Medium composition overturns the widely accepted sulfate-dependent repression of desulfurization phenotype in Rhodococcus qingshengii IGTS8.

Microbial desulfurization has been extensively studied as a promising alternative to the widely applied chemical desulfurization process. Sulfur removal from petroleum and its products becomes essential, as the environmental regulations become increasingly stringent. Rhodococcus qingshengii IGTS8 has gained ground as a naturally occurring model biocatalyst, due to its superior specific activity for desulfurization of dibenzothiophene (DBT). Recalcitrant organic sulfur compounds-DBT included-are preferentially removed by selective carbon-sulfur bond cleavage to avoid a reduction in the calorific value of the fuel. The process, however, still has not reached economically sustainable levels, as certain limitations have been identified. One of those bottlenecks is the repression of catalytic activity caused by ubiquitous sulfur sources such as inorganic sulfate, methionine, or cysteine. Herein, we report an optimized culture medium for wild-type stain IGTS8 that completely alleviates the sulfate-mediated repression of biodesulfurization activity without modification of the natural biocatalyst. Medium C not only promotes growth in the presence of several sulfur sources, including DBT, but also enhances biodesulfurization of resting cells grown in the presence of up to 5 mM sulfate. Based on the above, the present work can be considered as a step towards the development of a more viable commercial biodesulfurization process.

Advancing Desulfurization in the Model Biocatalyst Rhodococcus qingshengii IGTS8 via an In Locus Combinatorial Approach.

Biodesulfurization poses as an ideal replacement to the high cost hydrodesulfurization of the recalcitrant heterocyclic sulfur compounds, such as dibenzothiophene (DBT) and its derivatives. The increasingly stringent limits on fuel sulfur content intensify the need for improved desulfurization biocatalysts, without sacrificing the calorific value of the fuel. Selective sulfur removal in a wide range of biodesulfurization strains, as well as in the model biocatalyst Rhodococcus qingshengii IGTS8, occurs via the 4S metabolic pathway that involves the dszABC operon, which encodes enzymes that catalyze the generation of 2-hydroxybiphenyl and sulfite from DBT. Here, using a homologous recombination process, we generate two recombinant IGTS8 biocatalysts, harboring native or rearranged, nonrepressible desulfurization operons, within the native dsz locus. The alleviation of sulfate-, methionine-, and cysteine-mediated dsz repression is achieved through the exchange of the native promoter Pdsz, with the nonrepressible Pkap1 promoter. The Dsz-mediated desulfurization from DBT was monitored at three growth phases, through HPLC analysis of end product levels. Notably, an 86-fold enhancement of desulfurization activity was documented in the presence of selected repressive sulfur sources for the recombinant biocatalyst harboring a combination of three targeted genetic modifications, namely, a dsz operon rearrangement, a native promoter exchange, and a dszA-dszB overlap removal. In addition, transcript level comparison highlighted the diverse effects of our genetic engineering approaches on dsz mRNA ratios and revealed a gene-specific differential increase in mRNA levels. IMPORTANCE Rhodococcus is perhaps the most promising biodesulfurization genus and is able to withstand the harsh process conditions of a biphasic biodesulfurization process. In the present work, we constructed an advanced biocatalyst harboring a combination of three genetic modifications, namely, an operon rearrangement, a promoter exchange, and a gene overlap removal. Our homologous recombination approach generated stable biocatalysts that do not require antibiotic addition, while harboring nonrepressible desulfurization operons that present very high biodesulfurization activities and are produced in simple and low-cost media. In addition, transcript level quantification validated the effects of our genetic engineering approaches on recombinant strains' dsz mRNA ratios and revealed a gene-specific differential increase in mRNA levels. Based on these findings, the present work can pave the way for further strain and process optimization studies that could eventually lead to an economically viable biodesulfurization process.

Interplay between Sulfur Assimilation and Biodesulfurization Activity in Rhodococcus qingshengii IGTS8: Insights into a Regulatory Role of the Reverse Transsulfuration Pathway.

