The consensus sequence of Kluyveromyces lactis centromeres shows homology to functional centromeric DNA from Saccharomyces cerevisiae

Summary The nucleotide sequences of five of the six centromeres of the yeast Kluyveromyces lactis were determined. Mutual comparison of these sequences led to the following consensus: a short highly conserved box (5-ATCACGTGA-3) flanked by an AT-rich (±90%) stretch of ± 160 by followed by another conserved box (5-TNNTTTATGTTTCCGAAAATTAATAT-3).These three elements were named K1CDEI, K1CDEII, and K1CDEIII respectively, by analogy with the situation in Saccharomyces cerevisiae. In addition, a second 100 by AT-rich (±90%) element, named K1CDE0, was found ± 150 by upstream of K1CDEI. The sequences of both K1CDEI and K1CDEIII are highly conserved between K. lactis and S. cerevisiae; however, centromeres of K. lactis do not function in S. cerevisiae and vice versa. The most obvious differences between the centromeres of the two yeast species are the length of the AT-rich CDEII, which is 161–164 by in K. lactis versus 78–86 by in S. cerevisiae and the presence in K. lactis of K1CDEO, which is not found in S. cerevisiae.     

DNA deformability changes of single base pair mutants within CDE binding sites in S. Cerevisiae centromere DNA correlate with measured chromosomal loss rates and CDE binding site symmetries

Background  

The centromeres in yeast (S. cerevisiae) are organized by short DNA sequences (125 bp) on each chromosome consisting of 2 conserved elements: CDEI and CDEIII spaced by a CDEII region. CDEI and CDEIII are critical sequence specific protein binding sites necessary for correct centromere formation and following assembly with proteins, are positioned near each other on a specialized nucleosome. Hegemann et al. BioEssays 1993, 15: 451–460 reported single base DNA mutants within the critical CDEI and CDEIII binding sites on the centromere of chromosome 6 and quantitated centromere loss of function, which they measured as loss rates for the different chromosome 6 mutants during cell division. Olson et al. Proc Natl Acad Sci USA 1998, 95: 11163–11168 reported the use of protein-DNA crystallography data to produce a DNA dinucleotide protein deformability energetic scale (PD-scale) that describes local DNA deformability by sequence specific binding proteins. We have used the PD-scale to investigate the DNA sequence dependence of the yeast chromosome 6 mutants' loss rate data. Each single base mutant changes 2 PD-scale values at that changed base position relative to the wild type. In this study, we have utilized these mutants to demonstrate a correlation between the change in DNA deformability of the CDEI and CDEIII core sites and the overall experimentally measured chromosome loss rates of the chromosome 6 mutants.     

Mutational analysis of centromeric DNA elements of Klayveromyces lactis and their role in determining the species specificity of the highly homologous centromeres from K. lactis and Saccharomyces cerevisiae

The centromere of Kluyveromyces lactis was delimited to a region of approximately 280 bp, encompassing KICDEI, II, and III. Removal of 6 bp from the right side of KlCDEIII plus flanking sequences abolished centromere function, and removal of 5 bp of KICDEI and flanking sequences resulted in strongly reduced centromere function. Deletions of 20–80 bp from KlCDEII resulted in a decrease in plasmid stability, indicating that KlCDEII must have a certain length for proper centromere function. Centromeres of K. lactis do not function in Saccharomyces cerevisiae and vice versa. Adapting the length of K1CDEII to that of ScCDEII did not improve KlCEN function in S. cerevisiae, while doubling the ScCDEII length did not improve ScCEN function in K. lactis. Thus the difference in CDEII length is not in itself responsible for the species specificity of the centromeres from each of the two species of budding yeast. A chimeric K. lactis centromere with ScCDEIII instead of KlCDEIII was no longer functional in K. lactis, but did improve plasmid stability in S. cerevisiae, although to a much lower level then a wild-type ScCEN. This indicates that the exact CDEIII sequence is important, and suggests that the flanking AT-rich CDEII has to conform to specific sequence requirements.     

