Construction of Hansenula polymorpha strains with improved thermotolerance

The methylotrophic yeast Hansenula polymorpha has the potential to be used in the process of simultaneous saccharification and fermentation (SSF) of xylan derived xylose at elevated temperatures. To improve parameters of high‐temperature resistance and high‐temperature fermentation of H. polymorpha, strains carrying deletion of acid trehalase gene (ATH1) and overexpressing genes coding for heat‐shock proteins Hsp16p and Hsp104p were constructed. Results indicate that the corresponding recombinant strains have up to 12‐fold increased tolerance to heat‐shock treatment. The deletion of ATH1 gene and constitutive expression of HSP16 and HSP104 resulted in up to 5.8‐fold improvement of ethanol production from xylose at 50°C. Although the maximum ethanol concentration achieved from xylose was 0.9 g L⁻¹, our model H. polymorpha strains with elevated thermotolerance can be further modified by metabolic engineering to construct improved high‐temperature ethanol producers from this pentose. Biotechnol. Bioeng. 2009; 104: 911–919. © 2009 Wiley Periodicals, Inc.     

Hydration effects on the HET-s prion and amyloid-beta fibrillous aggregates, studied with three-dimensional molecular theory of solvation

We study the thermodynamic properties of the experimental fragments of the amyloid fibril made of the HET-s prion proteins (the infectious element of the filamentous fungus Podospora anserina) and of amyloid-β proteins (the major component of Alzheimer's disease-associated plaques) by using the three-dimensional molecular theory of solvation. The full quantitative picture of hydration effects, including the hydration thermodynamics and hydration structure around the fragments, is presented. For both the complexes, the hydration entropic effects dominate, which results in the entropic part offsetting the unfavorable energetic part of the free energy change upon the association. This is in accord with the fact that the hydrophobic cooperativity plays an essential role in the formation of amyloid fibrils. By calculating the partial molar volume of the proteins, we found that the volume change upon the association in both the systems is large and positive, with the implication that high pressure causes destabilization of the fibril. This observation is in good agreement with the recent experimental results. We also found that both the HET-s and amyloid-β pentamers have loose intermolecular packing with voids. The three-dimensional molecular theory of solvation predicts that water molecules can be locked in the interior cavities along the fibril axis for both the HET-s and amyloid-β proteins. We provide a detailed molecular picture of the structural water localized in the interior of the fibrils. Our results suggest that the interior hydration plays an important role in the structural stability of fibrils.     

Microsatellite Instability in Arabidopsis Increases with Plant Development

Plant development consists of the initial phase of intensive cell division followed by continuous genome endoreduplication, cell growth, and elongation. The maintenance of genome stability under these conditions is the main task performed by DNA repair and genome surveillance mechanisms. Our previous work showed that the rate of homologous recombination repair in older plants decreases. We hypothesized that this age-dependent decrease in the recombination rate is paralleled with other changes in DNA repair capacity. Here, we analyzed microsatellite stability using transgenic Arabidopsis (Arabidopsis thaliana) plants that carry the nonfunctional β-glucuronidase gene disrupted by microsatellite repeats. We found that microsatellite instability increased dramatically with plant age. We analyzed the contribution of various mechanisms to microsatellite instability, including replication errors and mistakes of DNA repair mechanisms such as mismatch repair, excision repair, and strand break repair. Analysis of total DNA polymerase activity using partially purified protein extracts showed an age-dependent decrease in activity and an increase in fidelity. Analysis of the steady-state RNA level of DNA replicative polymerases α, δ, Pol I-like A, and Pol I-like B and the expression of mutS homolog 2 (Msh2) and Msh6 showed an age-dependent decrease. An in vitro repair assay showed lower efficiency of nonhomologous end joining in older plants, paralleled by an increase in Ku70 gene expression. Thus, we assume that the more frequent involvement of nonhomologous end joining in strand break repair and the less efficient end-joining repair together with lower levels of mismatch repair activities may be the main contributors to the observed age-dependent increase in microsatellite instability.The genome of Arabidopsis (Arabidopsis thaliana) is extensively repetitive, which leads many to believe that Arabidopsis is subject to ancient autoploid events with many subsequent rearrangements and alterations (Meinke et al., 1998; Arabidopsis Genome Initiative, 2000; Blanc et al., 2000). Despite the highly reduplicated genome with the potential for a high degree of genetic redundancy, maintaining a consistent level of genome stability is critical. This is especially important when considering that plants do not have a predetermined germ line and that gametes are produced from meristematic cells that are products of many somatic cell divisions (Hays, 2002). Furthermore, as plants are sessile organisms, they are continuously exposed to various genotoxic elements such as heavy metals, reactive oxygen species, and UV irradiation. This constant exposure to harsh environmental conditions imposes a need for precise and efficient genome maintenance pathways, as the persistence of DNA damage and mutagenesis can decrease the fitness of current and future generations (Britt, 1996).DNA mutagenesis cannot solely be attributed to environmentally induced genotoxic stress, as DNA is prone to spontaneous or replication-induced mutagenesis. For example, transitions of 5-methylcytosine to thymine are common spontaneous mutations (Britt, 1996), while DNA replication and repair infidelity can induce numerous errors (Sia et al., 1997; Tuteja et al., 2001). Hundreds of mutations are introduced upon each genome replication due to DNA polymerase infidelity. Repetitive elements are particularly prone to this type of mutation due to replication slippage, which refers to DNA polymerase dissociation during the replication of short repetitive sequences followed by the separation and subsequent reassociation of the daughter strand in a different but identical repeat (Viguera et al., 2001). Polymerase reloading and the resumption of DNA synthesis can result in addition or subtraction of the repeated sequence. Microsatellites, the simple tandem repeats of one to six nucleotides (Viguera et al., 2001), are highly susceptible to replication slippage.The frequency at which these and other polymerase-derived errors persist depends largely on the DNA polymerase proofreading activity and the precision and fidelity of core DNA repair enzymes. Since many repair pathways involve DNA polymerase activity, many of them can potentially contribute to an increase in microsatellite instability. Mismatch repair (MMR) is a repair mechanism involved in the correction of replication errors. It is essential for the maintenance of repeated sequences, as mutations in MMR genes are associated with a substantial destabilization of microsatellites (Karran, 1996), and in humans, microsatellite instability increases with aging (Ben Yehuda et al., 2000; Krichevsky et al., 2004; Neri et al., 2005).The fidelity of different repair pathways can vary largely in the same or similar types of lesions. For example, single- and double-strand breaks can be repaired via homologous recombination (HR) or nonhomologous end joining (NHEJ) pathways (Britt, 1996; Tuteja et al., 2001; Kovalchuk et al., 2004; Boyko et al., 2006a). Of these pathways, HR is believed to be precise and largely error free, while NHEJ can induce numerous mutations ranging from single- to thousand-nucleotide insertions or deletions (Pelczar et al., 2003; Boyko et al., 2006b). It is unclear how either of these pathways is chosen for repair, but recent evidence from our laboratory suggests that the HR pathway is developmentally regulated, whereby NHEJ is up-regulated and HR is down-regulated with plant development (Boyko et al., 2006b). Currently, there is no information on whether other DNA repair pathways in plants are developmentally regulated.Previous publications suggest that aging human cells have a higher frequency of mutations in microsatellites (Ben Yehuda et al., 2000; Krichevsky et al., 2004; Neri et al., 2005). No such data exist for plants. Here, we investigated microsatellite stability during the development of Arabidopsis using the uidA (GUS) reporter gene inactivated by an artificially incorporated microsatellite (Azaiez et al., 2006). We found a strong increase in instability with plant maturity. We tested the contributions of various repair pathways to age-dependent microsatellite instability and suggest that these changes are primarily due to more frequent involvement of the NHEJ pathway in DNA repair.     