Biodesulfurization is a process that selectively removes sulfur from dibenzothiophene and its derivatives. Several natural biocatalysts harboring the highly conserved desulfurization operon dszABC, which is significantly repressed by methionine, cysteine, and inorganic sulfate, have been isolated. However, the available information on the metabolic regulation of gene expression is still limited. In this study, scarless knockouts of the reverse transsulfuration pathway enzyme genes cbs and metB were constructed in the desulfurizing strain Rhodococcus sp. strain IGTS8. We provide sequence analyses and report the enzymes' involvement in the sulfate- and methionine-dependent repression of biodesulfurization activity. Sulfate addition in the bacterial culture did not repress the desulfurization activity of the Δcbs strain, whereas deletion of metB promoted a significant biodesulfurization activity for sulfate-based growth and an even higher desulfurization activity for methionine-grown cells. In contrast, growth on cysteine completely repressed the desulfurization activity of all strains. Transcript level comparison uncovered a positive effect of cbs and metB gene deletions on dsz gene expression in the presence of sulfate and methionine, but not cysteine, offering insights into a critical role of cystathionine ß-synthase (CßS) and MetB in desulfurization activity regulation. IMPORTANCE Precise genome editing of the model biocatalyst Rhodococcus qingshengii IGTS8 was performed for the first time, more than 3 decades after its initial discovery. We thus gained insight into the regulation of dsz gene expression and biocatalyst activity, depending on the presence of two reverse transsulfuration enzymes, CßS and MetB. Moreover, we observed an enhancement of biodesulfurization capability in the presence of otherwise repressive sulfur sources, such as sulfate and l-methionine. The interconnection of cellular sulfur assimilation strategies was revealed and validated.

Biochemical and Thermodynamic Studies on a Novel Thermotolerant GH10 Xylanase from Bacillus safensis.

Xylanases have a broad range of applications in agro-industrial processes. In this study, we report on the discovery and characterization of a new thermotolerant GH10 xylanase from Bacillus safensis, designated as BsXyn10. The xylanase gene (bsxyn10) was cloned from Bacillus safensis and expressed in Escherichia coli. The reduced molecular mass of BsXyn10 was 48 kDa upon SDS-PAGE. Bsxyn10 was optimally active at pH 7.0 and 60 °C, stable over a broad range of pH (5.0-8.0), and also revealed tolerance toward different modulators (metal cations, EDTA). The enzyme was active toward various xylans with no activity on the glucose-based polysaccharides. KM, vmax, and kcat for oat spelt xylan hydrolysis were found to be 1.96 g·L-1, 58.6 µmole·min-1·(mg protein)-1, and 49 s-1, respectively. Thermodynamic parameters for oat spelt xylan hydrolysis at 60 °C were ΔS* = -61.9 J·mol-1·K-1, ΔH* = 37.0 kJ·mol-1 and ΔG* = 57.6 kJ·mol-1. BsXyn10 retained high levels of activity at temperatures up to 60 °C. The thermodynamic parameters (ΔH*D, ΔG*D, ΔS*D) for the thermal deactivation of BsXyn10 at a temperature range of 40-80 °C were: 192.5 ≤ ΔH*D ≤ 192.8 kJ·mol-1, 262.1 ≤ ΔS*D ≤ 265.8 J·mol-1·K-1, and 99.9 ≤ ΔG*D ≤ 109.6 kJ·mol-1. The BsXyn10-treated oat spelt xylan manifested the catalytic release of xylooligosaccharides of 2-6 DP, suggesting that BsXyn10 represents a promising candidate biocatalyst appropriate for several biotechnological applications.

Antibacterial Activity of Copper Nanoparticles against Xanthomonas campestris pv. vesicatoria in Tomato Plants.

Copper-based bactericides have appeared as a new tool in crop protection and offer an effective solution to combat bacterial resistance. In this work, two copper nanoparticle products that were previously synthesized and evaluated against major bacterial and fungal pathogens were tested on their ability to control the bacterial spot disease of tomato. Growth of Xanthomonas campestris pv. vesicatoria, the causal agent of the disease, was significantly suppressed by both nanoparticles, which had superior function compared to conventional commercial formulations of copper. X-ray fluorescence spectrometry measurements in tomato leaves revealed that bioavailability of copper is superior in the case of nanoparticles compared to conventional formulations and is dependent on synthesis rather than size. This is the first report correlating bioavailability of copper to nanoparticle efficacy.