Mutational analysis of centromeric DNA elements of Klayveromyces lactis and their role in determining the species specificity of the highly homologous centromeres from K. lactis and Saccharomyces cerevisiae

The centromere of Kluyveromyces lactis was delimited to a region of approximately 280 bp, encompassing KICDEI, II, and III. Removal of 6 bp from the right side of KlCDEIII plus flanking sequences abolished centromere function, and removal of 5 bp of KICDEI and flanking sequences resulted in strongly reduced centromere function. Deletions of 20–80 bp from KlCDEII resulted in a decrease in plasmid stability, indicating that KlCDEII must have a certain length for proper centromere function. Centromeres of K. lactis do not function in Saccharomyces cerevisiae and vice versa. Adapting the length of K1CDEII to that of ScCDEII did not improve KlCEN function in S. cerevisiae, while doubling the ScCDEII length did not improve ScCEN function in K. lactis. Thus the difference in CDEII length is not in itself responsible for the species specificity of the centromeres from each of the two species of budding yeast. A chimeric K. lactis centromere with ScCDEIII instead of KlCDEIII was no longer functional in K. lactis, but did improve plasmid stability in S. cerevisiae, although to a much lower level then a wild-type ScCEN. This indicates that the exact CDEIII sequence is important, and suggests that the flanking AT-rich CDEII has to conform to specific sequence requirements.     

Binding of the essential Saccharomyces cerevisiae kinetochore protein Ndc10p to CDEII

Chromosome segregation at mitosis depends critically on the accurate assembly of kinetochores and their stable attachment to microtubules. Analysis of Saccharomyces cerevisiae kinetochores has shown that they are complex structures containing >/=50 protein components. Many of these yeast proteins have orthologs in animal cells, suggesting that key aspects of kinetochore structure have been conserved through evolution, despite the remarkable differences between the 125-base pair centromeres of budding yeast and the Mb centromeres of animal cells. We describe here an analysis of S. cerevisiae Ndc10p, one of the four protein components of the CBF3 complex. CBF3 binds to the CDEIII element of centromeric DNA and initiates kinetochore assembly. Whereas CDEIII binding by Ndc10p requires the other components of CBF3, Ndc10p can bind on its own to CDEII, a region of centromeric DNA with no known binding partners. Ndc10p-CDEII binding involves a dispersed set of sequence-selective and -nonselective contacts over approximately 80 base pairs of DNA, suggesting formation of a multimeric structure. CDEII-like sites, active in Ndc10p binding, are also present along chromosome arms. We propose that a polymeric Ndc10p complex formed on CDEII and CDEIII DNA is the foundation for recruiting microtubule attachment proteins to kinetochores. A similar type of polymeric structure on chromosome arms may mediate other chromosome-spindle interactions.     

Probing the Architecture of a Simple Kinetochore Using DNA–Protein Crosslinking

In budding yeast, accurate chromosome segregation requires that one and only one kinetochore assemble per chromosome. In this paper, we report the use of DNA–protein crosslinking and nondenaturing gel analysis to study the structure of CBF3, a four-protein complex that binds to the essential CDEIII region of Saccharomyces cerevisiae centromeres. We find that three subunits of CBF3 are in direct contact with CDEIII over a region of DNA that spans 80 bp. A highly asymmetric core complex containing p58^CTF13 p64^CEP3 and p110^NDC10 in direct contact with DNA forms at the genetically defined center of CDEIII. This core complex spans ~56 bp of CEN3. An extended complex comprising the core complex and additional DNA-bound p110^NDC10 also forms. It spans ~80 bp of DNA. CBF3 makes sequence-specific and -nonspecific contacts with DNA. Both contribute significantly to the energy of CBF3–DNA interaction. Moreover, important sequence-specific contacts are made with bases that are not conserved among yeast centromeres. These findings provide a foundation for understanding the organization of the CBF3–centromere complex, a structure that appears to initiate the formation of microtubule attachment sites at yeast kinetochores. These results also have implications for understanding centromere-binding proteins in higher cells.     

