Significance of the KlLAC1 gene in glucosylceramide production by Kluyveromyces lactis

Each of the 12 genes involved in the synthesis of glucosylceramide was overexpressed in cells of Kluyveromyces lactis to construct a strain accumulating a high quantity of glucosylceramide. Glucosylceramide was doubled by the KlLAC1 gene, which encodes ceramide synthase, and not by 11 other genes, including the KlLAG1 gene, a homologue of KlLAC1 . Disruption of the KlLAC1 gene reduced the content below the detection level. Heterologous expression of the KlLAC1 gene in the cells of Saccharomyces cerevisiae caused the accumulation of ceramide, composed of C₁₈ fatty acid. The KlLAC1 protein preferred long-chain (C₁₈) fatty acids to very-long-chain (C₂₆) fatty acids for condensation with sphingoid bases and seemed to supply a ceramide moiety as the substrate for the formation of glucosylceramide. When the amino acid sequences of ceramide synthase derived from eight yeast species were compared, LAC1 proteins from five species producing glucosylceramide were clearly discriminated from those of the other three species and all LAG1 proteins. The LAC1 protein of K. lactis is the enzyme that plays a crucial role in the synthesis of glucosylceramide.     

KlADH3, a gene encoding a mitochondrial alcohol dehydrogenase, affects respiratory metabolism and cytochrome content in Kluyveromyces lactis

A Kluyveromyces lactis strain, harbouring KlADH3 as the unique alcohol dehydrogenase (ADH) gene, was used in a genetic screen on allyl alcohol to isolate mutants deregulated in the expression of this gene. Here we report the characterization of some mutants that lacked or had highly reduced amounts of KlAdh3p activity; in addition, these mutants showed alterations in glucose metabolism, reduced respiration and reduced cytochrome content. Our results confirm that the KlAdh3p activity contributes to the reoxidation of cytosolic NAD(P)H feeding the respiratory chain through KlNdi1p, the mitochondrial internal transdehydrogenase. The low levels of KlAdh3p in two of the mutants were associated with mutations in KlSDH1, one of the genes of complex II, suggesting signalling between the respiratory chain and expression of the KlADH3 gene.     

Galactose transport in Kluyveromyces lactis: major role of the glucose permease Hgt1

In Kluyveromyces lactis, galactose transport has been thought to be mediated by the lactose permease encoded by LAC12. In fact, a lac12 mutant unable to grow on lactose did not grow on galactose either and showed low and uninducible galactose uptake activity. The existence of other galactose transport systems, at low and at high affinity, had, however, been hypothesized on the basis of galactose uptake kinetics studies. Here we confirmed the existence of a second galactose transporter and we isolated its structural gene. It turned out to be HGT1, previously identified as encoding the high-affinity glucose carrier. Analysis of galactose transporter mutants, hgt1 and lac12, and the double mutant hgt1lac12, suggested that Hgt1 was the high-affinity and Lac12 was the low-affinity galactose transporter. HGT1 expression was strongly induced by galactose and insensitive to glucose repression. This could explain the rapid adaptation to galactose observed in K. lactis after a shift from glucose to galactose medium.     

Dosage suppression of the Kluyveromyces lactis zymocin by Saccharomyces cerevisiae ISR1 and UGP1

The Kluyveromyces lactis zymocin complex kills Saccharomyces cerevisiae cells in a process that involves tRNA cleavage by its tRNAse gamma-toxin subunit. In contrast to the gamma-toxin mode of action, the early steps of the zymocin response are less well characterized. Here, we present high-dosage suppressors of zymocin that encode a putative Pkc1-related kinase (ISR1) and UDP-glucose pyrophosphorylase (UGPase) (UGP1). Anti-UGPase Western blots and GAL10 - ISR1 overexpression suggest that zymocin suppression correlates with overproduction of UGPase or Isr1. As judged from protection against exo-zymocin and unaltered sensitivity to endogenous gamma-toxin, high-copy ISR1 and UGP1 operate in early, nontarget steps of the zymocin pathway. Consistent with a recent report on in vitro phosphorylation of Isr1 and UGPase by the CDK Pho85, high-copy ISR1 and UGP1 suppression of zymocin is abolished in a pho85 null mutant lacking CDK activity of Pho85. Moreover, suppression requires UGPase enzyme activity, and ISR1 overexpression also protects against CFW, a chitin-interfering poison. Our data agree with roles for UGPase in cell wall biosynthetic processes and for Isr1 in Pkc1-related cell wall integrity. In sum, high-copy ISR1 and UGP1 cells affect early steps of the zymocin response and potentially prevent the lethal K. lactis killer complex from establishing cell surface recognition and/or contact.     

Identification and characterization of a novel glucose-phosphorylating enzyme in Kluyveromyces lactis

Recent data suggest that hexokinase KlHxk1 (Rag5) represents the only glucose-phosphorylating enzyme of Kluyveromyces lactis, which also is required for glucose signalling. Long-term growth studies of a K. lactis rag5 mutant, however, reveal slow growth on glucose, but no growth on fructose. Isolation of the permissive glucose-phosphorylating enzyme, mass spectrometric tryptic peptide analysis and determination of basic kinetic data identify a novel glucokinase (KlGlk1) encoded by ORF KLLA0C01,155g. In accordance with the growth characteristics of the rag5 mutant, KlGlk1 phosphorylates glucose, but fails to act on fructose as a sugar substrate. Multiple sequence alignment indicates the presence of at least one glucokinase gene in all sequenced yeast genomes.     