Deciphering the biodesulfurization potential of two novel Rhodococcus isolates from a unique Greek environment.

Sustainable biodesulfurization (BDS) processes require the use of microbial biocatalysts that display high activity against the recalcitrant heterocyclic sulfur compounds and can simultaneously withstand the harsh conditions of contact with petroleum products, inherent to any industrial biphasic BDS system. In this framework, the functional microbial BDS-related diversity in a naturally oil-exposed ecosystem, was examined through a 4,6-dimethyl-dibenzothiophene based enrichment process. Two new Rhodococcus sp. strains were isolated, which during a medium optimization process revealed a significantly enhanced BDS activity profile when compared to the model strain R. qingshengii IGTS8. In biocatalyst stability studies conducted in biphasic mode using partially hydrodesulfurized diesel under various process conditions, the new strains also presented an enhanced stability phenotype. In these studies, it was also demonstrated for all strains, that the BDS activity losses were decoupled from the overall cells' viability, in addition to the fact that the use of whole-broth biocatalyst positively affected BDS performance.

Direct Antibiotic Activity of Bacillibactin Broadens the Biocontrol Range of Bacillus amyloliquefaciens MBI600.

Bacillus amyloliquefaciens is considered the most successful biological control agent due to its ability to colonize the plant rhizosphere and phyllosphere where it outgrows plant pathogens by competition, antibiosis, and inducing plant defense. Its antimicrobial function is thought to depend on a diverse spectrum of secondary metabolites, including peptides, cyclic lipopeptides, and polyketides, which have been shown to target mostly fungal pathogens. In this study, we isolated and characterized the catecholate siderophore bacillibactin by B. amyloliquefaciens MBI600 under iron-limiting conditions and we further identified its potential antibiotic activity against plant pathogens. Our data show that bacillibactin production restrained in vitro and in planta growth of the nonsusceptible (to MBI600) pathogen Pseudomonas syringae pv. tomato. Notably, it was also related to increased antifungal activity of MBI600. In addition to bacillibactin biosynthesis, iron starvation led to upregulation of specific genes involved in microbial fitness and competition. IMPORTANCE Siderophores have mostly been studied concerning their contribution to the fitness and virulence of bacterial pathogens. In the present work, we isolated and characterized for the first time the siderophore bacillibactin from a commercial bacterial biocontrol agent. We proved that its presence in the culture broth has significant biocontrol activity against nonsusceptible bacterial and fungal phytopathogens. In addition, we suggest that its activity is due to a new mechanism of action, that of direct antibiosis, rather than by competition through iron scavenging. Furthermore, we showed that bacillibactin biosynthesis is coregulated with the transcription of antimicrobial metabolite synthases and fitness regulatory genes that maximize competition capability. Finally, this work highlights that the efficiency and range of existing bacterial biocontrol agents can be improved and broadened via the rational modification of the growth conditions of biocontrol organisms.

Characterization of the Highly Efficient Acid-Stable Xylanase and ß-Xylosidase System from the Fungus Byssochlamys spectabilis ATHUM 8891 (Paecilomyces variotii ATHUM 8891).

Two novel xylanolytic enzymes, a xylanase and a ß-xylosidase, were simultaneously isolated and characterized from the extracellular medium of Byssochlamys spectabilis ATHUM 8891 (anamorph Paecilomyces variotii ATHUM 8891), grown on Brewer's Spent Grain as a sole carbon source. They represent the first pair of characterized xylanolytic enzymes of the genus Byssochlamys and the first extensively characterized xylanolytic enzymes of the family Thermoascaceae. In contrast to other xylanolytic enzymes isolated from the same family, both enzymes are characterized by exceptional thermostability and stability at low pH values, in addition to activity optima at temperatures around 65 °C and acidic pH values. Applying nano-LC-ESI-MS/MS analysis of the purified SDS-PAGE bands, we sequenced fragments of both proteins. Based on sequence-comparison methods, both proteins appeared conserved within the genus Byssochlamys. Xylanase was classified within Glycoside Hydrolase family 11 (GH 11), while ß-xylosidase in Glycoside Hydrolase family 3 (GH 3). The two enzymes showed a synergistic action against xylan by rapidly transforming almost 40% of birchwood xylan to xylose. The biochemical profile of both enzymes renders them an efficient set of biocatalysts for the hydrolysis of xylan in demanding biorefinery applications.

