#46. "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 and A. Schöler
Microbiome, vol. 5, article 118, (2017).   Open access at publisher's site  
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 non-exposed 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.

#45. "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 and G. Diallinas
Molecular Microbiology, vol. 105, pp. 426-439, (2017).   View in Journal   Request copy
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 nmeAΔ 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.

#44. "Enhancement of cellulosome-mediated deconstruction of cellulose by improving enzyme thermostability"
S. Moraïs, J. Stern, A. Kahn, A.P. Galanopoulou, S. Yoav, M. Shamshoum, M.A. Smith, D.G. Hatzinikolaou, F.H. Arnold and E.A. Bayer
Biotechnology for Biofuels, vol. 9, article 164, (2016).   Open access at publisher's site  
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 β-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.

#43. "Lignocellulose degradation potential of Basidiomycota from Thrace (NE Greece)".
A.G. Sergentani, Z. Gonou-Zagou, E. Kapsanaki-Gotsi and D.G. Hatzinikolaou
International Biodeterioration and Biodegradation, vol. 114, pp. 268-277, (2016).   View in Journal   Request copy
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-b-1,4-glucanase, endo-b-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, Fomitiporia 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.

#42. "3-Chloro-1,2-propanediol biodegradation by Ca-alginate immobilized Pseudomonas putida DSM 437 cells applying different processes: mass transfer effects"
A. Konti, D. Mamma, D.G. Hatzinikolaou and D. Kekos
Bioprocess and Biosystems Engineering, vol. 39, pp. 1597-1609, (2016).   View in Journal   Request copy
Abstract: 3-Chloro-1,2-propanediol (3-CPD) biodegradation by Ca-alginate immobilized Pseudomonas putida cells was performed in batch system, continuous stirred tank reactor (CSTR), and packed-bed reactor (PBR). Batch system exhibited higher biodegradation rates and 3-CPD uptakes compared to CSTR and PBR. The two continuous systems (CSTR and PBR) when compared at 200 mg/L 3-CPD in the inlet exhibited the same removal of 3-CPD at steady state. External mass-transfer limitations are found negligible at all systems examined, since the observable modulus for external mass transfer Ω<<1 and the Biot number Bi<1. Intra-particle diffusion resistance had a significant effect on 3-CPD biodegradation in all systems studied, but to a different extent. Thiele modulus was in the range of 2.5 in batch system, but it was increased at 11 when increasing cell loading in the beads, thus lowering significantly the respective effectiveness factor. Comparing the systems at the same cell loading in the beads PBR was less affected by internal diffusional limitations compared to CSTR and batch system, and, as a result, exhibited the highest overall effectiveness factor.

#41. "Insights into the functionality and stability of designer cellulosomes at elevated temperatures"
A.P. Galanopoulou, S. Moraïs, A. Georgoulis, E. Morag, E.A. Bayer and D.G. Hatzinikolaou
Applied Microbiology and Biotechnology, vol. 100, pp. 8731-8743, (2016).   View in Journal   Request copy
Abstract: Enzymatic breakdown of lignocellulose is a major limiting step in second generation biorefineries. Assembly of the necessary activities into designer cellulosomes increases the productivity of this step by enhancing enzyme synergy through the proximity effect. However, most cellulosomal components are obtained from mesophilic microorganisms, limiting the applications to temperatures up to 50 °C. We hypothesized that a scaffoldin, comprising modular components of mainly mesophilic origin, can function at higher temperatures when combined with thermophilic enzymes, and the resulting designer cellulosomes could be employed in higher temperature reactions. For this purpose, we used a tetravalent scaffoldin constituted of three cohesins of mesophilic origin as well as a cohesin and cellulose-binding module derived from the thermophilic bacterium Clostridium thermocellum. The scaffoldin was combined with four thermophilic enzymes from Geobacillus and Caldicellulosiruptor species, each fused with a dockerin whose specificity matched one of the cohesins. We initially verified that the biochemical properties and thermal stability of the resulting chimeric enzymes were not affected by the presence of the mesophilic dockerins. Then we examined the stability of the individual single-enzyme scaffoldin complexes and the full tetravalent cellulosome showing that all complexes are stable and functional for at least 6 h at 60 °C. Finally, within this time frame and conditions, the full complex appeared over 50% more efficient in the hydrolysis of corn stover compared to the free enzymes. Overall, the results support the utilization of scaffoldin components of mesophilic origin at relatively high temperatures and provide a framework for the production of designer cellulosomes suitable for high temperature biorefinery applications.

#40. "Metabolic engineering of Fusarium oxysporum to improve its ethanol-producing capability".
G.E. Anasontzis, E. Kourtoglou, S.G. Villas-Boâs, D.G. Hatzinikolaou and P. Christakopoulos
Frontiers in Microbiology, vol. 7, article 632, (2016).   Open access at publisher's site  
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 Saccharomyces 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 γ-aminobutyric acid shunt, and an excess in NADPH regeneration.