The Kluyveromyces lactis alpha1,6-mannosyltransferase KlOch1p is required for cell-wall organization and proper functioning of the secretory pathway

Mutants of Kluyveromyces lactis denominated vga (vanadate glycosylation affected) bear various combinations of glycosylation and cell-wall defects. The vga3 mutation of K. lactis was mapped in the KlOCH1 gene, encoding the functional homologue of the Saccharomyces cerevisiaealpha1,6-mannosyltransferase. Quantitative analysis of cell-wall components indicated a noticeable increase of chitin and beta1,6-glucans and a severe decrease of mannoproteins in the mutant cells as compared with the wild-type counterparts. Fine-structure determination of the beta1,6-glucan polymer indicated that, in the vga3-1 strain, the beta1,6-glucans are shorter and have more branches than in the wild-type strain. This suggests that cell-wall remodelling changes take place in K. lactis in the presence of glycosylation defects. Moreover, the vga3 cells showed a significantly improved capability of secreting heterologous proteins. Such a capability, accompanied by the highly reduced N-glycosylation, may be of biotechnological interest, especially when hyper-glycosylation of recombinant products must be avoided.     

Enhanced secretion of heterologous proteins in Kluyveromyces lactis by overexpression of the GDP-mannose pyrophosphorylase, KlPsa1p

GDP-mannose is the mannosyl donor for the glycosylation reactions and is synthesized by GDP-mannose pyrophosphorylase from GTP and d-mannose-1-phosphate; in Saccharomyces cerevisiae this enzyme is encoded by the PSA1/VIG9/SRB1 gene. We isolated the Kluyveromyces lactis KlPSA1 gene by complementing the osmotic growth defects of S. cerevisiae srb1/psa1 mutants. KlPsa1p displayed a high degree of similarity with other GDP-mannose pyrophosphorylases and was demonstrated to be the functional homologue of S. cerevisiae Psa1p. Phenotypic analysis of a K. lactis strain overexpressing the KlPSA1 gene revealed changes in the cell wall assembly. Increasing the KlPSA1 copy number restored the defects in O-glycosylation, but not those in N-glycosylation, that occur in K. lactis cells depleted for the hexokinase Rag5p. Overexpression of GDP-mannose pyrophosphorylase also enhanced heterologous protein secretion in K. lactis as assayed by using the recombinant human serum albumin and the glucoamylase from Arxula adeninivorans.     

The apyrase KlYnd1p of Kluyveromyces lactis affects glycosylation, secretion, and cell wall properties

The Kluyveromyces lactis ORF r_klactIV3,463 on chromosome IV, hereafter named KlYND1, encodes an endoapyrase that has nucleoside phosphatase activity with a lumenal orientation. The enzyme showed equally high activity towards GDP/UDP and ADP, and also showed activity, although to a lesser extent, towards GTP. No activity was detected with the other triphosphates and all monophosphates. The overexpression of KlYND1 in Klgda1Delta cells of K. lactis, devoid of the encoded GDPase/UDPase activity, suppressed the loss of O-glycosylation and cell wall-related defects described in such mutants, and suggests a partial overlap of function between the two genes, and therefore some redundancy. The overexpression of KlYND1 in wild-type cells enhanced the secretion of the recombinant human serum albumin and glucoamylase employed as reporters.     

Galactose induction in yeast involves association of Gal80p with Gal1p or Gal3p

Gal1p carries out two functions in the galactose pathway of yeast. It activates Gal4p by interacting with Gal80p – a function that can also served by Gal3p – and it catalyzes the formation of galactose-1-phosphate. Recently, we and others have presented biochemical evidence for complex formation between Gal1p and Gal80p. Here, we extend these data and present genetic evidence for an interaction between Gal1p and Gal80p in vivo, using a two-hybrid assay. Interaction between Gal1p and Gal80p depends on the presence of galactose, but not on the catalytic activity of Gal1p. A new class of Kluyveromyces lactis mutants was isolated, designated Klgal1-m, which have lost the derepressing activity but retain galactokinase activity, indicating that the two Gal1p activities are functionally independent. The KlGal1-m proteins are defective in their ability to interact with Gal80p in a two-hybrid assay. The locations of gal1-m mutations identify putative interaction sites in Gal1p and Gal80p. A dominant mutation, KlGAL1-d, leads to a high level of constitutive expression of genes of the galactose pathway. The behavior of chimeric proteins consisting of Gal3p and KlGal1p sequences indicates that both the N-terminal and C-terminal halves of KlGal1p are involved in specific interaction with KlGal80p. Received: 12 November 1998 / Accepted: 18 December 1998     

The Saccharomyces cerevisiaeSOM1 gene: heterologous complementation studies, homologues in other organisms, and association of the gene product with the inner mitochondrial membrane