Adaptive behavior of bacterial mechanosensitive channels is coupled to membrane mechanics

Mechanosensitive channel of small conductance (MscS), a tension-driven osmolyte release valve residing in the inner membrane of Escherichia coli, exhibits a complex adaptive behavior, whereas its functional counterpart, mechanosensitive channel of large conductance (MscL), was generally considered nonadaptive. In this study, we show that both channels exhibit similar adaptation in excised patches, a process that is completely separable from inactivation prominent only in MscS. When a membrane patch is held under constant pressure, adaptation of both channels is manifested as a reversible current decline. Their dose–response curves recorded with 1–10-s ramps of pressure are shifted toward higher tension relative to the curves measured with series of pulses, indicating decreased tension sensitivity. Prolonged exposure of excised patches to subthreshold tensions further shifts activation curves for both MscS and MscL toward higher tension with similar magnitude and time course. Whole spheroplast MscS recordings performed with simultaneous imaging reveal activation curves with a midpoint tension of 7.8 mN/m and the slope corresponding to ∼15-nm² in-plane expansion. Inactivation was retained in whole spheroplast mode, but no adaptation was observed. Similarly, whole spheroplast recordings of MscL (V23T mutant) indicated no adaptation, which was present in excised patches. MscS activities tried in spheroplast-attached mode showed no adaptation when the spheroplasts were intact, but permeabilized spheroplasts showed delayed adaptation, suggesting that the presence of membrane breaks or edges causes adaptation. We interpret this in the framework of the mechanics of the bilayer couple linking adaptation of channels in excised patches to the relaxation of the inner leaflet that is not in contact with the glass pipette. Relaxation of one leaflet results in asymmetric redistribution of tension in the bilayer that is less favorable for channel opening.     

Overexpression of bacterial xylose isomerase and yeast host xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast Hansenula polymorpha

The thermotolerant methylotrophic yeast Hansenula polymorpha is able to ferment xylose to ethanol. To improve characteristics of xylose fermentation, the recombinant strain Delta xyl1 Delta xyl2-ADelta xyl2-B, with deletions of genes encoding first enzymes of xylose utilization (NAD(P)H-dependent xylose reductase and NAD-dependent xylitol dehydrogenases, respectively), was constructed and used as a recipient for co-overexpression of the Escherichia coli xylA gene coding for xylose isomerase and endogenous XYL3 gene coding for xylulokinase. The expression of both genes was driven by the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase promoter. Xylose isomerase activities of obtained transformants amounted to approximately 80% of that of the bacterial host strain. Xylulokinase activities of the transformants increased twofold when compared with the parental strain. The recombinant strains displayed improved ethanol production during the fermentation of xylose.     

Conjugates of PNA with naphthalene diimide derivatives having a broad range of DNA affinities

Peptide nucleic acids (PNAs) are neutral DNA analogues, which bind single-stranded DNA (ssDNA) strongly and with high sequence specificity. However, binding efficiency is dependent on the purine content of the PNA strand. This property make more difficult application of PNA as hybridization probes in, e.g., PNA chips, since at a set temperature the hybridization of a fraction of the DNA targets to the PNA probes does not obey Watson-Crick binding rules. The polypurine PNAs, for example, bind the mismatch containing DNA targets stronger, than the pyrimidine rich PNAs their fully complementary targets. Herein we show that PNA-DNA binding efficiency can be finely tuned by the conjugation of derivatives of naphthalene diimide (NADI) to the N-terminus of PNA using polyamide linkers of different lengths. This approach can potentially be used for the design of PNA probes, which bind their DNA targets with similar affinity independently of the PNA sequence.