Production of glucoamylase in pyruvate decarboxylase deletion mutants of the yeast Kluyveromyces lactis

We have developed a novel Escherichia coli cell surface display system by employing PgsA as an anchoring motif. In our display system, C-terminal fusion to PgsA anchor protein from Bacillus subtilis was used. The enzymes selected for display were α-amylase (AmyA) from Streptococcus bovis 148 and lipase B (CALB) from Candida antarctica. The molecular mass values of AmyA and CALB are approximately 77 and 34 kDa, respectively. The enzymes were displayed on the surface as a fusion protein with a FLAG peptide tag at the C terminus. Both the PgsA-AmyA-FLAG and PgsA-CALB-FLAG fusion proteins were shown to be displayed by immunofluorescence labeling using anti-FLAG antibody. The displayed enzymes were active forms, and AmyA and CALB activities reached 990 U/g (dry cell weight) and 4.6 U/g (dry cell weight), respectively. AmyA-displaying E. coli cells grew utilizing cornstarch as the sole carbon source, while CALB-displaying E. coli cells catalyzed enantioselective transesterification, indicating that they are effective whole-cell biocatalysts. Since a target enzyme with a size of 77 kDa and an industrially useful lipase have been successfully displayed on the cell surface of E. coli for the first time, PgsA protein is probably a useful anchoring motif to display various enzymes.     

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.     

The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis. Implications for the substrate activation mechanism of this enzyme

The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis has been determined to 2.26 A resolution. Like other yeast enzymes, Kluyveromyces lactis pyruvate decarboxylase is subject to allosteric substrate activation. Binding of substrate at a regulatory site induces catalytic activity. This process is accompanied by conformational changes and subunit rearrangements. In the nonactivated form of the corresponding enzyme from Saccharomyces cerevisiae, all active sites are solvent accessible due to the high flexibility of loop regions 106-113 and 292-301. The binding of the activator pyruvamide arrests these loops. Consequently, two of four active sites become closed. In Kluyveromyces lactis pyruvate decarboxylase, this half-side closed tetramer is present even without any activator. However, one of the loops (residues 105-113), which are flexible in nonactivated Saccharomyces cerevisiae pyruvate decarboxylase, remains flexible. Even though the tetramer assemblies of both enzyme species are different in the absence of activating agents, their substrate activation kinetics are similar. This implies an equilibrium between the open and the half-side closed state of yeast pyruvate decarboxylase tetramers. The completely open enzyme state is favoured for Saccharomyces cerevisiae pyruvate decarboxylase, whereas the half-side closed form is predominant for Kluyveromyces lactis pyruvate decarboxylase. Consequently, the structuring of the flexible loop region 105-113 seems to be the crucial step during the substrate activation process of Kluyveromyces lactis pyruvate decarboxylase.     

Characterization of a nucleus-encoded chitinase from the yeast Kluyveromyces lactis

Endogenous proteins secreted from Kluyveromyces lactis were screened for their ability to bind to or to hydrolyze chitin. This analysis resulted in identification of a nucleus-encoded extracellular chitinase (KlCts1p) with a chitinolytic activity distinct from that of the plasmid-encoded killer toxin alpha-subunit. Sequence analysis of cloned KlCTS1 indicated that it encodes a 551-amino-acid chitinase having a secretion signal peptide, an amino-terminal family 18 chitinase catalytic domain, a serine-threonine-rich domain, and a carboxy-terminal type 2 chitin-binding domain. The association of purified KlCts1p with chitin is stable in the presence of high salt concentrations and pH 3 to 10 buffers; however, complete dissociation and release of fully active KlCts1p occur in 20 mM NaOH. Similarly, secreted human serum albumin harboring a carboxy-terminal fusion with the chitin-binding domain derived from KlCts1p also dissociates from chitin in 20 mM NaOH, demonstrating the domain's potential utility as an affinity tag for reversible chitin immobilization or purification of alkaliphilic or alkali-tolerant recombinant fusion proteins. Finally, haploid K. lactis cells harboring a cts1 null mutation are viable but exhibit a cell separation defect, suggesting that KlCts1p is required for normal cytokinesis, probably by facilitating the degradation of septum-localized chitin.     

Heterologous protein production in the yeast Kluyveromyces lactis

Kluyveromyces lactis is both scientifically and biotechnologically one of the most important non-Saccharomyces yeasts. Its biotechnological significance builds on its history of safe use in the food industry and its well-known ability to produce enzymes like lactase and bovine chymosin on an industrial scale. In this article, we review the various strains, genetic techniques and molecular tools currently available for the use of K. lactis as a host for protein expression. Additionally, we present data illustrating the recent use of proteomics studies to identify cellular bottlenecks that impede heterologous protein expression.     

