Budding Yeast Kluyveromyces Lactis Research Articles

CRISPR/Cas9-Based Genome Editing for Protein Expression and Secretion in Kluyveromyces lactis.

The budding yeast Kluyveromyces lactis has emerged as a promising microbial chassis in industrial biotechnology. However, a lack of efficient molecular genetic manipulation tools and strategies has hindered the development of K. lactis as a biomanufacturing platform. In this study, we developed and applied a CRISPR/Cas9-based genome editing method to K. lactis. Single-gene editing efficiency was increased to 80% by disrupting the nonhomologous end-joining-related gene KU80 and performing a series of process optimizations. Subsequently, the CRISPR/Cas9 system was explored based on different sgRNA delivery modes for simultaneous multigene editing. With the aid of the color indicator, the editing efficiencies of two and three genes reached 73.3 and 36%, respectively, in the KlΔKU80 strain. Furthermore, the CRISPR/Cas9 system was used for multisite integration to enhance lactase production and combinatorial knockout of TMED10 and HSP90 to characterize the extracellular secretion of lactase in K. lactis. Generally, genome editing is a powerful tool for constructing K. lactis cell factories for protein and chemical production.

Functional Interactions of Kluyveromyces lactis Telomerase Reverse Transcriptase with the Three-Way Junction and the Template Domains of Telomerase RNA.

The ribonucleoprotein telomerase contains two essential components: telomerase RNA (TER) and telomerase reverse transcriptase (TERT, Est2 in yeast). A small portion of TER, termed the template, is copied by TERT onto the chromosome ends, thus compensating for sequence loss due to incomplete DNA replication and nuclease action. Although telomerase RNA is highly divergent in sequence and length across fungi and mammals, structural motifs essential for telomerase function are conserved. Here, we show that Est2 from the budding yeast Kluyveromyces lactis (klEst2) binds specifically to an essential three-way junction (TWJ) structure in K. lactis TER, which shares a conserved structure and sequence features with the essential CR4-CR5 domain of vertebrate telomerase RNA. klEst2 also binds specifically to the template domain, independently and mutually exclusive of its interaction with TWJ. Furthermore, we present the high-resolution structure of the klEst2 telomerase RNA-binding domain (klTRBD). Mutations introduced in vivo in klTRBD based on the solved structure or in TWJ based on its predicted RNA structure caused severe telomere shortening. These results demonstrate the conservation and importance of these domains and the multiple protein–RNA interactions between Est2 and TER for telomerase function.

Crystal structure of the Ate1 arginyl-tRNA-protein transferase and arginylation of N-degron substrates

N-degron pathways are proteolytic systems that target proteins bearing N-terminal (Nt) degradation signals (degrons) called N-degrons. Nt-Arg of a protein is among Nt-residues that can be recognized as destabilizing ones by the Arg/N-degron pathway. A proteolytic cleavage of a protein can generate Arg at the N terminus of a resulting C-terminal (Ct) fragment either directly or after Nt-arginylation of that Ct-fragment by the Ate1 arginyl-tRNA-protein transferase (R-transferase), which uses Arg-tRNAArg as a cosubstrate. Ate1 can Nt-arginylate Nt-Asp, Nt-Glu, and oxidized Nt-Cys* (Cys-sulfinate or Cys-sulfonate) of proteins or short peptides. Ate1 genes of fungi, animals, and plants have been cloned decades ago, but a three-dimensional structure of Ate1 remained unknown. A detailed mechanism of arginylation is unknown as well. We describe here the crystal structure of the Ate1 R-transferase from the budding yeast Kluyveromyces lactis. The 58-kDa R-transferase comprises two domains that recognize, together, an acidic Nt-residue of an acceptor substrate, the Arg residue of Arg-tRNAArg, and a 3'-proximal segment of the tRNAArg moiety. The enzyme's active site is located, at least in part, between the two domains. In vitro and invivo arginylation assays with site-directed Ate1 mutants that were suggested by structural results yielded inferences about specific binding sites of Ate1. We also analyzed the inhibition of Nt-arginylation activity of Ate1 by hemin (Fe3+-heme), and found that hemin induced the previously undescribed disulfide-mediated oligomerization of Ate1. Together, these results advance the understanding of R-transferase and the Arg/N-degron pathway.

