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.

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.

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.

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.

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.

NmeA, a novel efflux transporter specific for nucleobases and nucleosides, contributes to metal resistance in Aspergillus nidulans.

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.

Insights into the functionality and stability of designer cellulosomes at elevated temperatures.

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.

Purine utilization proteins in the Eurotiales: cellular compartmentalization, phylogenetic conservation and divergence.

The purine utilization pathway has been thoroughly characterized in Aspergillus nidulans. We establish here the subcellular distribution of seven key intracellular enzymes, xanthine dehydrogenase (HxA), urate oxidase (UaZ), 5-hydroxy-isourate hydrolase (UaX), 2-oxo-4-hydroxy-4-carboxy ureido imidazoline decarboxylase (UaW), allantoinase (AlX), allantoicase (AaX), ureidoglycolate lyase (UglA), and the fungal-specific α-ketoglutarate Fe(II)-dependent dioxygenase (XanA). HxA, AlX, AaX, UaW and XanA are cytosolic, while UaZ, UaX and UglA are peroxisomal. Peroxisomal localization was confirmed by using appropriate pex mutants. The pathway is largely, but not completely conserved in the Eurotiomycetes, noticeably in some species AaX is substituted by an alternative enzyme of probable bacterial origin. UaZ and the urate-xanthine UapA and UapC transporters, are also localized in specific cells of the conidiophore. We show that metabolic accumulation of uric acid occurring in uaZ null mutations is associated with an increased frequency of appearance of morphologically distinct colony sectors, diminished conidiospore production, UV resistance and an altered response to oxidation stress, which may provide a rationale for the conidiophore-specific localization. The pathway-specific transcription factor UaY is localized in both the cytoplasm and nuclei under non-inducing conditions, but it rapidly accumulates exclusively to the nuclei upon induction by uric acid.

Constitutive homologous expression of phosphoglucomutase and transaldolase increases the metabolic flux of Fusarium oxysporum.

BACKGROUND: Fusarium oxysporum is among the few filamentous fungi that have been reported of being able to directly ferment biomass to ethanol in a consolidated bioprocess. Understanding its metabolic pathways and their limitations can provide some insights on the genetic modifications required to enhance its growth and subsequent fermentation capability. In this study, we investigated the hypothesis reported previously that phosphoglucomutase and transaldolase are metabolic bottlenecks in the glycolysis and pentose phosphate pathway of the F. oxysporum metabolism. RESULTS: Both enzymes were homologously overexpressed in F. oxysporum F3 using the gpdA promoter of Aspergillus nidulans for constitutive expression. Transformants were screened for their phosphoglucomutase and transaldolase genes expression levels with northern blot. The selected transformant exhibited high mRNA levels for both genes, as well as higher specific activities of the corresponding enzymes, compared to the wild type. It also displayed more than 20 and 15% higher specific growth rate upon aerobic growth on glucose and xylose, respectively, as carbon sources and 30% higher biomass to xylose yield. The determination of the relative intracellular amino and non-amino organic acid concentrations at the end of growth on glucose revealed higher abundance of most determined metabolites between 1.5- and 3-times in the recombinant strain compared to the wild type. Lower abundance of the determined metabolites of the Krebs cycle and an 68-fold more glutamate were observed at the end of the cultivation, when xylose was used as carbon source. CONCLUSIONS: Homologous overexpression of phosphoglucomutase and transaldolase in F. oxysporum was shown to enhance the growth characteristics of the strain in both xylose and glucose in aerobic conditions. The intracellular metabolites profile indicated how the changes in the metabolome could have resulted in the observed growth characteristics.

Assessment of the biomass hydrolysis potential in bacterial isolates from a volcanic environment: biosynthesis of the corresponding activities.

The biomass degrading enzymatic potential of 101 thermophilic bacterial strains isolated from a volcanic environment (Santorini, Aegean Sea, Greece) was assessed. 80 % of the strains showed xylanolytic activity in Congo Red plates, while only eight could simultaneously hydrolyze cellulose. Fifteen isolates were selected on the basis of their increased enzyme production, the majority of which was identified as Geobacilli through 16S rDNA analysis. In addition, the enzymatic profile was evaluated in liquid cultures using various carbon sources, a procedure that revealed lack of correlation on xylanase levels between the two cultivation modes and the inability of solid CMC cultures to fully unravel the cellulose degrading potential of the isolates. Strain SP24, showing more than 99 % 16S DNA similarity with Geobacillus sp. was further studied for its unique ability to simultaneously exhibit cellulase, xylanase, ß-glucosidase and ß-xylosidase activities. The first two enzymes were produced mainly extracellularly, while the ß-glycosidic activities were primarily detected in the cytosol. Maximum enzyme production by this strain was attained using a combination of wheat bran and xylan in the growth medium. Bioreactor cultures showed that aeration was necessary for both enhanced growth and enzyme production. Aeration had a strong positive effect on cellulase production while it negatively affected expression of ß-glucosidase. Xylanase and ß-xylosidase production was practically unaffected by aeration levels.

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.

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.

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.

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.

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.

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.

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.

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.

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.