Publications

Publications

Publications Between 2016 - 2020

60. "TITLE"
AUTHORS
Genomics, doi: (YEAR). Request copy
Abstract:TEXT

76. "The Nematicidal Potential of Bioactive Streptomyces Strains Isolated from Greek Rhizosphere Soils Tested on Arabidopsis Plants of Varying Susceptibility to Meloidogyne spp."
C. Meidani, A. Savvidis, E. Lampropoulou, A. Sagia, E. Katsifas, N. Monokrousos, D.G. Hatzinikolaou, A.D. Karagouni, E. Giannoutsou, I.D.S. Adamakis
Plants-Basel, doi:10.3390/plants9060699 (2020). Request copy
Abstract:A total of 461 indigenous Streptomycetes strains recovered from various Greek rhizosphere habitats were tested for their bioactivity. All isolates were examined for their ability to suppress the growth of 12 specific target microorganisms. Twenty-six were found to exert antimicrobial activity and were screened for potential nematicidal action. S. monomycini ATHUBA 220, S. colombiensis ATHUBA 438, S. colombiensis ATHUBA 431, and S. youssoufensis ATHUBA 546 were proved to have a nematicidal effect and thus were further sequenced. Batch culture supernatants and solvent extracts were assessed for paralysis on Meloidogyne javanica and Meloidogyne incognita second-stage juveniles (J2). The solvent extracts of S. monomycini ATHUBA 220 and S. colombiensis ATHUBA 438 had the highest paralysis rates, so these Streptomycetes strains were further on tested for nematodes' biological cycle arrest on two Arabidopsis thaliana plants; the wild type (Col-0) and the katanin mutantfra2, which is susceptible to M. incognita. Interestingly, S. monomycini ATHUBA 220 and S. colombiensis ATHUBA 438 were able to negatively affect the M. incognita biological cycle in Col-0 and fra2 respectively, and increased growth in Col-0 upon M. incognita infection. However, they were ineffective against M. javanica. Fra2 plants were also proved susceptible to M. javanica infestation, with a reduced growth upon treatments with the Streptomyces strains. The nematicidal action and the plant-growth modulating abilities of the selected Streptomycetes strains are discussed.

75. "Lipid production from indigenous Greek microalgae: a possible biodiesel source"
A.L. Savvides, K. Moisi, E.A. Katsifas, A.D. Karagouni, D.G. Hatzinikolaou,
Biotechnology Letters, doi:10.1007/s10529-019-02658-6 (2019). Request copy
Abstract:Objective: Microalgae gained interest for potential use as biodiesel producers, since they synthesize and accumulate significant quantities of lipids. The aim of this work was to isolate indigenous microalgae strains from Greek habitats, study their physicochemical growth conditions and finally select the best ones with respect to overall lipid production and profile. Results: Two sampling sites of marine aquatic ecosystems were selected in Attica prefecture, Greece in order to screen for novel wild type strains with lipid production capacity. Microalgae isolates (59) were obtained from the selected areas and were morphologically and molecularly characterized. Fatty acids were estimated through Flow Cytometry combined with BODIPY staining method. Four isolates were selected for their lipid production properties and were cultivated in 15 L tank cultures. The four isolates were also identified by 18S rDNA gene sequencing. Two of them, Chlorella sp. C9 and ACA17, exhibited both maximum biomass and lipid productivity. Optimization of growth conditions with respect to pH and initial NaNO3 concentration was performed for the two microalgae in 15 L cultures. Finally, 20 L fed batch cultures were set up using the optimum culture conditions. Lipid profiles were stabilized for both strains at dry biomass levels over 1gL(-1) and lipid content of 25% (w/w). Conclusions: Two Chlorella strains (ACA9 andACA17) were promising candidates for biodiesel production as they were easily grown in sea water in fed batch systems and produce lipids suitable for biodiesel, especially Chlorella sp. ACA9.