The Unfolded Protein Response Pathway in the Yeast Kluyveromyces lactis. A Comparative View among Yeast Species

Eukaryotic cells have evolved signalling pathways that allow adaptation to harmful conditions that disrupt endoplasmic reticulum (ER) homeostasis. When the function of the ER is compromised in a condition known as ER stress, the cell triggers the unfolded protein response (UPR) in order to restore ER homeostasis. Accumulation of misfolded proteins due to stress conditions activates the UPR pathway. In mammalian cells, the UPR is composed of three branches, each containing an ER sensor (PERK, ATF6 and IRE1). However, in yeast species, the only sensor present is the inositol-requiring enzyme Ire1. To cope with unfolded protein accumulation, Ire1 triggers either a transcriptional response mediated by a transcriptional factor that belongs to the bZIP transcription factor family or an mRNA degradation process. In this review, we address the current knowledge of the UPR pathway in several yeast species: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida glabrata, Cryptococcus neoformans, and Candida albicans. We also include unpublished data on the UPR pathway of the budding yeast Kluyveromyces lactis. We describe the basic components of the UPR pathway along with similarities and differences in the UPR mechanism that are present in these yeast species.

Synergistic cooperation promotes multicellular performance and unicellular free-rider persistence

The evolution of multicellular life requires cooperation among cells, which can be undermined by intra-group selection for selfishness. Theory predicts that selection to avoid non-cooperators limits social interactions among non-relatives, yet previous evolution experiments suggest that intra-group conflict is an outcome, rather than a driver, of incipient multicellular life cycles. Here we report the evolution of multicellularity via two distinct mechanisms of group formation in the unicellular budding yeast Kluyveromyces lactis. Cells remain permanently attached following mitosis, giving rise to clonal clusters (staying together); clusters then reversibly assemble into social groups (coming together). Coming together amplifies the benefits of multicellularity and allows social clusters to collectively outperform solitary clusters. However, cooperation among non-relatives also permits fast-growing unicellular lineages to ‘free-ride’ during selection for increased size. Cooperation and competition for the benefits of multicellularity promote the stable coexistence of unicellular and multicellular genotypes, underscoring the importance of social and ecological context during the transition to multicellularity.

Centromere-specific histone Cse4 by the chaperone Scm3

A specialized nucleosome is a component of all eukaryotic kineto-chores. The core of this nucleosome contains a centromere-specific histone, CENP-A (the Cse4 gene product in budding yeast), instead of the usual H3. Assembly of a centromeric nucleosome depends on a specific chaperone, called Scm3 in yeast and HJURP in higher eukaryotes. We describe here the structure of a complex formed by an N-terminal fragment of Scm3 with the histone-fold domains of Cse4, and H4, all prepared as recombinant proteins derived from the budding yeast Kluyveromyces lactis. The contacts of Scm3 with Cse4 explain its selectivity for the centromere-specific histone; key residues at the interface are conserved in HJURP, indicating a common mechanism for centromeric histone deposition. We also report the structure of a (Cse4:H4)2 heterotetramer; comparison with the structure of the Scm3:Cse4:H4 complex shows that tetramer formation and DNA binding require displacement of Scm3 from the nucleosome core. The two structures together suggest that specific contacts between the chaperone and Cse4, rather than an altered overall structure of the nucleosome core, determine the selective presence of Cse4 at centromeres.

