Thermophilic Research Articles

Heterologous expression of a highly thermostable L-asparaginase from Thermococcus zilligii in Aspergillus niger for efficient reduction of acrylamide in French fries

L-asparaginase (L-ASNase) can hydrolyze L-asparagine, a precursor to acrylamide, thereby reducing toxic acrylamide formation in fried foods. Currently, commercial L-ASNases are primarily produced by wild-type (WT) filamentous fungi; however, these enzymes often exhibit rapid activity loss during high-temperature processing due to limited thermal stability. In this study, we screened a thermostable L-ASNase gene from thermophile bacteria and expressed it in Aspergillus niger to reduce acrylamide content in French fries. Initially, four genes encoding thermostable L-ASNases were selected and integrated into the A. niger genome via non-homologous end joining. Among these, the L-ASNase gene tzi from Thermococcus zilligii was successfully expressed in A. niger, yielding an extracellular activity of 114 U·mg−1. The recombinant enzyme (An-Tzi) displayed the same optimal temperature and pH as its WT counterpart but exhibited superior catalytic efficiency, likely due to the efficient post-translational modifications in A. niger. To further enhance expression, the tzi gene was integrated into the amylase (amyA) locus of the A. niger genome using the CRISPR-Cas9 system, resulting in increased activity of 128 U·mg−1. Additionally, various lengths of the highly expressed glucoamylase (glaA) protein from A. niger AG11 were fused to the N-terminus of the Tzi. Notably, fusing the 500-amino-acid catalytic domain of glaA led to a substantial 3.3-fold increase in enzyme activity. Despite the metabolic stress induced by high-level expression of glaA, supplementing the culture medium with metal ions and sophorose resulted in an extracellular activity of 486.74 U·mg−1, the highest reported yield of L-ASNase in shake flasks. Finally, applying the An-Tzi to French fries achieved a 32 % greater reduction in acrylamide compared to the commercial enzyme. Overall, the recombinant A. niger strain expressing thermostable An-Tzi demonstrates significant potential for industrial applications targeting acrylamide reduction in fried and baked foods.

Effect of rhamnolipid on the performance of compound thermophilic bacteria agent pretreatment system for waste sludge hydrolysis.

This study innovatively introduced rhamnolipid (RL) to compound thermophilic bacteria (TB) agent pretreatment system for further accelerating the waste sludge hydrolysis and substrates transformation. The results showed that combined pretreatment was beneficial for the sludge extracellular polymers (EPS) rupture and dissolved organic matters (DOM) release. In the optimal dosage of 40mg/g SS RL, the activities of protease and α-glucosidase increased by 20.7% and 33.3% than that without RL addition, respectively. The addition of RL enhanced efficient contacts between hydrolases and organic substrates, and excitation emission matrix (EEM) spectrum revealed that combined pretreatment with 40mg/g SS RL could achieve higher soluble microbial by-products occupancy (54%) and lower fulvic acid-like substances (6%) occupancy in DOM, promoting the waste sludge biodegradability. High organics availability conducted to more shifts in microbial community structure, compared with TB agent pretreatment, the relative abundance of genus Geobacillus and norank_f__Synergistaceae were enhanced by 29.08 and 0.33 times in combined pretreatment system, respectively, which was conducive to sludge hydrolysis and subsequent anaerobic fermentation process.

Physicochemical and aromatic properties of iron-enriched tomato paste during storage

In this study, tomato paste was fortified with iron compounds at 25, 50, and 75 ppm concentrations. The effect of adding these micronutrient iron concentrations on the paste’s physical, mechanical, aromatic, and chemical properties was evaluated every 15 days over a 60-day, storage period. The results indicated a gradual decrease in pH, total soluble solids (TSS), and taste index, alongside an increase in total acidity (TA) for all treatments throughout the storage period. The highest lycopene content observed in the Fe-25 % fortified samples at 43.05 mg/kg and the lowest in the control samples at 21.57 mg/kg. Positive values for a* and b* at the beginning and throughout the storage period confirmed the dominance of red and yellow hues in the tomato paste. Viscosity gradually decreased over time. During storage, growth and changes in mold spores and acid-resistant thermophilic bacteria were limited until the 30th day, and total microorganism counts were restricted until the 45th day. The overall accuracy of the support vector machines (C-SVM) and linear discriminant analysis (LDA) method in distinguishing samples into four groups (control, fortified with 25, 50, and 75 ppm iron) were 38 % and 62 % Overall, it can be concluded that increasing the iron micronutrient enhances the nutritional value and positively affects the physical, chemical, and microbial properties and the retention of aromatic compounds in tomato paste.

