L-Asparaginase Conjugates from the Hyperthermophilic Archaea Thermococcus sibiricus with Improved Biocatalytic Properties.

This study investigated the effect of polycationic and uncharged polymers (and oligomers) on the catalytic parameters and thermostability of L-asparaginase from Thermococcus sibiricus (TsA). This enzyme has potential applications in the food industry to decrease the formation of carcinogenic acrylamide during the processing of carbohydrate-containing products. Conjugation with the polyamines polyethylenimine and spermine (PEI and Spm) or polyethylene glycol (PEG) did not significantly affect the secondary structure of the enzyme. PEG contributes to the stabilization of the dimeric form of TsA, as shown by HPLC. Furthermore, neither polyamines nor PEG significantly affected the binding of the L-Asn substrate to TsA. The conjugates showed greater maximum activity at pH 7.5 and 85 °C, 10-50% more than for native TsA. The pH optima for both TsA-PEI and TsA-Spm conjugates were shifted to lower pH ranges from pH 10 (for the native enzyme) to pH 8.0. Additionally, the TsA-Spm conjugate exhibited the highest activity at pH 6.5-9.0 among all the samples. Furthermore, the temperature optimum for activity at pH 7.5 shifted from 90-95 °C to 80-85 °C for the conjugates. The thermal inactivation mechanism of TsA-PEG appeared to change, and no aggregation was observed in contrast to that of the native enzyme. This was visually confirmed and supported by the analysis of the CD spectra, which remained almost unchanged after heating the conjugate solution. These results suggest that TsA-PEG may be a more stable form of TsA, making it a potentially more suitable option for industrial use.

New Biocides Based on N4-Alkylcytidines: Effects on Microorganisms and Application for the Protection of Cultural Heritage Objects of Painting.

The rapid increase in the antibiotic resistance of microorganisms, capable of causing diseases in humans as destroying cultural heritage sites, is a great challenge for modern science. In this regard, it is necessary to develop fundamentally novel and highly active compounds. In this study, a series of N4-alkylcytidines, including 5- and 6-methylcytidine derivatives, with extended alkyl substituents, were obtained in order to develop a new generation of antibacterial and antifungal biocides based on nucleoside derivatives. It has been shown that N4-alkyl 5- or 6-methylcytidines effectively inhibit the growth of molds, isolated from the paintings in the halls of the Ancient Russian Paintings of the State Tretyakov Gallery, Russia, Moscow. The novel compounds showed activity similar to antiseptics commonly used to protect works of art, such as benzalkonium chloride, to which a number of microorganisms have acquired resistance. It was also shown that the activity of N4-alkylcytidines is comparable to that of some antibiotics used in medicine to fight Gram-positive bacteria, including resistant strains of Staphylococcus aureus and Mycobacterium smegmatis. N4-dodecyl-5- and 6-methylcytidines turned out to be the best. This compound seems promising for expanding the palette of antiseptics used in painting, since quite often the destruction of painting materials is caused by joint fungi and bacteria infection.

5-Substituted Uridines with Activity against Gram-Positive Bacteria.

The emergence of drug-resistant strains of pathogenic microorganisms necessitates the creation of new drugs. A series of uridine derivatives containing an extended substituent at the C-5 position as well as C-5 alkyloxymethyl, alkylthiomethyl, alkyltriazolylmethyl, alkylsulfinylmethyl and alkylsulfonylmethyl uridines were obtained in order to explore their antimicrobial properties and solubility. It has been shown that new ribonucleoside derivatives have an order of magnitude better solubility in water compared to their 2'-deoxy analogues and effectively inhibit the growth of a number of Gram-positive bacteria, including resistant strains of Mycobacterium smegmatis (MIC=15-200 µg/mL) and Staphylococcus aureus (MIC=25-100 µg/mL). Their activity is comparable to that of some antibiotics used in medicine.

Fungal BGCs for Production of Secondary Metabolites: Main Types, Central Roles in Strain Improvement, and Regulation According to the Piano Principle.

