is regulated in part by interaction with the small GTPase Rho1p. This small G protein is in turn rendered inactive (GDP-bound) or is activated (GTP-bound) by the influence of GTPase activating proteins (GAPs) and the GDP/GTP exchange factors (GEFs), respectively. We report here on the isolation of a gene from Kluyveromyces lactis, KlROM2, which encodes a member of the latter protein family. The nucleotide sequence contains an open reading frame of 1227 amino acids, with an overall identity of 57% to the Rom2 protein of S. cerevisiae. Four conserved sequence motifs could be identified: a RhoGEF domain, a DEP sequence, a CNH domain and a less conserved pleckstrin homology (PH) sequence. Klrom2 null mutants show a lethal phenotype, which indicates that the gene may encode the only functional GEF regulating the cellular integrity pathway in K. lactis. Conditional genomic expression of KlROM2 resulted in sensitivity towards caffeine and Calcofluor white as typical phenotypes of mutants defective in this pathway. Overexpression of KIROM2 from multicopy plasmids under the control of the ScGAL1 promoter severely impaired growth in both S. cerevisiae and in K. lactis. The fact that the lethal phenotype was not prevented in mpk1 deletion mutants indicates that growth inhibition is not simply caused by hyperactivation of the Pkc1p signal transduction pathway.     

Mating type locus-dependent stability of the Kluyveromyces linear pGKL plasmids in Saccharomyces cerevisiae

The linear killer plasmids, pGKL1 and pGKL2, from Kluyveromyces lactis stably replicated in mitochondrial DNA-deficient (rho 0) MATa or MAT alpha haploids of Saccharomyces cerevisiae, but were unstable and frequently lost in rho 0 MATa/MAT alpha diploids, suggesting that the replication of pGKL plasmids was under the control of the MAT locus. In MATa/MAT alpha cells of S. cerevisiae, the MAT alpha gene product (alpha 2) is combined with the MATa gene product (a1) and the resultant protein, a1-alpha 2, acts to repress the expression of haploid specific genes. Experiments showed that the K. lactis linear plasmids were stably maintained in rho 0 mata1/MAT alpha diploids, indicating that the a1-alpha 2 repressor interfered with the stability of pGKL2. It was revealed by computer analysis that the consensus sequence homologous to the a1-alpha 2 repressor binding site occurred within the coding regions of pGKL2 genes which were presumed to be essential for the plasmid replication. Since the plasmids were stably maintained in diploids of K. lactis, the mating type control must not be working there.     

Isolation and sequence analysis of a K. lactis chromosomal DNA element able to autonomously replicate in S. cerevisiae and K. lactis

We have undertaken a search for autonomously replicating (ARSs) from Kluyveromyces lactis chromosomal DNA able to sustain plasmid replication in K. lactis and in Saccharomyces cerevisiae. The discovery of such sequences might be interesting for the comparison of ARSs from different sources and possibly useful for the construction of multivalent vectors. HindIII fragments from K. lactis chromosomal DNA were inserted in the YIp5 plasmid (lacking an origin of replication) and the resulting chimaeric plasmids were selected for the ability to transform S. cerevisiae. Four plasmids were identified and further analysed. Two contained the same 1.8 kb K. lactis fragment and transformed both K. lactis and S. cerevisiae with the same efficiency and stability, whereas the third transformed only S. cerevisiae and the fourth transformed K. lactis with a higher efficiency than S. cerevisiae. A detailed study was performed on the 1.8 kb fragment which exhibited ARS function in both yeasts. The fragment was subcloned using different restriction enzymes and Bal31 exonuclease. Subclones were tested for ARS function. ARS activities in the two yeasts were localized in the same 100 bp region. Sequencing demonstrated the presence in this region of the dodecanucleotide 5'ATTTATTGTTTT3' differing from the ARS core consensus of S. cerevisiae only by a T insertion. A similar nucleotide sequence is present in the putative replication origin of the 2 mu-like plasmid pKD1 which stably replicates in K. lactis. Homologies with ARSs from S. cerevisiae were also found in the regions flanking the above-mentioned dodecanucleotide.     

Identification of tet(M) in two Lactococcus lactis strains isolated from a Spanish traditional starter-free cheese made of raw milk and conjugative transfer of tetracycline resistance to lactococci and enterococci

Specific PCR and sequencing showed that a tet(M) gene was present in two tetracycline-resistant Lactococcus lactis strains isolated from a raw milk, starter-free cheese. Hybridisation experiments using as a probe an internal segment of the gene obtained by PCR associated tet(M) with plasmids of around the same size (30 kbp) in both strains. Molecular analysis of the tetracycline resistance loci, including the upstream and downstream regions of the genes, showed them to be identical to one other and to the tet(M) encoded by the conjugative transposon Tn916. Amplification of Tn916-derived segments suggested the transposon was complete in the two L. lactis strains. Further, curing of the tetracycline resistance was accompanied by a reduction in size of the plasmids comparable to that expected for Tn916. Tetracycline resistance could be transferred by conjugation to plasmid-free Lactococcus and Enterococcus strains. However, no plasmid DNA was detected among the transconjugants while both tet(M) and transposon-related sequences were amplified by PCR. This suggested that only the transposon was mobilized.     