Microbial bioprospecting for lignocellulose degradation at a unique Greek environment.

Bacterial systems have gained wide attention for depolymerization of lignocellulosic biomass, due to their high functional diversity and adaptability. To achieve the full microbial exploitation of lignocellulosic residues and the cost-effective production of bioproducts within a biorefinery, multiple metabolic pathways and enzymes of various specificities are required. In this work, highly diverse aerobic, mesophilic bacteria enriched from Keri Lake, a pristine marsh of increased biomass degradation and natural underground oil leaks, were explored for their metabolic versatility and enzymatic potential towards lignocellulosic substrates. A high number of Pseudomonas species, obtained from enrichment cultures where organosolv lignin served as the sole carbon and energy source, were able to assimilate a range of lignin-associated aromatic compounds. Comparatively more complex bacterial consortia, including members of Actinobacteria, Proteobacteria, Bacilli, Sphingobacteria, and Flavobacteria, were also enriched from cultures with xylan or carboxymethyl cellulose as sole carbon sources. Numerous individual isolates could target diverse structural lignocellulose polysaccharides by expressing hydrolytic activities on crystalline or amorphous cellulose and xylan. Specific isolates showed increased potential for growth in lignin hydrolysates prepared from alkali pretreated agricultural wastes. The results suggest that Keri isolates represent a pool of effective lignocellulose degraders with significant potential for industrial applications in a lignocellulose biorefinery.

Doing synthetic biology with photosynthetic microorganisms.

The use of photosynthetic microbes as synthetic biology hosts for the sustainable production of commodity chemicals and even fuels has received increasing attention over the last decade. The number of studies published, tools implemented, and resources made available for microalgae have increased beyond expectations during the last few years. However, the tools available for genetic engineering in these organisms still lag those available for the more commonly used heterotrophic host organisms. In this mini-review, we provide an overview of the photosynthetic microbes most commonly used in synthetic biology studies, namely cyanobacteria, chlorophytes, eustigmatophytes and diatoms. We provide basic information on the techniques and tools available for each model group of organisms, we outline the state-of-the-art, and we list the synthetic biology tools that have been successfully used. We specifically focus on the latest CRISPR developments, as we believe that precision editing and advanced genetic engineering tools will be pivotal to the advancement of the field. Finally, we discuss the relative strengths and weaknesses of each group of organisms and examine the challenges that need to be overcome to achieve their synthetic biology potential.

Bactericides Based on Copper Nanoparticles Restrain Growth of Important Plant Pathogens.

Copper nanoparticles (CuNPs) can offer an alternative to conventional copper bactericides and possibly slow down the development of bacterial resistance. This will consequently lower the accumulation rate of copper to soil and water and lower the environmental and health burden imposed by copper application. Physical and chemical methods have been reported to synthesize CuNPs but their use as bactericides in plants has been understudied. In this study, two different CuNPs products have been developed, CuNP1 and CuNP2 in two respective concentrations (1500 ppm or 300 ppm). Both products were characterized using Dynamic Light Scattering, Transmission Electron Microscopy, Attenuated Total Reflection measurements, X-ray Photoelectron Spectroscopy, X-ray Diffraction and Scattering, and Laser Doppler Electrophoresis. They were evaluated for their antibacterial efficacy in vitro against the gram-negative species Agrobacterium tumefaciens, Dickeya dadantii, Erwinia amylovora, Pectobacterium carotovorum, Pseudomonas corrugata, Pseudomonas savastanoi pv. savastanoi, and Xanthomonas campestris pv. campestris. Evaluation was based on comparisons with two commercial bactericides: Kocide (copper hydroxide) and Nordox (copper oxide). CuNP1 inhibited the growth of five species, restrained the growth of P. corrugata, and had no effect in X. c. pv campestris. MICs were significantly lower than those of the commercial formulations. CuNP2 inhibited the growth of E. amylovora and restrained growth of P. s. pv. savastanoi. Again, its overall activity was higher compared to commercial formulations. An extensive in vitro evaluation of CuNPs that show higher potential compared to their conventional counterpart is reported for the first time and suggests that synthesis of stable CuNPs can lead to the development of low-cost sustainable commercial products.