“Poisoning” yeast telomeres distinguishes between redundant telomere capping pathways

In most eukaryotes, telomeres are composed of tandem arrays of species-specific DNA repeats ending with a G-rich 3' overhang. In budding yeast, Cdc13 binds this overhang and recruits Ten1-Stn1 and the telomerase protein Est1 to protect (cap) and elongate the telomeres, respectively. To dissect and study the various pathways employed to cap and maintain the telomere end, we engineered telomerase to incorporate Tetrahymena telomeric repeats (G₄T₂) onto the telomeres of the budding yeast Kluyveromyces lactis. These heterologous repeats caused telomere-telomere fusions, cell cycle arrest at G2/M, and severely reduced viability--the hallmarks of telomere uncapping. Fusing Cdc13 or Est1 to universal minicircle sequence binding protein (UMSBP), a small protein that binds the single-stranded G₄T₂ repeats, rescued the cell viability and restored telomere capping, but not telomerase-mediated telomere maintenance. Surprisingly, Cdc13-UMSBP-mediated telomere capping was dependent on the homologous recombination factor Rad52, while Est1-UMSBP was not. Thus, our results distinguish between two, redundant, telomere capping pathways.

Functional Analysis of the Single Est1/Ebs1 Homologue in Kluyveromyces lactis Reveals Roles in both Telomere Maintenance and Rapamycin Resistance

Est1 and Ebs1 in Saccharomyces cerevisiae are paralogous proteins that arose through whole-genome duplication and that serve distinct functions in telomere maintenance and translational regulation. Here we present our functional analysis of the sole Est1/Ebs1 homologue in the related budding yeast Kluyveromyces lactis (named KlEst1). We show that similar to other Est1s, KlEst1 is required for normal telomere maintenance in vivo and full telomerase primer extension activity in vitro. KlEst1 also associates with telomerase RNA (Ter1) and an active telomerase complex in cell extracts. Both the telomere maintenance and the Ter1 association functions of KlEst1 require its N-terminal domain but not its C terminus. Analysis of clusters of point mutations revealed residues in both the N-terminal TPR subdomain and the downstream helical subdomain (DSH) that are important for telomere maintenance and Ter1 association. A UV cross-linking assay was used to establish a direct physical interaction between KlEst1 and a putative stem-loop in Ter1, which also requires both the TPR and DSH subdomains. Moreover, similar to S. cerevisiae Ebs1 (ScEbs1) (but not ScEst1), KlEst1 confers rapamycin sensitivity and may be involved in nonsense-mediated decay. Interestingly, unlike telomere regulation, this apparently separate function of KlEst1 requires its C-terminal domain. Our findings provide insights on the mechanisms and evolution of Est1/Ebs1 homologues in budding yeast and present an attractive model system for analyzing members of this multifunctional protein family.

Structure of the budding yeast Saccharomyces cerevisiae centromeric histones Cse4-H4 complexed with the chaperone Scm3

Based on the crystal structure of the budding yeast Kluyveromyces lactis (Kl) Scm3–Cse4–H4 complex and the proteolysis results of the budding yeast Saccharomyces cerevisiae (Sc) Scm3–Cse4–H4 complex, Cho and Harrison (1) questioned our NMR structure of the Sc Scm3–Cse4–H4 complex (2), stating that “The construct deleted helix I of H4, however, and this deletion, together with omission of about 20 residues in the N-terminal part of the conserved region of Scm3, appears to have generated severe distortions in about half the structure, leading to incorrect conclusions about histone conformational differences and about a number of the Scm3-Cse4 contacts.”