Filamentous fungi are one of the most important producers of secondary metabolites. Some of them can have a toxic effect on the human body, leading to diseases. On the other hand, they are widely used as pharmaceutically significant drugs, such as antibiotics, statins, and immunosuppressants. A single fungus species in response to various signals can produce 100 or more secondary metabolites. Such signaling is possible due to the coordinated regulation of several dozen biosynthetic gene clusters (BGCs), which are mosaically localized in different regions of fungal chromosomes. Their regulation includes several levels, from pathway-specific regulators, whose genes are localized inside BGCs, to global regulators of the cell (taking into account changes in pH, carbon consumption, etc.) and global regulators of secondary metabolism (affecting epigenetic changes driven by velvet family proteins, LaeA, etc.). In addition, various low-molecular-weight substances can have a mediating effect on such regulatory processes. This review is devoted to a critical analysis of the available data on the "turning on" and "off" of the biosynthesis of secondary metabolites in response to signals in filamentous fungi. To describe the ongoing processes, the model of "piano regulation" is proposed, whereby pressing a certain key (signal) leads to the extraction of a certain sound from the "musical instrument of the fungus cell", which is expressed in the production of a specific secondary metabolite.

Enhancing the Catalytic Activity of Thermo-Asparaginase from Thermococcus sibiricus by a Double Mesophilic-like Mutation in the Substrate-Binding Region.

L-asparaginases (L-ASNases) of microbial origin are the mainstay of blood cancer treatment. Numerous attempts have been performed for genetic improvement of the main properties of these enzymes. The substrate-binding Ser residue is highly conserved in L-ASNases regardless of their origin or type. However, the residues adjacent to the substrate-binding Ser differ between mesophilic and thermophilic L-ASNases. Based on our suggestion that the triad, including substrate-binding Ser, either GSQ for meso-ASNase or DST for thermo-ASNase, is tuned for efficient substrate binding, we constructed a double mutant of thermophilic L-ASNase from Thermococcus sibiricus (TsA) with a mesophilic-like GSQ combination. In this study, the conjoint substitution of two residues adjacent to the substrate-binding Ser55 resulted in a significant increase in the activity of the double mutant, reaching 240% of the wild-type enzyme activity at the optimum temperature of 90 °C. The mesophilic-like GSQ combination in the rigid structure of the thermophilic L-ASNase appears to be more efficient in balancing substrate binding and conformational flexibility of the enzyme. Along with increased activity, the TsA D54G/T56Q double mutant exhibited enhanced cytotoxic activity against cancer cell lines with IC90 values from 2.8- to 7.4-fold lower than that of the wild-type enzyme.

Thermo-L-Asparaginases: From the Role in the Viability of Thermophiles and Hyperthermophiles at High Temperatures to a Molecular Understanding of Their Thermoactivity and Thermostability.

L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine, food industry, and diagnostics. Among various organisms expressing L-ASNases, thermophiles and hyperthermophiles produce enzymes with superior performances-stable and heat resistant thermo-ASNases. This review is an attempt to take a broader view on the thermo-ASNases. Here we discuss the position of thermo-ASNases in the large family of L-ASNases, their role in the heat-tolerance cellular system of thermophiles and hyperthermophiles, and molecular aspects of their thermoactivity and thermostability. Different types of thermo-ASNases exhibit specific L-asparaginase activity and additional secondary activities. All products of these enzymatic reactions are associated with diverse metabolic pathways and are important for mitigating heat stress. Thermo-ASNases are quite distinct from typical mesophilic L-ASNases based on structural properties, kinetic and activity profiles. Here we attempt to summarize the current understanding of the molecular mechanisms of thermo-ASNases' thermoactivity and thermostability, from amino acid composition to structural-functional relationships. Research of these enzymes has fundamental and biotechnological significance. Thermo-ASNases and their improved variants, cloned and expressed in mesophilic hosts, can form a large pool of enzymes with valuable characteristics for biotechnological application.

Spermidine and 1,3-Diaminopropane Have Opposite Effects on the Final Stage of Cephalosporin C Biosynthesis in High-Yielding Acremonium chrysogenum Strain.