Sulfate Alters the Competition Among Microbiome Members of Sediments Chronically Exposed to Asphalt.

Sulfate-reducing microorganisms (SRMs) often compete with methanogens for common substrates. Due to thermodynamic reasons, SRMs should outcompete methanogens in the presence of sulfate. However, many studies have documented coexistence of these microbial groups in natural environments, suggesting that thermodynamics alone cannot explain the interactions among them. In this study, we investigated how SRMs compete with the established methanogenic communities in sediment from a long-term, electron acceptor-depleted, asphalt-exposed ecosystem and how they affect the composition of the organic material. We hypothesized that, upon addition of sulfate, SRMs (i) outcompete the methanogenic communities and (ii) markedly contribute to transformations of the organic material. We sampled sediments from the test and proximate control sites under anoxic conditions and incubated them in seawater medium with or without sulfate. Abundance and activity pattern of SRMs and methanogens, as well as the total prokaryotic community, were followed for 6 weeks by using qPCR targeting selected marker genes. Some of these genes were also subjected to amplicon sequencing to assess potential shifts in diversity patterns. Alterations of the organic material in the microcosms were determined by mass spectrometry. Our results indicate that the competition of SRMs with methanogens upon sulfate addition strongly depends on the environment studied and the starting microbiome composition. In the asphalt-free sediments (control), the availability of easily degradable organic material (mainly plant-derived) allows SRMs to use a larger variety of substrates, reducing interspecies competition with methanogens. In contrast, the abundant presence of recalcitrant compounds in the asphalt-exposed sediment was associated with a strong competition between SRMs and methanogens, ultimately detrimental for the latter. Our data underpin the importance of the quality of bioavailable organic materials in anoxic environments as a driver for microbial community structure and function.

Catalytic and thermodynamic properties of an acidic α-amylase produced by the fungus Paecilomyces variotii ATHUM 8891.

An extracellular acid stable α-amylase from Paecilomyces variotii ATHUM 8891 (PV8891 α-amylase) was purified to homogeneity applying ammonium sulfate fractionation, ion exchange and gel filtration chromatography and exhibited a reduced molecular weight of 75 kDa. The purified enzyme was optimally active at pH 5.0 and 60 °C and stable in acidic pH (3.0-6.0). K m, v max and k cat for starch hydrolysis were found 1.1 g L-1, 58.5 µmole min-1 (mg protein)-1, and 73.1 s-1, respectively. Amylase activity was marginally enhanced by Ca2+ and Fe2+ ions while Cu2+ ions strongly inhibited it. Thermodynamic parameters determined for starch hydrolysis (Ε α, ΔH*, ΔG*, ΔS*, Δ G E - S ∗ and Δ G E - T ∗ ) suggests an effective capacity of PV8891 α-amylase towards starch hydrolysis. Thermal stability of PV8891 α-amylase was assessed at different temperatures (30-80 οC). Thermodynamic parameters ( E a d , ΔH*, ΔG*, ΔS*) as well as the integral activity of a continuous system for starch hydrolysis by the PV8891 α-amylase revealed satisfactory thermostability up to 60 °C. The acidic nature and its satisfactory performance at temperatures lower than the industrially used amylases may represent potential applications of PV8891 α-amylase in starch processing industry.

Τhe Nematicidal Potential of Bioactive Streptomyces Strains Isolated from Greek Rhizosphere Soils Tested on Arabidopsis Plants of Varying Susceptibility to Meloidogyne spp.