74. Evaluation of Thermal Stability of Cellulosomal Hydrolases and Their Complex Formation A. Kahn, A.P. Galanopoulou, D.G. Hatzinikolaou, S. Morais, E.A. Bayer CELLULASES: METHODS AND PROTOCOLS, doi:10.1007/978-1-4939-7877-9_12 (2018) Abstract: Enzymatic breakdown of plant biomass is an essential step for its utilization in biorefinery applications, and the products could serve as substrates for the sustainable and environmentally friendly production of fuels and chemicals. Toward this end, the incorporation of enzymes into polyenzymatic cellulosome complexes- able to specifically bind to and hydrolyze crystalline cellulosic materials, such as plant biomassis known to increase the efficiency and the overall hydrolysis performance of a cellulase system. Despite their relative abundance in various mesophilic anaerobic cellulolytic bacteria, there are only a few reports of cellulosomes of thermophilic origin. However, since various biorefinery processes are favored by elevated temperatures, the development of thermophilic designer cellulosomes could be of great importance. Owing to the limited number of thermophilic cellulosomes, designer cellulosomes, composed of mixtures of mesophilic and thermophilic components, have been constructed. As a result, the overall thermal profile of the individual parts and the resulting complex has to be extensively evaluated. Here, we describe a practical guide for the determination of temperature stability for cellulases in the cellulosome complexes. The approach is also appropriate for other related enzymes, notably xylanases as well as other glycoside hydrolases. We provide detailed experimental procedures for the evaluation of the thermal stability of the individual designer cellulosome components and their complexes as well as protocols for the assessment of complex integrity at elevated temperatures.

73. More than 2500 years of oil exposure shape sediment microbiomes with the potential for syntrophic degradation of hydrocarbons linked to methanogenesis A. Michas, G. Vestergaard, K. Trautwein, P. Avramidis, D.G. Hatzinikolaou, C.E. Vorgias, H. Wilkes, R. Rabus, M. Schloter, A. Scholer Microbiome, doi:10.1186/s40168-017-0337-8 (2017) Abstract: Background: Natural oil seeps offer the opportunity to study the adaptation of ecosystems and the associated microbiota to long-term oil exposure. In the current study, we investigated a land-to-sea transition ecosystem called "Keri Lake" in Zakynthos Island, Greece. This ecosystem is unique due to asphalt oil springs found at several sites, a phenomenon already reported 2500 years ago. Sediment microbiomes at Keri Lake were studied, and their structure and functional potential were compared to other ecosystems with oil exposure histories of various time periods. Results: Replicate sediment cores (up to 3-m depth) were retrieved from one site exposed to oil as well as a nonexposed control site. Samples from three different depths were subjected to chemical analysis and metagenomic shotgun sequencing. At the oil-exposed site, we observed high amounts of asphalt oil compounds and a depletion of sulfate compared to the non-exposed control site. The numbers of reads assigned to genes involved in the anaerobic degradation of hydrocarbons were similar between the two sites. The numbers of denitrifiers and sulfate reducers were clearly lower in the samples from the oil-exposed site, while a higher abundance of methanogens was detected compared to the non-exposed site. Higher abundances of the genes of methanogenesis were also observed in the metagenomes from other ecosystems with a long history of oil exposure, compared to short-term exposed environments. Conclusions: The analysis of Keri Lake metagenomes revealed that microbiomes in the oil-exposed sediment have a higher potential for methanogenesis over denitrification/sulfate reduction, compared to those in the non-exposed site. Comparison with metagenomes from various oil-impacted environments suggests that syntrophic interactions of hydrocarbon degraders with methanogens are favored in the ecosystems with a long-term presence of oil.