The small nuclear gene SOM1 of Saccharomyces cerevisiae was isolated as a multicopy suppressor of a mutation in the IMP1 gene, which encodes the mitochondrial inner membrane peptidase subunit 1 (Imp1). Analysis revealed that Som1 and Imp1 are components of a mitochondrial protein export system, and interaction between these two proteins is indicated by the genetic suppression data. Here we describe the identification of a gene from Kluyveromyces lactis, which restores respiratory function to a S. cerevisiae SOM1 deletion mutant at 28° C. The sequence of the K. lactis gene predicts a protein product of 8.1-kDa, comprising 71 amino acid residues, with a putative mitochondrial signal sequence at its N-terminus. The protein is 50% identical to its S.cerevisiae counterpart. The expression pattern of a homologous sequence in Leishmania major suggests a more general role for SOM1 in mitochondrial biogenesis and protein sorting. The various Som1 proteins exhibit a highly conserved region and a remarkable pattern of cysteine residues. A protein of the expected size was transcribed and translated in vitro. The Som1 protein was detected in fractions of S. cerevisiae enriched for mitochondria and found to be associated with the inner mitochondrial membrane. Received: 22 July 1997 / Accepted: 27 October 1997     

Molecular cloning of the neutral trehalase gene from Kluyveromyces lactis and the distinction between neutral and acid trehalases

We cloned the Kluyveromyces lactis KlNTH1 gene, which encodes neutral trehalase. It showed 65.2% and 68.5% identity at nucleotide and amino acid sequence level, respectively, with the Saccharomyces cerevisiae NTH1 gene. Multiple alignment of the predicted trehalase protein sequences from yeasts, bacteria, insects, and mammals revealed two major domains of conservation. Only the yeast trehalases displayed in an N-terminal extension two consensus sites for cAMP-dependent protein phosphorylation and a putative Ca²⁺-binding sequence. Gene disruption of the KlNTH1 gene abolished neutral trehalase activity and clearly revealed a trehalase activity with an acid pH optimum. It also resulted in a high constitutive trehalose level. Expression of the KlNTH1 gene in an S. cerevisiae nth1Δ mutant resulted in rapid activation of the heterologous trehalase upon addition of glucose to cells growing on a nonfermentable carbon source and upon addition of a nitrogen source to cells starved for nitrogen in a glucose-containing medium. In K. lactis, the same responses were observed except that rapid activation by glucose was observed only in early-exponential-phase cells. Inactivation of K. lactis neutral trehalase by alkaline phosphatase and activation by cAMP in cell extracts are consistent with control of the enzyme by cAMP-dependent protein phosphorylation. Received: 19 March 1996 / Accepted: 15 October 1996     

牛凝乳酶原基因的合成及其在乳酸克鲁维酵母中的表达

凝乳酶在奶酪加工中应用广泛,为获得高活性的凝乳酶制剂,采用乳酸克鲁维酵母为宿主,首次对经密码子优化的牛凝乳酶原基因进行表达。利用DNAWorks3.0软件辅助设计,用两步PCR法合成了小牛凝乳酶原基因(GenBank Accession No.AA30448)。将该基因插入酵母表达载体pKLAC1,构建了重组载体pKLAC1-Prochy,并用电脉冲法将线性化的重组质粒转化到乳酸克鲁维酵母GG799中。通过含1%酪蛋白的YEPD平板活性筛选,PCR鉴定,最后获得了一株多拷贝整合的基因工程菌chy1。该菌株可分泌表达牛凝乳酶原,经SDS-PAGE分析,证明重组牛凝乳酶原的分子量约为41kDa,符合预期大小,酸化处理后为36kDa,证明可以正确自我剪切。液体培养96h后,酶活最高达到99.67SU/mL。分别以半乳糖和葡萄糖为碳源的条件下表达,其酶活性差异不大,说明在发酵期间,可以不经过半乳糖诱导即可产生高水平的牛凝乳酶原产物。该工程菌的获得为进一步优化产酶条件及放大工艺提供了条件,并为凝乳酶的工业化生产奠定了基础。     

An approach to the hypoxic and oxidative stress responses in Kluyveromyces lactis by analysis of mRNA levels

Genome duplication, after the divergence of Saccharomyces cerevisiae from Kluyveromyces lactis along evolution, has been proposed as a mechanism of yeast evolution from strict aerobics, such as Candida albicans, to facultatives/fermentatives, such as S. cerevisiae. This feature, together with the preponderance of respiration and the use of the pentose phosphate pathway in glucose utilization, makes K. lactis a model yeast for studies related to carbon and oxygen metabolism. In this work, and based on the knowledge of the sequence of the genome of K. lactis, obtained by the Génolevures project, we have constructed DNA arrays from K. lactis including a limited amount of selected probes. They are related to the aerobiosis-hypoxia adaptation and to the oxidative stress response, and have been used to test changes in mRNA levels in response to hypoxia and oxidative stress generated by H(2)O(2). The study was carried out in both wild-type and rag2 mutant K. lactis strains in which glycolysis is blocked at the phosphoglucose isomerase step. This approach is the first analysis carried out in K. lactis for the majority of the genes selected.