Temperature and osmotic stress dependence of the thermodynamics for binding linker histone H10, Its carboxyl domain (H10-C) or globular domain (H10-G) to B-DNA

Linker histones (H1) are the basic proteins in higher eukaryotes that are responsible for the final condensation of chromatin. In contrast to the nucleosome core histone proteins, the role of H1 in compacting DNA is not clearly understood. In this study ITC was used to measure the binding constant, enthalpy change, and binding site size for the interactions of H1⁰, or its C-terminal (H1⁰-C) and globular (H1⁰-G) domains to highly polymerized calf-thymus DNA at temperatures from 288 K to 308 K. Heat capacity changes, ΔC_p, for these same H1⁰ binding interactions were estimated from the temperature dependence of the enthalpy changes. The enthalpy changes for binding H1⁰, H1⁰-C, or H1⁰-G to CT-DNA are all endothermic at 298 K, becoming more exothermic as the temperature is increased. The ΔH for binding H1⁰-G to CT-DNA is exothermic at temperatures above approximately 300 K. Osmotic stress experiments indicate that the binding of H1⁰ is accompanied by the release of approximately 35 water molecules.We estimate from our naked DNA titration results that the binding of the H1⁰ to the nucleosome places the H1⁰ protein in close contact with approximately 41 DNA bp. The breakdown is that the H1⁰ carboxyl terminus interacts with 28 bp of linker DNA on one side of the nucleosome, the H1⁰ globular domain binds directly to 7 bp of core DNA, and shields another 6 linker DNA bases, 3 bp on either side of the nucleosome where the linker DNA exits the nucleosome core.     

Chromatin structures of Kluyveromyces lactis centromeres in K. lactis and Saccharomyces cerevisiae

We have investigated the chromatin structure of Kluyveromyces lactis centromeres in isolated nuclei of K. lactis and Saccharomyces cerevisiae by using micrococcal nuclease and DNAse I digestion. The protected region found in K. lactis is approximately 270 bp long and encompasses the centromeric DNA elements, KlCDEI, KlCDEII, and KlCDEIII, but not KlCDE0. Halving KlCDEII to 82 bp impaired centromere function and led to a smaller protected structure (210 bp). Likewise, deletion of 5 bp from KlCDEI plus adjacent flanking sequences resulted in a smaller protected region and a decrease in centromere function. The chromatin structures of KlCEN2 and KlCEN4 present on plasmids were found to be similar to the structures of the corresponding centromeres in their chromosomal context. A different protection pattern of KlCEN2 was detected in S. cerevisiae, suggesting that KlCEN2 is not properly recognized by at least one of the centromere binding proteins of S. cerevisiae. The difference is mainly found at the KlCDEIII side of the structure. This suggests that one of the components of the ScCBF3-complex is not able to bind to KlCDEIII, which could explain the species specificity of K. lactis and S. cerevisiae centromeres.     

A 125-base-pair CEN6 DNA fragment is sufficient for complete meiotic and mitotic centromere functions in Saccharomyces cerevisiae.

Saccharomyces cerevisiae centromeres contain a conserved region ranging from 111 to 119 base pairs (bp) in length, which is characterized by the three conserved DNA elements CDEI, CDEII, and CDEIII. We isolated a 125-bp CEN6 DNA fragment (named ML CEN6) containing only these conserved elements and assayed it completely separated from its chromosomal context on circular minichromosomes and on a large linear chromosome fragment. The results show that this 125-bp CEN6 DNA fragment is by itself sufficient for complete mitotic and meiotic centromere functions.     

PVA-gel (Lentikats®) as an effective matrix for yeast strain immobilization aimed at heterologous protein production

Three different yeast strains, namely Saccharomyces cerevisiae mnn1mnn9, Kluyveromyces lactis JA6/GAA and Zygosaccharomyces bailii [pZ₃klIL-1β], were entrapped in polyvinyl alcohol (PVA) gel particles, obtained following the commercially available immobilization kit named Lentikat^®. After immobilization in the PVA-gel particles, yeast cells remained viable: colonization of the gel matrix reached up 100 mg d.w. of cells cm⁻³ gel for all the strains examined.Lentikats^® of K. lactis JA6/GAA and Z. bailii [pZ₃klIL-1β] showed to be suitable for the continuous production of glucoamylase and interleukin 1β, respectively, when employed under non-selective conditions. They were of easy handiness and showed excellent mechanical properties during prolonged operation in stirred tank reactors.     

Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae.

Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.     