Intraspecific gene expression variability in the yeast Kluyveromyces lactis revealed by micro-array analysis

Using the Genolevures sequencing data, we developed an expression micro-array for the yeast Kluyveromyces lactis consisting of 482 genes, mainly involved in central metabolism, compound transport facilitators and stress response. The array was validated using the LAC/GAL system. By comparing gene expression in the laboratory reference strain CBS2359 and in an industrial strain B1, we demonstrated the influence of two carbon sources, glucose and lactose, on the expression of genes involved in the respiratory and in the fermentative metabolic pathways. We also showed that the two strains, although both originating from dairies, display unexpected differences in gene expression on each type of carbon source.     

The Kluyveromyces lactis KEX1 gene encodes a subtilisin-type serine proteinase

KEX1 is a chromosomal gene required for the production of the killer toxin encoded by the linear DNA plasmid pGKL-1 of Kluyveromyces lactis. The nucleotide sequence of the cloned KEX1 gene has been determined. The deduced structure of the KEX1 protein, 700 amino acids long, indicated that it contained an internal domain with a striking homology to the sequences of the subtilisin-type proteinases, and a probable transmembrane domain near the carboxyl terminus. The results confirm the hypothesis that the product of the gene KEX1 of K. lactis is a proteinase involved in the processing of the toxin precursor.     

Autoregulation of yeast pyruvate decarboxylase gene expression requires the enzyme but not its catalytic activity.

In the yeast, Saccharomyces cerevisiae, pyruvate decarboxylase (Pdc) is encoded by the two isogenes PDC1 and PDC5. Deletion of the more strongly expressed PDC1 gene stimulates the promoter activity of both PDC1 and PDC5, a phenomenon called Pdc autoregulation. Hence, pdc1Delta strains have high Pdc specific activity and can grow on glucose medium. In this work we have characterized the mutant alleles pdc1-8 and pdc1-14, which cause strongly diminished Pdc activity and an inability to grow on glucose. Both mutant alleles are expressed as detectable proteins, each of which differs from the wild-type by a single amino acid. The cloned pdc1-8 and pdc1-14 alleles, as well as the in-vitro-generated pdc1-51 (Glu51Ala) allele, repressed expression of PDC5 and diminished Pdc specific activity. Thus, the repressive effect of Pdc1p on PDC5 expression seems to be independent of its catalytic activity. A pdc1-8 mutant was used to isolate spontaneous suppressor mutations, which allowed expression of PDC5. All three mutants characterized had additional mutations within the pdc1-8 allele. Two of these mutations resulted in a premature translational stop conferring phenotypes virtually indistinguishable from those of a pdc1Delta mutation. The third mutation, pdc1-803, led to a deletion of two amino acids adjacent to the pdc1-8 mutation. The alleles pdc1-8 and pdc1-803 were expressed in Escherichia coli and purified to homogeneity. In the crude extract, both proteins had 10% residual activity, which was lost during purification, probably due to dissociation of the cofactor thiamin diphosphate (ThDP). The defect in pdc1-8 (Asp291Asn) and the two amino acids deleted in pdc1-803 (Ser296 and Phe297) are located within a flexible loop in the beta domain. This domain appears to determine the relative orientation of the alpha and gamma domains, which bind ThDP. Alterations in this loop may also affect the conformational change upon substrate binding. The mutation in pdc1-14 (Ser455Phe) is located within the ThDP fold and is likely to affect binding and/or orientation of the cofactor in the protein. We suggest that autoregulation is triggered by a certain conformation of Pdc1p and that the mutations in pdc1-8 and pdc1-14 may lock Pdc1p in vivo in a conformational state which leads to repression of PDC5.     

