Enhancement of cloned gene product synthesis via autoselection in recombinant Saccharomyces cerevisiae

Saccharomyces cerevisiae autoselection strains with mutations in the ura3, fur1, and urid-k genes have been obtained through a sequential isolation procedure. This autoselection system is an extension of one described by Loison et al. The mutations effectively block both the pyrimidine biosynthetic and salvage pathways and in combination are lethal to the host. Therefore, a plasmidencoded URA3 gene is essential for cell viability regardless of the growth conditions, and complex (traditionally nonselective) media can be employed without the risk of plasmid loss. The effects of medium enrichment on growth and cloned gene product synthesis were examined in batch culture for two autoselection strains. The plasmid gene product beta-galactosidase was under the control of the yeast GAL1 promoter, and two methods of induction were employed; one strain was induced via temperature shift while the other was induced by galactose addition. Three nutrient media were investigated: a lean selective medium (SD), a richer semidefined medium (SDC), and a rich complex medium (YPD). The results demonstrated the improvements in cloned gene productivity possible when the growth medium is enriched, with up to 10-fold increases in beta-galactosidase productivity observed. Plasmid instability and mutation reversion were not problems for the autoselection strains, even in uracil-containing medium. Short-term plasmid stabilities were approximately 90% in all three media tested. During continuous culture of the autoselection temperature-sensitive strain, long-term plasmid stability was excellent and beta-galactosidase expression remained high after more than 25 residence times under inducing conditions. In contrast, both beta-galactosidase specific activity and plasmid stability decreased linearly with time for an analogous nonautoselection strain. The introduced fur1 and uridk mutations were very stable; after more than 50 generations of growth in complex medium, stability values of 99-100% were measured. (c) 1993 Wiley & Sons, Inc.     

An efficient method to optimize Kluyveromyces lactis gene targeting

Kluyveromyces lactis strains impaired in the nonhomologous end-joining pathway are relevant tools for the homologous integration of exogenous DNA into the genome, as in the mutant strains, close to 100% of the integrants are targeted to the homologous locus, compared with a few per cent for the wild-type recipient. Using a loxP-kanMX-loxP cassette together with a Cre-recombinase plasmid, a nej1∷loxP mutant strain suitable for multiple gene disruption has been constructed. Furthermore, using this strain, PCR-generated constructs with only 50 bp of homologous flanking sequences resulted in efficient exogenous DNA targeting.     

Disruption of the MNN10 gene enhances protein secretion in Kluyveromyces lactis and Saccharomyces cerevisiae

Screening for genes affecting super-secreting phenotype of the over-secreting mutant of Kluyveromyces lactis resulted in isolation of the gene named KlMNN10, sharing high homology with Saccharomyces cerevisiae MNN10. The disruption of the KlMNN10 in Kluyveromyces lactis, as well as of MNN10 and MNN11 in Saccharomyces cerevisiae, conferred the super-secreting phenotype. MNN10 isolated from Saccharomyces cerevisiae suppressed the super-secretion phenotype in Kluyveromyces lactis klmnn10, as did the homologous KlMNN10. The genes MNN10 and MNN11 of Saccharomyces cerevisiae encode mannosyltransferases responsible for the majority of the alpha-1,6-polymerizing activity of the mannosyltransferase complex. These data agree with the view that the structure of glycoproteins in a yeast cell wall strongly influences the release of homologous and heterologous proteins in the medium. The set of genes namely the suppressors of the over-secreting phenotype, could be attractive for further analysis of gene functions, over-secreting mechanisms and for construction of new strains optimized for heterologous protein secretion. KlMNN10 has EMBL accession no. AJ575132.     

Application of medium optimization tools for improving recombinant human interferon gamma production from Kluyveromyces lactis

The present study is focused upon improving biomass of Kluyveromyces lactis cells expressing recombinant human interferon gamma (hIFN-γ), with the aim of augmenting hIFN-γ concentration using statistical and artificial intelligence approach. Optimization of medium components viz., lactose, yeast extract, and trace elements were performed with Box–Behnken design (BBD) and artificial neural network linked genetic algorithm (ANN-GA) for maximizing biomass of recombinant K. lactis (objective function). The studies resulted over 1.5-fold improvement in the biomass concentration in a medium composed of 80?g/L lactose, 10.353?g/L yeast extract, and 15?mL/L trace elements as compared with initial biomass value. In the same study hIFN-γ concentration reached 881?µg/L which was 2.28-fold higher as compared with initial hIFN-γ concentration obtained in unoptimized medium. Further the batch fermentation study displayed mixed growth associated kinetics with the maximum hIFN-γ production rate of 1.1?mg/L. BBD and ANN-GA, both optimization techniques predicted a higher lactose concentration was clearly beneficial for augmenting K. lactis biomass which in turn increased hIFN-γ concentration.     