The P170 expression system enhances hyaluronan molecular weight and production in metabolically-engineered Lactococcus lactis

Recombinant Lactococcus lactis strains based on the P170 expression system were developed for hyaluronan (HA) production, by incorporating genes from the has operon of Streptococcus zooepidemicus and compared with nisin-inducible recombinant L. lactis strains containing the hasABC and hasABD constructs. It was found across all batch and fed-batch experimental studies that HA concentration and molecular weight (MW) were higher for the P170 expression systems than the corresponding NICE-based strains. The highest hyaluronan MW was obtained for all constructs in batch studies at 60 g/L initial glucose concentration, the highest being 2.94 MDa for the P170 strains with hasABC construct (L. lactis APJ3). In fed-batch studies with constant feed rate, the L. lactis APJ3 gave better HA yield (0.03 g/g) than the NICE-based strain. A higher hyaluronan MW was obtained for all strains in pulse fed-batch compared to constant feed experiments, the highest being 2.52 MDa for L. lactis APJ3.     

Efficient production of 2,3-butanediol in Saccharomyces cerevisiae by eliminating ethanol and glycerol production and redox rebalancing

2,3-Butanediol is a promising valuable chemical that can be used in various areas as a liquid fuel and a platform chemical. Here, 2,3-butanediol production in Saccharomyces cerevisiae was improved stepwise by eliminating byproduct formation and redox rebalancing. By introducing heterologous 2,3-butanediol biosynthetic pathway and deleting competing pathways producing ethanol and glycerol, metabolic flux was successfully redirected to 2,3-butanediol. In addition, the resulting redox cofactor imbalance was restored by overexpressing water-forming NADH oxidase (NoxE) from Lactococcus lactis. In a flask fed-batch fermentation with optimized conditions, the engineered adh1Δadh2Δadh3Δadh4Δadh5Δgpd1Δgpd2Δ strain overexpressing Bacillus subtilis α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD), S. cerevisiae 2,3-butanediol dehydrogenase (Bdh1), and L. lactis NoxE from a single multigene-expression vector produced 72.9 g/L 2,3-butanediol with the highest yield (0.41 g/g glucose) and productivity (1.43 g/(L·h)) ever reported in S. cerevisiae.     

Enantioselective synthesis of ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE) from ethyl-3-oxo-3-phenylpropanoate using recombinant fatty acid synthase (FAS2) from Kluyveromyces lactis KCTC 7133 in Pichia pastoris GS115

Various yeast strains were examined for the microbial reduction of ethyl-3-oxo-3-phenylpropanoate (OPPE) to ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE), which is the chiral intermediate for the synthesis of a serotonin uptake inhibitor, Fluoxetine. Kluyveromyces lactis KCTC 7133 was found as the most efficient strain in terms of high yield (83% at 50 mM) and high optical purity ee > 99% of S-HPPE. Based on the protein purification, activity analysis and the genomic analysis, a fatty acid synthase (FAS) was identified as the responsible β-ketoreductase. To increase the productivity, a recombinant Pichia pastoris GS115 over-expressing FAS2 (α-subunit of FAS) of K. lactis KCTC7133 was constructed. In the optimized media condition, the recombinant P. pastoris functionally over-expressed the FAS2. Recombinant P. pastoris showed 2.3-fold higher reductase activity compared with wild type P. pastoris. With the recombinant P. pastoris, the 91% yield of S-HPPE was achieved at 50 mM OPPE maintaining the high optical purity of the product (ee > 99%).     

Codon optimization through a two-step gene synthesis leads to a high-level expression of Aspergillus niger lip2 gene in Pichia pastoris

Aspergillus niger lipases are important biocatalysts for a broad range of industrial applications. To enhance the expression level of a newly cloned lipase gene lip2 of A. niger in Pichia pastoris, we applied codon optimization and synthesized the full length codon-optimized gene by a two-step gene synthesis strategy. This strategy consists of an assembly PCR for several small DNA fragments and enzymatic digestion and ligation steps to ligate these fragments into the full-length gene. First, the full-length lip2 gene was divided into three fragments F1 (237 bp), F2 (238 bp) and F3 (422 bp) with the additions of proper restriction sites, and separately amplified by assembly PCR reactions. Second, three PCR amplified fragments were digested and ligated into the full-length lip2 gene. In the two-step gene synthesis, synthesis of smaller DNA fragments resulted in a significant lower level of nonspecific mismatching among oligonucleotides and a very low mutational rate of the PCR products, demonstrating the superiority of the method. When compared with the originally cloned lip2 gene of A. niger, the new codon optimized lip2 gene expressed at a significantly higher level in yeasts after methanol induction for 72 h, and both the enzyme activity and protein content reached maximal levels of 191 U/ml and 154 mg/1, with 11.6- and 5.3-fold increases, respectively.     