72. NmeA, a novel efflux transporter specific for nucleobases and nucleosides, contributes to metal resistance in Aspergillus nidulans S. Balaska, V. Myrianthopoulos, M. Tselika, D.G. Hatzinikolaou, E. Mikros, G. Diallinas Molecular Microbiology, doi:10.1111/mmi.13708 (2017) Abstract: Through Minos transposon mutagenesis we obtained A. nidulans mutants resistant to 5-fluorouracil due to insertions into the upstream region of the uncharacterized gene nmeA, encoding a Major Facilitator Superfamily (MFS) transporter. Minos transpositions increased nmeA transcription, which is otherwise extremely low under all conditions tested. To dissect the function of NmeA we used strains overexpressing or genetically lacking the nmeA gene. Strains overexpressing NmeA are resistant to toxic purine analogues, but also, to cadmium, zinc and borate, whereas an isogenic nmeAD null mutant exhibits increased sensitivity to these compounds. We provide direct evidence that nmeA overexpression leads to efflux of adenine, xanthine, uric acid and allantoin, the latter two being intermediate metabolites of purine catabolism that are toxic when accumulated cytoplasmically due to relevant genetic lesions. By using a functional GFP-tagged version we show that NmeA is a plasma membrane transporter. Homology modeling and docking approaches identified a single purine binding site and a tentative substrate translocation trajectory in NmeA. Orthologues of NmeA are present in all Aspergilli and other Eurotiomycetes, but are absent from other fungi or non-fungal organisms. NmeA is thus the founding member of a new class of transporters essential for fungal success under specific toxic conditions.

71. Enhancement of cellulosome-mediated deconstruction of cellulose by improving enzyme thermostability S. Morais, J. Stern, A. Kahn, A.P. Galanopoulou, S. Yoav, M. Shamshoum, M.A. Smith, D.G. Hatzinikolaou, F.H. Arnold, E.A. Bayer Biotechnology for Biofuels, doi:10.1186/s13068-016-0577-z (2016) Abstract: Background: The concerted action of three complementary cellulases from Clostridium thermocellum, engineered to be stable at elevated temperatures, was examined on a cellulosic substrate and compared to that of the wild-type enzymes. Exoglucanase Cel48S and endoglucanase Cel8A, both key elements of the natural cellulosome from this bacterium, were engineered previously for increased thermostability, either by SCHEMA, a structure-guided, site-directed protein recombination method, or by consensus-guided mutagenesis combined with random mutagenesis using error-prone PCR, respectively. A thermostable beta-glucosidase BglA mutant was also selected from a library generated by error-prone PCR that will assist the two cellulases in their methodic deconstruction of crystalline cellulose. The effects of a thermostable scaffoldin versus those of a largely mesophilic scaffoldin were also examined. By improving the stability of the enzyme subunits and the structural component, we aimed to improve cellulosome-mediated deconstruction of cellulosic substrates. Results: The results demonstrate that the combination of thermostable enzymes as free enzymes and a thermostable scaffoldin was more active on the cellulosic substrate than the wild-type enzymes. Significantly, "thermostable" designer cellulosomes exhibited a 1.7-fold enhancement in cellulose degradation compared to the action of conventional designer cellulosomes that contain the respective wild-type enzymes. For designer cellulosome formats, the use of the thermostabilized scaffoldin proved critical for enhanced enzymatic performance under conditions of high temperatures. Conclusions: Simple improvement in the activity of a given enzyme does not guarantee its suitability for use in an enzyme cocktail or as a designer cellulosome component. The true merit of improvement resides in its ultimate contribution to synergistic action, which can only be determined experimentally. The relevance of the mutated thermostable enzymes employed in this study as components in multienzyme systems has thus been confirmed using designer cellulosome technology. Enzyme integration via a thermostable scaffoldin is critical to the ultimate stability of the complex at higher temperatures. Engineering of thermostable cellulases and additional lignocellulosic enzymes may prove a determinant parameter for development of state-of-the-art designer cellulosomes for their employment in the conversion of cellulosic biomass to soluble sugars.