The addition of exogenous polyamines increases the production of antibiotic cephalosporin C (CPC) in Acremonium chrysogenum high-yielding (HY) strain during fermentation on a complex medium. However, the molecular basis of this phenomenon is still unknown. In the current study, we developed a special synthetic medium on which we revealed the opposite effect of polyamines. The addition of 1,3-diaminopropane resulted in an increase in the yield of CPC by 12-15%. However, the addition of spermidine resulted in a decrease in the yield of CPC by 14-15% and accumulation of its metabolic pathway precursor, deacetylcephalosporin C (DAC); the total amount of cephems (DAC and CPC) was the same as after the addition of DAP. This indicates that spermidine, but not 1,3-diaminopropane, affects the final stage of CPC biosynthesis, associated with the acetylation of its precursor. In both cases, upregulation of biosynthetic genes from beta-lactam BGCs occurred at the same level as compared to the control; expression of transport genes was at the control level. The opposite effect may be due to the fact that N1-acetylation is much more efficient during spermidine catabolism than for 1,3-diaminopropane. The addition of spermidine, but not 1,3-diaminopropane, depleted the pool of acetyl coenzyme A by more than two-fold compared to control, which could lead to the accumulation of DAC.

The Application of Chitosan for Protection of Cultural Heritage Objects of the 15-16th Centuries in the State Tretyakov Gallery.

Microorganisms are one of the main factors in the deterioration of cultural heritage, in particular art paintings. The antiseptics currently used in painting have significant limitations due to insufficient effectiveness or increased toxicity and interaction with art materials. In this regard, the actual challenge is the search for novel materials that effectively work against microorganisms in the composition with painting materials and do not change their properties. Chitosan has pronounced antimicrobial properties but was not used previously as an antiseptic for paintings. In our study we developed a number of mock layers based on sturgeon glue, supplemented which chitosan (molecular weight 25 kDa or 45 kDa), standard antiseptics for paintings (positive controls) or without additives (negative control). According to Fourier transform infrared spectroscopy and atomic force microscopy, the addition of chitosan did not significantly affect the optical and surface properties of this material. The ability of chitosan to effectively protect paintings was shown after inoculation on the created mock-up layers of 10 fungi-destructors of tempera painting, previously isolated from cultural heritage of the of the 15-16th centuries in the State Tretyakov Gallery, on the created mock layers. Our study demonstrated the principled opportunity of using chitosan in the composition of painting materials to prevent biodeterioration for the first time.

Biotransformation of Androstenedione by Filamentous Fungi Isolated from Cultural Heritage Sites in the State Tretyakov Gallery.

The transformation of steroids by microorganisms is widely used in medical biotechnology. A huge group of filamentous fungi is one of the most promising taxa for screening new biocatalytic reactions in order to obtain pharmaceutically significant steroids. In this work, we screened 10 filamentous fungi-destructors of egg tempera for the ability to biotransform androst-4-en-3,17-dione (AD) during cultivation in a liquid nutrient medium or in a buffer solution. These taxonomically unrelated strains, belonging to the classes Eurotiomycetes, Dothideomycetes and Sordariomycetes, are dominant representatives of the microbiome from halls where works of tempera painting are stored in the State Tretyakov Gallery (STG, Moscow, Russia). Since the binder of tempera paints, egg yolk, contains about 2% cholesterol, these degrading fungi appear to be a promising group for screening for steroid converting activity. It turned out that all the studied fungi-destructors are able to transform AD. Some strains showed transformation efficiency close to the industrial strain Curvularia lunata RNCIM F-981. In total, 33 steroids formed during the transformation of AD were characterized, for 19 of them the structure was established by gas chromatography/mass spectrometry analysis. In this work, we have shown for the first time that fungi-destructors of tempera paintings can efficiently transform steroids.

Highly Active Thermophilic L-Asparaginase from Melioribacter roseus Represents a Novel Large Group of Type II Bacterial L-Asparaginases from Chlorobi-Ignavibacteriae-Bacteroidetes Clade.

L-asparaginase (L-ASNase) is a biotechnologically relevant enzyme for the pharmaceutical, biosensor and food industries. Efforts to discover new promising L-ASNases for different fields of biotechnology have turned this group of enzymes into a growing family with amazing diversity. Here, we report that thermophile Melioribacter roseus from Ignavibacteriae of the Bacteroidetes/Chlorobi group possesses two L-ASNases-bacterial type II (MrAII) and plant-type (MrAIII). The current study is focused on a novel L-ASNase MrAII that was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 70 °C and pH 9.3, with a high L-asparaginase activity of 1530 U/mg and L-glutaminase activity ~19% of the activity compared with L-asparagine. The kinetic parameters KM and Vmax for the enzyme were 1.4 mM and 5573 µM/min, respectively. The change in MrAII activity was not significant in the presence of 10 mM Ni2+, Mg2+ or EDTA, but increased with the addition of Cu2+ and Ca2+ by 56% and 77%, respectively, and was completely inhibited by Zn2+, Fe3+ or urea solutions 2-8 M. MrAII displays differential cytotoxic activity: cancer cell lines K562, Jurkat, LnCap, and SCOV-3 were more sensitive to MrAII treatment, compared with normal cells. MrAII represents the first described enzyme of a large group of uncharacterized counterparts from the Chlorobi-Ignavibacteriae-Bacteroidetes clade.