Regulation of cytokinesis in the milk yeast Kluyveromyces lactis

Cytokinesis in yeast and mammalian cells is a highly coordinated process mediated by the constriction of an actomyosin ring. In yeasts, it is accompanied by the formation of a chitinous primary septum. Although much is known about the regulation of cytokinesis in budding yeast, overlapping functions of redundant genes complicates genetic analyses. Here, we investigated the effects of various deletion mutants on cytokinesis in the milk yeast Kluyveromyces lactis. To determine the spatiotemporal parameters of cytokinesis components, live-cell imaging of fluorophor-tagged KlMyo1 and a new Lifeact probe for KlAct1 was employed. In contrast to Saccharomyces cerevisiae, where deletion of ScMYO1 is lethal, Klmyo1 deletion was temperature-sensitive. Transmission and scanning electron microscopy demonstrated that the Klmyo1 deletion cells had a defect in the formation of the primary septum and in cell separation; this result was confirmed by FACS analyses. Deletion of KlCYK3 was lethal, whereas in S. cerevisiae a cyk3 deletion is synthetically lethal with hof1 deletion. Growth of Klhof1 mutants was osmoremedial at 25 °C, as it is in S. cerevisiae. CYK3 and HOF1 genes cross-complemented in both species, suggesting that they are functional homologs. Inn1, a common interactor for these two regulators, was essential in both yeasts and the encoding genes did not cross-complement. The C2 domain of the Inn1 homologs conferred species specificity. Thus, our work establishes K. lactis as a model yeast to study cytokinesis with less genetic redundancy than S. cerevisiae. The viability of Klmyo1 deletions provides an advantage over budding yeast to study actomyosin-independent cytokinesis. Moreover, the lethality of Klcyk3 null mutants suggests that there are fewer functional redundancies with KlHof1 in K. lactis.     

Genetics and molecular physiology of the yeast Kluyveromyces lactis

With the recent development of powerful molecular genetic tools, Kluyveromyces lactis has become an excellent alternative yeast model organism for studying the relationships between genetics and physiology. In particular, comparative yeast research has been providing insights into the strikingly different physiological strategies that are reflected by dominance of respiration over fermentation in K. lactis versus Saccharomyces cerevisiae. Other than S. cerevisiae, whose physiology is exceptionally affected by the so-called glucose effect, K. lactis is adapted to aerobiosis and its respiratory system does not underlie glucose repression. As a consequence, K. lactis has been successfully established in biomass-directed industrial applications and large-scale expression of biotechnically relevant gene products. In addition, K. lactis maintains species-specific phenomena such as the "DNA-killer system, " analyses of which are promising to extend our knowledge about microbial competition and the fundamentals of plasmid biology.     

Molecular genetics of phosphofructokinase in the yeast Kluyveromyces lactis

We have undertaken a study of phosphofructokinase (PFK; E.C. 2.7.1.11) in the yeast Kluyveromyces lactis. Like other eukaryotic PFKs, the K. lactis enzyme is activated by the allosteric effectors AMP and fructose-2,6-bisphosphate. PFK activity is induced in cells grown on glucose as compared to ethanol-grown cells, in contrast to the constitutive expression of PFK in Saccharomyces cerevisiae. We show here that phosphofructokinase of the yeast K. lactis is composed of two non-identical types of sub-units, encoded by the genes KIPFK1 and KIPFK2. We have cloned and sequenced both genes. KIPFK1 and KIPFK2 encode the α- and the β-PFK subunits with deduced molecular weights of 109.336 Da and 104.074Da, respectively. Sequence analysis indicates that the genes evolved from a double duplication event. Null mutants in either of the genes lack detectable PFK activity in vitro and the respective subunits cannot be detected on Western blots. In contrast to the situation in S. cerevisiae, Klpfk1 Klpfk2 double mutants retain the ability to grow on glucose. However. Klpfk2 mutants and the double mutants do not grow on glucose, when respiration is blocked. These data suggest that the pentose phosphate pathway and respiration play a substantial role in glucose utilization by K. lactis. The K. lactis PFK genes can be expressed independently in S. cerevisiae and each of them complements the glucose-negative phenotype of pfk1 pfk2 double deletion mutants in this yeast. Expression of both K. lactis PFK genes simultaneously in S. cerevisiae pfk double deletion mutants complements for PFK activity. However, expression of a combination of PFK genes from K. lactis and S. cerevisiae does not lead to the production of a functional enzyme.     

Lactose utilization by Saccharomyces cerevisiae strains expressing Kluyveromyces lactis LAC genes

Whey generated in cheese manufacture continues being an industrial problem without a satisfactory solution. Genetic modification of the yeast S. cerevisiae to obtain strains able to utilize lactose, is a prerequisite for the utilization of this yeast to convert cheese whey into useful fermentation products (i.e. biomass, heterologous protein and other recombinant products). Although the construction of S. cerevisiae Lac(+) strains has been achieved by different strategies, most of these strains have unsuitable characteristics, such as genetic instability of the Lac phenotype or diauxic growth. In previous communications we have described the construction of genetically stable strains of S. cerevisiae that assimilate lactose with a high efficiency. These strains carry multiple copies of Kluyveromyces lactis LAC4 and LAC12 genes, which code for a beta-galactosidase and a lactose permease, respectively. In this work we report additional results about the effect of gene dosage, and analyze the performance of a selected strain in the bioconversion of cheese whey. Additionally, we describe the construction of a new strain, which combines the Lac(+) phenotype with additional properties of biotechnological interest: flocculence, and the ability to hydrolyze starch.