70. Metabolic Engineering of Fusarium oxysporum to Improve Its Ethanol-Producing Capability G.E. Anasontzis, E. Kourtoglou, S.G. Villas-Boas, D.G. Hatzinikolaou, P. Christakopoulos Frontiers In Microbiology, doi:10.3389/fmicb.2016.00632 (2016) Abstract: Fusarium oxysporum is one of the few filamentous fungi capable of fermenting ethanol directly from plant cell wall biomass. It has the enzymatic toolbox necessary to break down biomass to its monosaccharides and, under anaerobic and microaerobic conditions, ferments them to ethanol. Although these traits could enable its use in consolidated processes and thus bypass some of the bottlenecks encountered in ethanol production from lignocellulosic material when Saccharornyces cerevisiae is used namely its inability to degrade lignocellulose and to consume pentoses-two major disadvantages of F. oxysporum compared to the yeast its low growth rate and low ethanol productivity hinder the further development of this process. We had previously identified phosphoglucomutase and transaldolase, two major enzymes of glucose catabolism and the pentose phosphate pathway, as possible bottlenecks in the metabolism of the fungus and we had reported the effect of their constitutive production on the growth characteristics of the fungus. In this study, we investigated the effect of their constitutive production on ethanol productivity under anaerobic conditions. We report an increase in ethanol yield and a concomitant decrease in acetic acid production. Metabolomics analysis revealed that the genetic modifications applied did not simply accelerate the metabolic rate of the microorganism; they also affected the relative concentrations of the various metabolites suggesting an increased channeling toward the chorismate pathway, an activation of the gamma-aminobutyric acid shunt, and an excess in NADPH regeneration.

69. Fungi in Consolidated Bioprocessing of Lignocellulosic Materials A.P. Galanopoulou, D.G. Hatzinikolaou Fungal Applications In Sustainable Environmental Biotechnology, doi:10.1007/978-3-319-42852-9_11 (2016) Abstract: Second generation biorefineries are based on the efficient exploitation of the carbon stored in lignocellulosic biomass. Many fungal genera have the capacity to enzymatically degrade lignocellulose, while other can transform the resulting degradation products into compounds of economical value, such as biofuels. The possibility of performing both tasks in a single vessel and, ideally, by a single microbial strain is described by the term consolidated bioprocessing (CBP). This strategy is in principle applicable to the production of a broad range of compounds using plant biomass as raw material. In this chapter we will discuss the progress, problems, and prospects of CBP systems that exploit fungal species as their main biocatalyst. Since most of the relevant research has been conducted toward the production of bioethanol, our approach will focus on the efforts to either engineer ethanologenic strains for cellulase and hemicellulase production or to increase the fermentative capacity of natural biomass degraders. Finally, we will give an overview of fungal lignocellulose CBP processes for the production of additional higher value-added chemicals.

68. Lignocellulose degradation potential of Basidiomycota from Thrace (NE Greece) A.G. Sergentani, Z. Gonou-Zagou, E. Kapsanaki-Gotsi, D.G. Hatzinikolaou International Biodeterioration & Biodegradation, doi:10.1016/j.ibiod.2016.07.004 (2016) Abstract: Eighty fungal strains isolated from Northeastern Greece were evaluated for their biomass-degrading ability, both on solid and liquid cultures. Remazol Brilliant Blue R- and MnCl2-glucose plates were used for assessing ligninolytic potential. Cellulolytic and xylanolytic capabilities were evaluated with the use of Azo-CMC and Azo-Xylan, respectively, a choice that proved useful for the parallel, detection of lignin degradation activities. Twenty eight strains were selected to study the production of the corresponding enzymatic activities in wheat-bran based liquid media. Most isolates produced relatively high extracellular endo-beta-1,4-glucanase, endo-beta-1,4-xylanase and laccase activities, whereas neither lignin nor manganese-dependent peroxidase activity was detected. The pH dependence of the measured activities was also determined. The highest cellulase and xylanase levels were detected in Trametes pubescens, while Cerrena unicolor proved to be the best laccase producer. The present work points out the great potential of Agaricomycetes (Basidiomycota) for lignocellulose degradation applications, and for many species, such as Agaricus impudicus, Coprinellus micaceus, Daedaleopsis tricolor, Fotnitiporia pseudopunctata, Gymnopus foetidus, Neolentinus cyathiformis and Psathyrella fagetophila, represents the first report on the-production of lignocellulolytic enzymes. Concerning fungal diversity, the species Coprinopsis spelaiophila, D. tricolor and F pseudopunctata are reported for the first time from Greece. The strains of A. impudicus, C spelaiophila, G. foetidus and P. fagetophila are unique as pure culture isolates.