Recognition of the centromere-specific histone Cse4 by the chaperone Scm3

A specialized nucleosome is a component of all eukaryotic kinetochores. The core of this nucleosome contains a centromere-specific histone, CENP-A (the Cse4 gene product in budding yeast), instead of the usual H3. Assembly of a centromeric nucleosome depends on a specific chaperone, called Scm3 in yeast and HJURP in higher eukaryotes. We describe here the structure of a complex formed by an N-terminal fragment of Scm3 with the histone-fold domains of Cse4, and H4, all prepared as recombinant proteins derived from the budding yeast Kluyveromyces lactis. The contacts of Scm3 with Cse4 explain its selectivity for the centromere-specific histone; key residues at the interface are conserved in HJURP, indicating a common mechanism for centromeric-histone deposition. We also report the structure of a (Cse4:H4)(2) heterotetramer; comparison with the structure of the Scm3:Cse4:H4 complex shows that tetramer formation and DNA-binding require displacement of Scm3 from the nucleosome core. The two structures together suggest that specific contacts between the chaperone and Cse4, rather than an altered overall structure of the nucleosome core, determine the selective presence of Cse4 at centromeres.

Challenging the Gβγ Paradigm

In the budding yeast Kluyveromyces lactis , the β subunit of the G protein can function without the γ subunit in the mating response.

Switching the mechanism of mating type switching: a domesticated transposase supplants a domesticated homing endonuclease

Programmed DNA rearrangements are critical for the development of many organisms and, intriguingly, can be catalyzed by domesticated mobile genetic elements. In this issue of Genes & Development, Barsoum and colleagues (pp. 33-44) demonstrate that, in the budding yeast Kluyveromyces lactis, a DNA rearrangement associated with mating type switching requires a domesticated transposase and occurs through a mechanism distinct from that in the related yeast, Saccharomyces cerevisiae. Thus, mechanisms for mating type switching have evolved multiple times, indicating the relative ease with which mobile genetic elements can be captured.

Pseudoknot structures with conserved base triples in telomerase RNAs of ciliates

Telomerase maintains the integrity of telomeres, the ends of linear chromosomes, by adding G-rich repeats to their 3′-ends. Telomerase RNA is an integral component of telomerase. It contains a template for the synthesis of the telomeric repeats by the telomerase reverse transcriptase. Although telomerase RNAs of different organisms are very diverse in their sequences, a functional non-template element, a pseudoknot, was predicted in all of them. Pseudoknot elements in human and the budding yeast Kluyveromyces lactis telomerase RNAs contain unusual triple-helical segments with AUU base triples, which are critical for telomerase function. Such base triples in ciliates have not been previously reported. We analyzed the pseudoknot sequences in 28 ciliate species and classified them in six different groups based on the lengths of the stems and loops composing the pseudoknot. Using miniCarlo, a helical parameter-based modeling program, we calculated 3D models for a representative of each morphological group. In all cases, the predicted structure contains at least one AUU base triple in stem 2, except for that of Colpidium colpoda, which contains unconventional GCG and AUA triples. These results suggest that base triples in a pseudoknot element are a conserved feature of all telomerases.

Crystallization and preliminary X-ray diffraction studies of hexokinase KlHxk1 from Kluyveromyces lactis.

Glucose acts as both a carbon source and a hormone-like regulator of gene expression in eukaryotic organisms from yeast to man. Phosphorylation of glucose is executed by hexokinases, which represent a class of multifunctional enzymes that, in addition to their contribution to the uptake and initiation of metabolism of glucose, fructose and mannose, are involved in glucose signalling. The genome of the budding yeast Kluyveromyces lactis encodes a single hexokinase (KlHxk1) and a single glucokinase (KlGlk1). KlHxk1 exists in a monomer-homodimer equilibrium which is presumed to play a role in metabolic regulation. In order to evaluate the physiological significance of KlHxk1 dimerization on a molecular level, the enzyme was crystallized and subjected to X-ray structure analysis. Crystallization employing ammonium sulfate, diammonium phosphate or polyethylene glycol 6000 at pH values of 8.0-9.5 gave seven different crystal forms of KlHxk1. Crystallographic data to 1.66 A resolution were obtained using synchrotron radiation. Structure determination of KlHxk1 in various packing environments will reveal the full architecture of the homodimeric enzyme and complete our mechanistic understanding of the catalytic and regulatory functions of the enzyme.