Nucleotide sequence of the cytochrome oxidase subunit 2 and val-tRNA genes and surrounding sequences from Kluyveromyces lactis K8 mitochondrial DNA

The nucleotide sequence of the cytochrome oxidase subunit 2 (cox2) and val-tRNA genes and surrounding regions from Kluyveromyces lactis mitochondrial DNA is reported. Analysis of the coding region shows that the codons CUN (Thr), CGN (Arg) and AUA (Met) are absent in this gene. A single sequence, ATATAAGTAA, identical to the baker's yeast mtRNA polymerase recognition site, was detected upstream of val-tRNA. This sequence is absent from regions between val-tRNA-cox2 and cox2-cox1. In addition a sequence AATAATATTCTT, identical to the mRNA processing site in other yeast mitochondrial genomes is present 32-43 bp downstream to the TAA stop codon for the cox2 gene. Another short conserved sequence of 5 bp, TCTAA, is present upstream of the coding regions of cox2 genes in several yeasts, including K. lactis, but is not present upstream of other genes. Comparison of cox2 sequences from other organisms indicates that the mitochondrial DNA of K. lactis is closely related to that of Saccharomyces cerevisiae.     

Plasmids with the Cre-recombinase and the dominant nat marker, suitable for use in prototrophic strains of Saccharomyces cerevisiae and Kluyveromyces lactis

Two plasmids are described which can be used to remove the "loxP-markerMX-loxP" cassettes in strains lacking the ura3 mutation. Both contain the Cre-recombinase under control of the GAL1 promoter and the natMX cassette with the dominant marker nat, which gives yeasts resistance to the antibiotic ClonNat. pNatCre contains ARSH and CEN6 for maintenance in Saccharomyces cerevisiae. pKlNatCre has a Kluyveromyces lactis replication origin and centromere in addition.     

高效表达木糖醇脱氢酶基因酿酒酵母的构建及木酮糖发酵的初步研究

通过RT-PCR方法克隆得到Candida tropicalis木糖醇脱氢酶基因xyl2,将该基因连入酵母表达载体pYES2的诱导型启动子GAL1下,构建表达质粒pYES2-xyl2;同时用从Pichia pastoris中克隆获取的甘油醛磷酸脱氢酶基因GAP换下GAL1基因,构建含组成型启动子GAP基因的表达质粒pYES2-GAP-xyl2;通过电转化法将其依次转入酿酒酵母S.cerevisiae INVSc1,山梨醇培养基上筛选的转化子经木糖醇梯度驯化培养,筛选出1株耐木糖醇浓度为20%的酿酒酵母重组菌株ZCX4和1株在半乳糖诱导下耐木糖醇浓度为15%的重组菌株YDX2。酶活测定表明,重组菌株ZCX4比酶活0.621 U/mg(蛋白),是YDX2比酶活的2.29倍。摇瓶发酵结果显示,重组菌株ZCX4木糖醇消耗76.46 g/L,木糖醇消耗率为76.46%,是重组菌株YDX2木糖醇消耗率的1.63倍,说明木糖醇脱氢酶实现了高效表达。     

The alcohol dehydrogenase system in the yeast, Kluyveromyces lactis

We have studied the alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis. Southern hybridization to the Saccharomyces cerevisiae ADH2 gene indicates four probable structural ADH genes in K. lactis. Two of these genes have been isolated from a genomic bank by hybridization to ADH2. The nucleotide sequence of one of these genes shows 80% and 50% sequence identity to the ADH genes of S. cerevisiae and Schizosaccharomyces pombe respectively. One K. lactis ADH gene is preferentially expressed in glucose-grown cells and, in analogy to S. cerevisiae, was named K1ADH1. The other gene, homologous to K1ADH1 in sequence, shows an amino-terminal extension which displays all of the characteristics of a mitochondrial targeting presequence. We named this gene K1ADH3. The two genes have been localized on different chromosomes by Southern hybridization to an orthogonal-field-alternation gel electrophoresis-resolved K. lactis genome. ADH activities resolved by gel electrophoresis revealed several ADH isozymes which are differently expressed in K. lactis cells depending on the carbon source.     