Polyamines Upregulate Cephalosporin C Production and Expression of ß-Lactam Biosynthetic Genes in High-Yielding Acremonium chrysogenum Strain.

The high-yielding production of pharmaceutically significant secondary metabolites in filamentous fungi is obtained by random mutagenesis; such changes may be associated with shifts in the metabolism of polyamines. We have previously shown that, in the Acremonium chrysogenum cephalosporin C high-yielding strain (HY), the content of endogenous polyamines increased by four- to five-fold. Other studies have shown that the addition of exogenous polyamines can increase the production of target secondary metabolites in highly active fungal producers, in particular, increase the biosynthesis of ß-lactams in the Penicillium chrysogenum Wis 54-1255 strain, an improved producer of penicillin G. In the current study, we demonstrate that the introduction of exogenous polyamines, such as spermidine or 1,3-diaminopropane, to A. chrysogenum wild-type (WT) and HY strains, leads to an increase in colony germination and morphological changes in a complete agar medium. The addition of 5 mM polyamines during fermentation increases the production of cephalosporin C in the A. chrysogenum HY strain by 15-20% and upregulates genes belonging to the beta-lactam biosynthetic cluster. The data obtained indicate the intersection of the metabolisms of polyamines and beta-lactams in A. chrysogenum and are important for the construction of improved producers of secondary metabolites in filamentous fungi.

A Novel L-Asparaginase from Hyperthermophilic Archaeon Thermococcus sibiricus: Heterologous Expression and Characterization for Biotechnology Application.

L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine and food industry. Drawbacks of current commercial L-ASNases stimulate the search for better-producing sources of the enzyme, and extremophiles are especially attractive in this view. In this study, a novel L-asparaginase originating from the hyperthermophilic archaeon Thermococcus sibiricus (TsA) was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 90 °C and pH 9.0 with a specific activity of 2164 U/mg towards L-asparagine. Kinetic parameters KM and Vmax for the enzyme are 2.8 mM and 1200 µM/min, respectively. TsA is stable in urea solutions 0-6 M and displays no significant changes of the activity in the presence of metal ions Ni2+, Cu2+, Mg2+, Zn2+ and Ca2+ and EDTA added in concentrations 1 and 10 mmol/L except for Fe3+. The enzyme retains 86% of its initial activity after 20 min incubation at 90 °C, which should be enough to reduce acrylamide formation in foods processed at elevated temperatures. TsA displays strong cytotoxic activity toward cancer cell lines K562, A549 and Sk-Br-3, while normal human fibroblasts WI-38 are almost unsensitive to it. The enzyme seems to be a promising candidate for further investigation and biotechnology application.

Discovery of novel N4-alkylcytidines as promising antimicrobial agents.

The emergence of drug-resistant strains of pathogenic microorganisms necessitates the creation of new drugs. In order to find new compounds that effectively inhibit the growth of pathogenic bacteria and fungi, we synthesized a set of N4-derivatives of cytidine, 2'-deoxycytidine and 5-metyl-2'-deoxycytidine bearing extended N4-alkyl and N4-phenylalkyl groups. The derivatives demonstrate activity against a number of Gram-positive bacteria, including Mycobacterium smegmatis (MIC = 24-200 µM) and Staphylococcus aureus (MIC = 50-200 µM), comparable with the activities of some antibiotics in medical use. The most promising compound appeared to be N4-dodecyl-5-metyl-2'-deoxycytidine 4h with activities of 24 and 48 µM against M. smegmatis and S. aureus, respectively, and high inhibitory activity of 0.5 mM against filamentous fungi that can, among other things, damage works of art, such as tempera painting. Noteworthy, some of other synthesized compounds are active against fungal growth with the inhibitory concentration in the range of 0.5-3 mM.