A Triple Helix within a Pseudoknot Is a Conserved and Essential Element of Telomerase RNA

Telomerase copies a short template within its integral telomerase RNA onto eukaryotic chromosome ends, compensating for incomplete replication and degradation. Telomerase action extends the proliferative potential of cells, and thus it is implicated in cancer and aging. Nontemplate regions of telomerase RNA are also crucial for telomerase function. However, they are highly divergent in sequence among species, and their roles are largely unclear. Using in silico three-dimensional modeling, constrained by mutational analysis, we propose a three-dimensional model for a pseudoknot in telomerase RNA of the budding yeast Kluyveromyces lactis. Interestingly, this structure includes a U-A.U major-groove triple helix. We confirmed the triple-helix formation in vitro using oligoribonucleotides and showed that it is essential for telomerase function in vivo. While triplex-disrupting mutations abolished telomerase function, triple compensatory mutations that formed pH-dependent G-C.C(+) triples restored the pseudoknot structure in a pH-dependent manner and partly restored telomerase function in vivo. In addition, we identified a novel type of triple helix that is formed by G-C.U triples, which also partly restored the pseudoknot structure and function. We propose that this unusual structure, so far found only in telomerase RNA, provides an essential and conserved telomerase-specific function.

Efficient gene targeting in Kluyveromyces lactis.

Integration of a DNA fragment in a host genome requires the action of a double-strand break (DSB) repair mechanism. Homologous recombination (HR) is initiated by binding of Rad52p to DNA ends and results in targeted integration. Binding of the Ku heterodimer (Ku70p/Ku80p) results in random integration via non-homologous end joining (NHEJ). In contrast to Saccharomyces cerevisiae, the budding yeast Kluyveromyces lactis shows variable, but in general low, gene targeting efficiency. To study and to improve gene targeting efficiency, K. lactis has been used as a model. The KlRAD51, KlRAD52 and KlKU80 genes have been isolated and deletion mutants for these genes have been constructed. Efficiency of gene targeting was determined at the KlADE2 locus using targeting constructs with different lengths of homologous flanking sequences. In wild-type K. lactis, the gene targeting efficiency ranged from 0% with 50 to 88% with 600 bp flanks. The Klku80 mutant, however, showed >97% gene targeting efficiency independently of the size of the homologous flanks. These results demonstrate that deletion of the NHEJ mechanism results in a higher gene targeting efficiency. Furthermore, increased gene targeting efficiency was achieved by the transformation of wild-type K. lactis with the KlADE2 deletion construct in the presence of excess small DNA fragments. Using this method, PCR-generated deletion constructs containing only 50 bp of homologous flanking sequences resulted in efficient targeted gene replacement.

Activity of the Kluyveromyces lactis Pdr5 multidrug transporter is modulated by the Sit4 protein phosphatase.

A possible role for posttranslational modifications in regulating the activity of ATP-binding cassette (ABC) transporters has not been well established. In this study, the drug efflux ABC transporter gene KlPDR5 was isolated from the budding yeast Kluyveromyces lactis, and it was found that the encoded KlPdr5 drug pump is posttranslationally regulated by the type 2A-related Ser/Thr protein phosphatase, Sit4p. The KlPdr5 transporter is a protein of 1,525 amino acids sharing 63.8% sequence identity with its Saccharomyces cerevisiae counterpart, ScPdr5p. Overexpression of the KlPDR5 gene confers resistance to oligomycin, antimycin, econazole, and ketoconazole, whereas cells with a disrupted allele of KlPDR5 are hypersensitive to the drugs and have a decreased capacity to carry out efflux of the anionic fluorescent dye rhodamine 123. It was found that a chromosomal disruption of KlPDR5 abolishes the drug-resistant phenotype associated with sit4 mutations and that a synergistic hyperresistance to the drugs can be created by overexpressing KlPDR5 in sit4 mutants. These data strongly indicate that the multidrug-resistant phenotype of sit4 mutants is mediated by negatively modulating the activity of KlPdr5p. As the transcriptional level of KlPDR5 and the steady-state level of KlPdr5p are not significantly affected by mutations in SIT4, the regulation by Sit4p appears to be a posttranslational process.