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus for acetic acid production

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus was carried out for high yield of acetic acid. Acetic acid production process was divided into three stages. The first stage was the growth of S. cerevisiae and ethanol production, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. The second stage was the co-culture of S. cerevisiae and A. pasteurianus, fermentation temperature and aeration rate were maintained at 34 °C and 0.4 vvm, respectively. The third stage was the growth of A. pasteurianus and production of acetic acid, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. Inoculation volume of A. pasteurianus and S. cerevisiae was 16% and 0.06%, respectively. The average acetic acid concentration was 52.51 g/L under these optimum conditions. To enhance acetic acid production, a glucose feeding strategy was subsequently employed. When initial glucose concentration was 90 g/L and 120 g/L glucose was fed twice during fermentation, acetic acid concentration reached 66.0 g/L.     

Ciliogenesis in cryopreserved mammalian tracheal epithelial cells cultured at the air–liquid interface

To determine air–liquid interface (ALI) culture derived from cryopreserved mammalian tracheal ciliated cells is a viable ciliated cell model for the investigations of regulatory mechanisms of ciliary beat frequency (CBF), two studies were performed using ovine and porcine tracheae obtained from local slaughterhouses. The protease-digested tracheal ciliated cells were harvested and cultured at the ALI using collagen-coated, porous membrane inserts. In study 1, the ALI culturing protocols were established using non-cryopreserved ovine tracheal ciliated cells. Ciliogenesis was documented with immuno-histology and electron micrographs. Vigorous beating cilia were video-recorded. CBF was measured by laser light scattering. The functional integrity of the autonomic receptors of the ciliated cells was confirmed with the stimulatory responses of CBF using luminal methacholine and basolateral terbutaline. In study 2, porcine tracheal ciliated cells stored in liquid nitrogen for a minimum of 4 weeks were used. The cryopreserved cells were thawed and cultured using the ALI protocol established in study 1. After two months, cilia outgrowths were confirmed using video microscopy and scanning electron micrograph (SEM). The trans-epithelial resistances were 28.5 kΩ (n = 4). Luminal applications of 1 μM and 10 μM methacholine stimulated CBF from a baseline of 7.4 ± 0.2 Hz to 8.4 ± 0.8 Hz and 7.7 ± 0.4 Hz, respectively (n = 5). Basolateral applications of 1 μM and 10 μM terbutaline stimulated CBF from a baseline of 7.5 ± 0.3 Hz to 8.2 ± 0.4 Hz and 8.0 ± 0.4 Hz, respectively (n = 5). These data demonstrated that a ciliated cell bank can be established using cryopreserved ciliated cells for pulmonary drug discovery and toxicological screening.     

Factors required for the binding of reassembled yeast kinetochores to microtubules in vitro

Kinetochores are structures that assemble on centromeric DNA and mediate the attachment of chromosomes to the microtubules of the mitotic spindle. The protein components of kinetochores are poorly understood, but the simplicity of the S. cerevisiae kinetochore makes it an attractive candidate for molecular dissection. Mutations in genes encoding CBF1 and CBF3, proteins that bind to yeast centromeres, interfere with chromosome segregation in vivo. To determine the roles played by these factors and by various regions of centromeric DNA in kinetochore function, we have developed a method to partially reassemble kinetochores on exogenous centromeric templates in vitro and to visualize the attachment of these reassembled kinetochore complexes to microtubules. In this assay, single reassembled complexes appear to mediate microtubule binding. We find that CBF3 is absolutely essential for this attachment but, contrary to previous reports (Hyman, A. A., K. Middleton, M. Centola, T.J. Mitchison, and J. Carbon. 1992. Microtubule- motor activity of a yeast centromere-binding protein complex. Nature (Lond.). 359:533-536) is not sufficient. Additional cellular factors interact with CBF3 to form active microtubule-binding complexes. This is mediated primarily by the CDEIII region of centromeric DNA but CDEII plays an essential modulatory role. Thus, the attachment of kinetochores to microtubules appears to involve a hierarchy of interactions by factors that assemble on a core complex consisting of DNA-bound CBF3.