The critical role of plasma membrane H+-ATPase activity in cephalosporin C biosynthesis of Acremonium chrysogenum.

The filamentous fungus Acremonium chrysogenum is the main industrial producer of cephalosporin C (CPC), one of the major precursors for manufacturing of cephalosporin antibiotics. The plasma membrane H+-ATPase (PMA) plays a key role in numerous fungal physiological processes. Previously we observed a decrease of PMA activity in A. chrysogenum overproducing strain RNCM 408D (HY) as compared to the level the wild-type strain A. chrysogenum ATCC 11550. Here we report the relationship between PMA activity and CPC biosynthesis in A. chrysogenum strains. The elevation of PMA activity in HY strain through overexpression of PMA1 from Saccharomyces cerevisiae, under the control of the constitutive gpdA promoter from Aspergillus nidulans, results in a 1.2 to 10-fold decrease in CPC production, shift in beta-lactam intermediates content, and is accompanied by the decrease in cef genes expression in the fermentation process; the characteristic colony morphology on agar media is also changed. The level of PMA activity in A. chrysogenum HY OE::PMA1 strains has been increased by 50-100%, up to the level observed in WT strain, and was interrelated with ATP consumption; the more PMA activity is elevated, the more ATP level is depleted. The reduced PMA activity in A. chrysogenum HY strain may be one of the selected events during classical strain improvement, aimed at elevating the ATP content available for CPC production.

Detection of potential biodeterioration risks for tempera painting in 16th century exhibits from State Tretyakov Gallery.

In this study, we investigated biodeterioration of materials used in tempera painting by analyzing the structure of the microbiome in ancient tempera paintings exhibited in State Tretyakov Gallery, Moscow, Russia. Samples were obtained from 16th-century paintings, including a grand Russian Orthodox icon "The Church Militant" (all exhibits were without visible signs of biodeterioration), and from surrounding walls and ceilings (with vast zones of visible microbial growth). A number of microorganisms isolated from visible signs of environmental bio-damage were also detected in tempera paintings kept in temperature- and humidity-controlled conditions unfavorable for the growth of microflora. To determine the biodegrading potential of the microbiome for tempera paintings, we developed a set of mock layers from paintwork materials used in tempera painting of 16th century and their modern analogues and inoculated them with cultures containing filamentous fungi and bacteria. The susceptibility to microbial degradation of individual tempera painting materials was examined by micro-Fourier Transform Infrared (FTIR) spectroscopy, which enabled detection of even invisible signs of biodeterioration. The results indicate that the microorganisms isolated from paintings and surrounding areas in the museum are capable of causing significant damage of various tempera materials, among which varnishes were the most resistant; however, the addition of antiseptic (sodium pentachlorophenolate) can inhibit microbial growth on sturgeon glue.

Hydroxylamine Analogue of Agmatine: Magic Bullet for Arginine Decarboxylase.

The biogenic polyamines, spermine, spermidine (Spd) and putrescine (Put) are present at micro-millimolar concentrations in eukaryotic and prokaryotic cells (many prokaryotes have no spermine), participating in the regulation of cellular proliferation and differentiation. In mammalian cells Put is formed exclusively from L-ornithine by ornithine decarboxylase (ODC) and many potent ODC inhibitors are known. In bacteria, plants, and fungi Put is synthesized also from agmatine, which is formed from L-arginine by arginine decarboxylase (ADC). Here we demonstrate that the isosteric hydroxylamine analogue of agmatine (AO-Agm) is a new and very potent (IC50 3•10-8 M) inhibitor of E. coli ADC. It was almost two orders of magnitude less potent towards E. coli ODC. AO-Agm decreased polyamine pools and inhibited the growth of DU145 prostate cancer cells only at high concentration (1 mM). Growth inhibitory analysis of the Acremonium chrysogenum demonstrated that the wild type (WT) strain synthesized Put only from L-ornithine, while the cephalosporin C high-yielding strain, in which the polyamine pool is increased, could use both ODC and ADC to produce Put. Thus, AO-Agm is an important addition to the set of existing inhibitors of the enzymes of polyamine biosynthesis, and an important instrument for investigating polyamine biochemistry.