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 mutant fra2, 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.

XynDZ5: A New Thermostable GH10 Xylanase.

Xylanolytic enzymes have a broad range of applications in industrial biotechnology as biocatalytic components of various processes and products, such as food additives, bakery products, coffee extraction, agricultural silage and functional foods. An increasing market demand has driven the growing interest for the discovery of xylanases with specific industrially relevant characteristics, such as stability at elevated temperatures and in the presence of other denaturing factors, which will facilitate their incorporation into industrial processes. In this work, we report the discovery and biochemical characterization of a new thermostable GH10 xylanase, termed XynDZ5, exhibiting only 26% amino acid sequence identity to the closest characterized xylanolytic enzyme. This new enzyme was discovered in an Icelandic hot spring enrichment culture of a Thermoanaerobacterium species using a recently developed bioinformatic analysis platform. XynDZ5 was produced recombinantly in Escherichia coli, purified and characterized biochemically. This analysis revealed that it acts as an endo-1,4-ß-xylanase that performs optimally at 65-75°C and pH 7.5. The enzyme is capable of retaining high levels of catalytic efficiency after several hours of incubation at high temperatures, as well as in the presence of significant concentrations of a range of metal ions and denaturing agents. Interestingly, the XynDZ5 biochemical profile was found to be atypical, as it also exhibits significant exo-activity. Computational modeling of its three-dimensional structure predicted a (ß/α)8 TIM barrel fold, which is very frequently encountered among family GH10 enzymes. This modeled structure has provided clues about structural features that may explain aspects of its catalytic performance. Our results suggest that XynDZ5 represents a promising new candidate biocatalyst appropriate for several high-temperature biotechnological applications in the pulp, paper, baking, animal-feed and biofuel industries.

Lipid production from indigenous Greek microalgae: a possible biodiesel source.

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. ΑCΑ9 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 1 g L-1 and lipid content of 25% (w/w). CONCLUSIONS: Two Chlorella strains (ACA9 and ACA17) 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.

Creation of a functional hyperthermostable designer cellulosome.

BACKGROUND: Renewable energy has become a field of high interest over the past decade, and production of biofuels from cellulosic substrates has a particularly high potential as an alternative source of energy. Industrial deconstruction of biomass, however, is an onerous, exothermic process, the cost of which could be decreased significantly by use of hyperthermophilic enzymes. An efficient way of breaking down cellulosic substrates can also be achieved by highly efficient enzymatic complexes called cellulosomes. The modular architecture of these multi-enzyme complexes results in substrate targeting and proximity-based synergy among the resident enzymes. However, cellulosomes have not been observed in hyperthermophilic bacteria. RESULTS: Here, we report the design and function of a novel hyperthermostable "designer cellulosome" system, which is stable and active at 75 °C. Enzymes from Caldicellulosiruptor bescii, a highly cellulolytic hyperthermophilic anaerobic bacterium, were selected and successfully converted to the cellulosomal mode by grafting onto them divergent dockerin modules that can be inserted in a precise manner into a thermostable chimaeric scaffoldin by virtue of their matching cohesins. Three pairs of cohesins and dockerins, selected from thermophilic microbes, were examined for their stability at extreme temperatures and were determined stable at 75 °C for at least 72 h. The resultant hyperthermostable cellulosome complex exhibited the highest levels of enzymatic activity on microcrystalline cellulose at 75 °C, compared to those of previously reported designer cellulosome systems and the native cellulosome from Clostridium thermocellum. CONCLUSION: The functional hyperthermophilic platform fulfills the appropriate physico-chemical properties required for exothermic processes. This system can thus be adapted for other types of thermostable enzyme systems and could serve as a basis for a variety of cellulolytic and non-cellulolytic industrial objectives at high temperatures.

Evaluation of Thermal Stability of Cellulosomal Hydrolases and Their Complex Formation.

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 biomass-is 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.

More than 2500 years of oil exposure shape sediment microbiomes with the potential for syntrophic degradation of hydrocarbons linked to methanogenesis.

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.