Telomere fusions caused by mutating the terminal region of telomeric DNA.

Mutations in the template region of a telomerase RNA gene can lead to the corresponding sequence alterations appearing in newly synthesized telomeric repeats. We analyzed a set of mutations in the template region of the telomerase RNA gene (TER1) of the budding yeast Kluyveromyces lactis that were predicted to lead to synthesis of mutant telomeric repeats with disrupted binding of the telomeric protein Rap1p. We showed previously that mutating the left side of the 12-bp consensus Rap1p binding site led to immediate and severe telomere elongation. Here, we show that, in contrast, mutating either the right side of the site or both sides together leads initially to telomere shortening. On additional passaging, certain mutants of both categories exhibit telomere-telomere fusions. Often, six new Bal-31-resistant, telomere repeat-containing bands appeared, and we infer that each of the six K. lactis chromosomes became circularized. These fusions were not stable, appearing occasionally to resolve and then reform. We demonstrate directly that a linear minichromosome introduced into one of the fusion mutant strains circularized by means of end-to-end fusions of the mutant repeat tracts. In contrast to the chromosomal circularization reported previously in Schizosaccharomyces pombe mutants defective in telomere maintenance, the K. lactis telomere fusions retained their telomeric DNA repeat sequences.

Regulation of primary carbon metabolism in Kluyveromyces lactis.

In the recent past, through advances in development of genetic tools, the budding yeast Kluyveromyces lactis has become a model system for studies on molecular physiology of so-called "Nonconventional Yeasts." The regulation of primary carbon metabolism in K. lactis differs markedly from Saccharomyces cerevisiae and reflects the dominance of respiration over fermentation typical for the majority of yeasts. The absence of aerobic ethanol formation in this class of yeasts represents a major advantage for the "cell factory" concept and large-scale production of heterologous proteins in K. lactis cells is being applied successfully. First insight into the molecular basis for the different regulatory strategies is beginning to emerge from comparative studies on S. cerevisiae and K. lactis. The absence of glucose repression of respiration, a high capacity of respiratory enzymes and a tight regulation of glucose uptake in K. lactis are key factors determining physiological differences to S. cerevisiae. A striking discrepancy exists between the conservation of regulatory factors and the lack of evidence for their functional significance in K. lactis. On the other hand, structurally conserved factors were identified in K. lactis in a new regulatory context. It seems that different physiological responses result from modified interactions of similar molecular modules.

Positive and Negative Control of Multidrug Resistance by the Sit4 Protein Phosphatase in Kluyveromyces lactis

The nuclear gene encoding the Sit4 protein phosphatase was identified in the budding yeast Kluyveromyces lactis. K. lactis cells carrying a disrupted sit4 allele are resistant to oligomycin, antimycin, ketoconazole, and econazole but hypersensitive to paromomycin, sorbic acid, and 4-nitroquinoline-N-oxide (4-NQO). Overexpression of SIT4 leads to an elevation in resistance to paromomycin and to lesser extent tolerance to sorbic acid, but it has no detectable effect on resistance to 4-NQO. These observations suggest that the Sit4 protein phosphatase has a broad role in modulating multidrug resistance in K. lactis. Expression or activity of a membrane transporter specific for paromomycin and the ABC pumps responsible for 4-NQO and sorbic acid would be positively regulated by Sit4p. In contrast, the function of a Pdr5-type transporter responsible for ketoconazole and econazole extrusion, and probably also for efflux of oligomycin and antimycin, is likely to be negatively regulated by the phosphatase. Drug resistance of sit4 mutants was shown to be mediated by ABC transporters as efflux of the anionic fluorescent dye rhodamine 6G, a substrate for the Pdr5-type pump, is markedly increased in sit4 mutants in an energy-dependent and FK506-sensitive manner.