Expression of the Arxula adeninivorans glucoamylase gene in Kluyveromyces lactis

 The glucoamylase gene of the yeast Arxula adeninivorans was expressed in Kluyveromyces lactis by using the GAP promoter from Saccharomyces cerevisiae and a multicopy plasmid vector. The transformants secreted 90.1% of the synthesized glucoamylase into the culture medium. The secreted glucoamylase activities are about 20 times higher in comparison to those of Saccharomyces cerevisiae transformants using the same promoter. Secreted glucoamylase possesses identical N-terminal amino acid sequences to those secreted by A. adeninivorans showing that cleavage of the N-terminal signal peptide takes place at the same site. Biochemical characteristics of glucoamylase expressed by K. lactis and A. adeninivorans are very similar. Received: 12 June 1995/Received revision: 17 July 1995/Accepted: 26 July 1995     

Structure of Kluyveromyces lactis subtelomeres: duplications and gene content

We have constructed a map of the duplicated regions of Kluyveromyces lactis subtelomeres. Seven out of 12 subtelomeres contain an almost identical 9 kb long segment starting from the end. This segment is bordered by a long terminal repeat element. Two of the subtelomeres share sequence similarity that extends over a total of 20 kb. The other subtelomeres also contain duplicated regions of 1-6 kb. Nonduplicated regions contain unique genes and genes from paralog families. All duplicated segments are in the same orientation with respect to the telomere, probably as a result of genetic exchange. We map the only two copies of retrotransposons in the genome, in subtelomeres. Low-complexity gene sequences that encode threonine- and serine-rich peptides are associated with the subtelomeres of K. lactis, as in Saccharomyces cerevisiae. The ubiquity of these sequences in hemiascomycete genomes, and the propensity they have to encode proteins with extracellular localization, make these genes ideal candidates for fast evolving 'contingency' genes involved in the adaptation of a species to its environment.     

扣囊复膜孢酵母α-淀粉酶基因在乳酸克鲁维酵母中的表达与重组酶性质

通过PCR方法从扣囊复膜孢酵母基因组DNA中克隆获得α-淀粉酶基因成熟肽编码区（SfA）,插入乳酸克鲁维酵母表达载体pKLACl的d因子信号肽下游,构建重组表达载体pKLACl-SfA。重组载体转化乳酸克鲁维酵母GG799,筛选获得表达α-淀粉酶SfA水平较高的重组茵。酶活检测和SDS．PAGE电泳检测均显示,重组茵分泌重组酶SfA到发酵液中。酶学性质研究表明：SfA最适温度为45℃,最适pH5．0,在pH4．5～5．5、50℃条件下保持稳定。Ca2＋等二价金属离子对SfA酶活有激活作用,EDTA强烈的抑制SfA活性。HPLC分析显示SfA水解糊精获得麦芽寡糖和少量葡萄糖,其中麦芽三糖是主要产物,占水解产物总量的52％。     

RAG3 gene and transcriptional regulation of the pyruvate decarboxylase gene in Kluyveromyces lactis

The RAG3 gene has been cloned from a Kluyveromyces lactis genomic library by complementation of the rag3 mutation, which shows impaired fermentative growth on glucose in the presence of respiratory inhibitors. From the nucleotide sequence of the cloned DNA, which contained an open reading frame of 765 codons, the predicted protein is 49.5% identical to the Pdc2 protein of Saccharomyces cerevisiae, a regulator of pyruvate decarboxylase in this yeast. Measurement of the pyruvate decarboxylase activity in the original rag3–1 mutant and in the null mutant confirmed that the RAG3 gene is involved in pyruvate decarboxylase synthesis in K. lactis. The effect is exerted at the mRNA level of the pyruvate decarboxylase structural gene KIPDCA. Despite analogies between the RAG3 gene of K. lactis and the PDC2 gene of S. cerevisiae, these genes were unable to reciprocally complement.     

pH influences growth and plasmid stability of recombinant Lactococcus lactis subsp. lactis

Continuous cultures of a recombinant Lactococcus lactis subsp. lactis strain were performed to display the effect of the fermentation pH on the specific growth rate and plasmid stability. The proportion of plasmid pIL252 bearing cells decreased exponentially with the number of generations. The influence of the pH on the rate of loss of plasmid pIL252 and on the specific growth rate of L. lactis IL2682 was described by second order polynomial equations. Optimal pH for the growth and plasmid stability were 6.39 and 6.41 respectively. © Rapid Science Ltd. 1998     

Evidence for an inducible glucose transport system in Kluyveromyces lactis.