Cloning and analysis of the Kluyveromyces lactis TRP1 gene: a chromosomal locus flanked by genes encoding inorganic pyrophosphatase and histone H3

The TRP1 gene of the yeast Kluyveromyces lactis has been cloned from a genomic library by complementation of the Saccharomyces cerevisiae trp1-289 mutation. The gene was located within the clone by transposon mutagenesis and the coding region identified by DNA sequencing. This has indicated that K. lactis TRP1 encodes a 210-amino acid polypeptide which shows 53% identity to the homologous S. cerevisiae protein. The K. lactis TRP1 gene has been disrupted by substituting the S. cerevisiae URA3 gene for a large part of the TRP1 coding sequence. Replacement of the chromosomal TRP1 locus with this construction has enabled the production of non-reverting trp1- strains of K. lactis, while a genetic analysis of the disrupted allele confirmed that the TRP1 gene had been cloned. DNA sequencing has also shown that the K. lactis TRP1 sequence is flanked by genes encoding inorganic pyrophosphatase and histone H3, which we have designated IPP and HHT1 respectively. Hybridization studies have shown that in common with S. cerevisiae, K. lactis has two copies of the histone H3 gene. Each H3 gene is closely linked to a gene encoding histone H4 and in both yeast species the IPP gene is tightly linked to one of the histone gene pairs.     

Genome organization of the linear cytoplasmic element pPE1B from Pichia etchellsii.

The linear cytoplasmic element pPE1B from Pichia etchellsii CBS2011 (synonym Debaryomyces etchellsii) was totally sequenced. It consists of 12835 bp and has a remarkable high A+T content of 77.3%. The termini of pPE1B were found to consist of inversely orientated identical nucleotide repetitions 161 base pairs long, to which proteins are probably covalently linked at the 5' ends. Ten putative genes (open reading frames, ORFs) were identified, covering 96.5% of the total sequence. The predicted polypeptides correspond to proteins encoded by ORFs 2-11 of the linear plasmids pGKL2 of Kluyveromyces lactis and pSKL of Saccharomyces kluyveri. ORF1, existing on both latter elements, is lacking on pPE1B. An upstream conserved sequence motif (UCS) is located at the expected distance from the start codon of each of the 10 ORFs. As the arbitrarily chosen UCS6 was able to drive expression of a reporter gene in the heterologous pGKL-encoded killer system of K. lactis, extranuclear promoter function is probable. The almost congruent genome organization of pPE1B and other autonomous linear yeast plasmids sequenced so far, i.e. pGKL2 and pSKL, suggests a common, presumably viral, ancestor.     

Physical separation and functional interaction of Kluyveromyces lactis and Saccharomyces cerevisiae ARS elements derived from killer plasmid DNA

Two DNA fragments which have autonomously replicating sequence (ARS) activity in both Saccharomyces cerevisiae and Kluyveromyces lactis have been isolated from the K. lactis kl killer plasmid. One fragment (Kla1) is 700 base pairs (bp) in length and plasmids carrying it are mitotically unstable in both hosts. In K. lactis, this instability leads to colonies having a 'nibbled' phenotype when grown on selective media and appears to be the result of inefficient plasmid segregation. The other fragment (Kla2) is an artificial junction fragment of 1100 bp which was produced during the cloning procedure. Kla2 has been divided into two sub-fragments Kla2A and Kla2B which have, respectively, ARS activity in K. lactis and S. cerevisiae but not the other species. This indicates that these two closely related yeasts have different sequence requirements for ARS activity. Kla2B contains a perfect match to the S. cerevisiae ARS consensus but Kla2A does not. Both Kla2A and Kla1 share a 10 bp sequence as the sole region of homology between them. This sequence, 5'TCATAATATA3', is tentatively offered as defining the ARS consensus sequence for K. lactis.     

Isolation and nucleotide sequence of a gene encoding tRNA nucleotidyltransferase from Kluyveromyces lactis

A gene (KlCCA1) encoding ATP(CTP):tRNA specific tRNA nucleotidyltransferase (EC 2.7.7.25) was isolated from Kluyveromyces lactis by complementation of the Saccharomyces cerevisiae cca1-1 mutation. Sequencing of a 2665 bp EcoRI-SpeI restriction fragment revealed an open reading frame potentially encoding a protein of 489 amino acids with 57% sequence similarity to its S. cerevisiae homologue. Southern hybridization revealed a single copy of KlCCA1 in the K. lactis genome. KlCCA1 was able to complement both the mitochondrial and cytosolic defects in the cca1-1 mutant, suggesting that, as in S. cerevisiae, the K. lactis gene encodes a sorting isozyme that is targeted to mitochondria and the nucleus and/or cytosol. An altered KlCCA1 gene encoding a tRNA nucleotidyltransferase that lacked its first 35 amino acids was able to complement the nuclear/cytosolic but not the mitochondrial defect in the S. cerevisiae cca1-1 mutant, suggesting that the 35 amino-terminal amino acids are necessary for targeting to mitochondria but are not required for enzyme activity. Our results suggest that the mechanisms for production and distribution of mitochondrial and nuclear/cytosolic tRNA nucleotidyltransferase in K. lactis differ from those seen in S. cerevisiae.