To find the cause of delayed glucose oxidation in succinate-grown Kluyveromyces lactis, glucose transport was studied in glucose- and in succinate-grown cells. The initial rate of 2-deoxyglucose (2-dGlc) accumulation, as well as the appearance of 2-deoxyglucose 6-phosphate, was higher in the glucose-grown cells. In both cell types, 2-dGlc was apparently transported in the free form to be phosphorylated intracellularly. In glucose-grown cells the level of free 2-dGlc in the pool was always less than the external concentration. Exchange transport in starved, poisoned cells loaded with unlabeled 2-dGlc was 140-fold greater in glucose- than in succinate-grown cells, probably beacuse of the presence of an inducible transport component. The development of the increased rate of transport in a succinate-grown uracil-requiring auxotroph after transfer to glucose depends on the presence of uracil.     

High-level secretion of correctly processed recombinant human interleukin-1 beta in Kluyveromyces lactis.

The lactose-assimilating yeast, Kluyveromyces lactis, has been developed as a microbial host for the synthesis and secretion of human proteins. Here, we report the use of multi-copy vectors based on the 2 mu-like plasmid pKD1 from Kluyveromyces drosophilarum [Chen et al., Nucleic Acids Res. 14 (1986) 4471-4481] for the secretion of recombinant human interleukin-1 beta (reIL-1 beta). High levels of reIL-1 beta were secreted into the growth medium when the structural gene was fused in-frame to a synthetic secretion signal derived from the 'pre'-region of the K. lactis killer toxin. N-terminal sequencing of the excreted protein showed highly efficient (greater than 95%) maturation of the signal sequence. Synthesis as prepro-IL-1 beta, the 'pro'-sequence being derived from the human serum albumin-encoding gene, resulted in equally efficient secretion of mature IL-1 beta. Cytoplasmic production of Met-IL-1 beta, without a secretion signal, was found to be toxic to K. lactis. As in Saccharomyces cerevisiae [Baldari et al., EMBO J. 6 (1987) 229-234], but unlike native human IL-1 beta, K. lactis reIL-1 beta is glycosylated. This glycosylation led to a 95% loss of its biological activity. Removal of the carbohydrate chains by endo-beta-N-acetyl-glucosamidase H treatment fully restored the biological activity. A modified form of IL-1 beta (Asn7----Gln7), in which the unique site for Asn-linked glycosylation was deleted, exhibited the same biological activity as native IL-1 beta. The level of secretion of mature recombinant IL-1 beta ws glycosylation-independent.     

Genetic and biochemical characterization of the galactose gene cluster in Kluyveromyces lactis.

We isolated and identified mutant strains of Kluyveromyces lactis that are defective for the Leloir pathway enzymes galactokinase, transferase, and epimerase, and we termed these loci GAL1 , GAL7 , and GAL10 , respectively. Genetic data indicate that these three genes are tightly linked, having an apparent order of GAL7 - GAL10 - GAL1 . This same gene order has been observed in Saccharomyces cerevisiae. Strains harboring gal7 mutations have elevated levels of beta-galactosidase, coded by an unlinked gene, galactokinase, and epimerase activities under uninduced conditions. We investigated the genetic basis of this constitutive gene expression and found no recombinants between the constitutive and Gal- phenotypes among 76 tetrads, suggesting that either GAL7 or a tightly linked gene codes for a regulatory function. This is the second gene that has been shown to specifically coregulate expression of the genes coding for beta-galactosidase and the Leloir pathway enzymes.     

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.     

Partial-pKD1 plasmids provide enhanced structural stability for heterologous protein production in Kluyveromyces lactis

The stability of pKD1-based vectors in the yeast Kluyveromyces lactis was investigated during short- and long-term culture. The vectors carried an expression/secretion cassette consisting of the Saccharomyces cerevisiaeSUC2 gene under the control of the S. cerevisiaeα-factor promoter and leader. The first set of vectors contained the entire pKD1 sequence linearized at either the unique EcoRI or the unique SphI site of the pKD1 plasmid. During long-term sequential batch culture in selective medium with either vector, invertase activity rapidly dropped while the plasmid-bearing population increased from 60% to 100%. This apparently contradictory behavior was due to structural instability. The enzyme restriction patterns of recovered plasmid DNA retained the pKD1 band while the band containing the SUC2 cassette had decreased substantially in size. To overcome this structural instability, a vector carrying the pKD1 replication origin and the cis-acting stability locus (lacking the inverted repeats) was employed in a pKD1⁺ (but otherwise isogenic) strain. With this plasmid, invertase activity remained constant (for at least 70 generations). While the new vector was significantly more stable, initial invertase activity was substantially lower than that for the vectors containing the full pKD1 sequence. Southern hybridization confirmed that this decrease was primarily due to reduced copy number. The results indicate that full-pKD1 vectors may be preferred for batch culture, while partial-pKD1 vectors are more suitable for long-term (e.g. fed-batch or continuous) culture. Received: 24 June 1997 / Received revision: 14 November 1997 / Accepted: 29 November 1997     

A plasmid selection system in Lactococcus lactis and its use for gene expression in L. lactis and human kidney fibroblasts

We report the development of a nonantibiotic and nonpathogenic host-plasmid selection system based on lactococcal genes and threonine complementation. We constructed an auxotrophic Lactococcus lactis MG1363Deltathr strain which carries deletions in two genes encoding threonine biosynthetic enzymes. To achieve plasmid-borne complementation, we then constructed the minimal cloning vector, pJAG5, based on the genes encoding homoserine dehydrogenase-homoserine kinase (the hom-thrB operon) as a selective marker. Using strain MG1363Deltathr, selection and maintenance of cells carrying pJAG5 were obtained in threonine-free defined media. Compared to the commonly used selection system based on erythromycin resistance, the designed complementation system offers a competitive and stable plasmid selection system for the production of heterologous proteins in L. lactis. The potential of pJAG5 to deliver genes for expression in eukaryotes was evaluated by insertion of a mammalian expression unit encoding a modified green fluorescent protein. The successful delivery and expression of genes in human kidney fibroblasts indicated the potential of the designed nonantibiotic host-plasmid system for use in genetic immunization.     

Development of a LAC4 promoter-based gratuitous induction system in Kluyveromyces lactis

A gratuitous induction system based on the strong, indigenous LAC4 promoter was developed for Kluyveromyces lactis. To prevent consumption of the inducer galactose, a strain with a gal1-209 mutation was employed; this mutation disables the galactokinase function but retains the regulatory function for induction. The Escherichia coli lacZ gene (encoding beta-galactosidase) is functional in K. lactis and was used as the reporter gene downstream of the LAC4 promoter on a multicopy plasmid. The gal1-209 strain exhibited several unexpected phenomena, including partial consumption of the inducer galactose (although at a much slower rate relative to GAL1 strains) and growth inhibition at high concentrations of galactose. These unusual characteristics, however, did not prevent the successful construction of a strong gratuitous induction system. Due to the low rate of inducer consumption for the gratuitous strain, very low concentrations of galactose (1:20 galactose:glucose) resulted in high-level induction. Under these conditions, beta-galactosidase specific and volumetric activities were 4.2- and 5.5-fold higher, respectively, than those for the "GAL1" nongratuitous strain. This research demonstrated the improved productivity possible via LAC4 promoter-based gratuitous induction (and thus a more stable inducer concentration). The effects of various carbon source concentrations on growth and induction were also determined.     

Glucoamylase by recombinant Kluyveromyces lactis cells: production and modelling of a fed batch bioreactor

A cultural system, aimed at the production of glucoamylase with cells of a non-conventional yeast transformed for the enzyme expression, Kluyveromyces lactis JA6-GAA was realised. Glucoamylase production was accomplished in a reactor operating in fed batch mode to avoid limitations with respect to oxygen transfer, and achieve high cell density. A mathematical model able to describe batch and fed batch operations was developed. The theoretical and experimental approach permitted to catch sight of possible physiological changes in the producer strain and set up a suitable fed-batch run to achieve a higher cell density.