The Natural Whey Starter Used in the Production of Grana Padano and Parmigiano Reggiano PDO Cheeses: A Complex Microbial Community

Natural whey starter (NWS) is an undefined complex culture used in the production of Grana Padano and Parmigiano Reggiano PDO cheeses. The aim of this review is to discuss, in light of the latest research results, the role of NWS as a primary player in the cheese-making process, considering the microbial community scenario. NWS is traditionally produced by fermenting part of the whey collected at the end of a previous cheese-making process. The method used to produce NWS, based on the back-slopping principle, favors the selection of a microbiota composed mainly of thermophilic lactic acid bacteria. This method of preparation induces the survival of several different species and biotypes. The presence of such a mixture of strains facilitates the development of a natural starter characterized by a remarkable ability to adapt to non-standardized cheese-making parameters. NWS is a microbial community whose activity is not simply the result of the sum of the activities of individual microorganisms, but rather the activity of the community as a whole, in which each individual bacterial cell responds to the presence of the others. According to this traditional protocol, the NWS becomes the ‘microbiological bond’ between cheeses over time.

Physical and Biochemical Properties of N-Acetyl Transferase RimL of the Hyperthermophilic Bacteria Thermus thermophilus

Bacterial N-terminal acetyltransferases (NATs) are involved in the biosynthesis/degradation of antibiotics. The RimL enzyme from E. coli provides it with resistance to the antibiotic microcin C. To date, the NATs of pathogenic bacteria have been well studied, but there is no data on the NATs of thermophilic bacteria. The purpose of the work is to study the physicochemical properties and specificity of a new NAT — RimL from Thermus thermophilus. We cloned the RimL ORF (TTHA1799) and developed a method for purifying the enzyme. The stability of RimL to pH, high temperatures and denaturing agents was studied using the protein intrinsic fluorescence method. We have obtained a thermophilic enzyme that can be used in biotechnology for the acetylation of proteins and peptides under non-standard conditions.

The Transcriptomic Response of Cells of the Thermophilic Bacterium Geobacillus icigianus to Terahertz Irradiation.

As areas of application of terahertz (THz) radiation expand in science and practice, evidence is accumulating that this type of radiation can affect not only biological molecules directly, but also cellular processes as a whole. In this study, the transcriptome in cells of the thermophilic bacterium Geobacillus icigianus was analyzed immediately after THz irradiation (0.23 W/cm2, 130 μm, 15 min) and at 10 min after its completion. THz irradiation does not affect the activity of heat shock protein genes and diminishes the activity of genes whose products are involved in peptidoglycan recycling, participate in redox reactions, and protect DNA and proteins from damage, including genes of chaperone protein ClpB and of DNA repair protein RadA, as well as genes of catalase and kinase McsB. Gene systems responsible for the homeostasis of transition metals (copper, iron, and zinc) proved to be the most sensitive to THz irradiation; downregulation of these systems increased significantly 10 min after the end of the irradiation. It was also hypothesized that some negative effects of THz radiation on metabolism in G. icigianus cells are related to disturbances in activities of gene systems controlled by metal-sensitive transcription factors.

Characterization of psychrotrophic and thermoduric bacteria in raw milk using a multi-omics approach.

Psychrotrophic and thermoduric bacteria are the main reasons for the spoilage of dairy products. This study aims to address the composition and function of psychrotrophic and thermoduric bacteria in eight groups of raw milk samples obtained from Heilongjiang Province and Inner Mongolia (China). Microbial enumeration showed an average total bacterial count of 4.63 log c.f.u. ml-1 and psychrotrophic bacterial counts of 4.82 log c.f.u. ml-1. The mean counts of mesophilic and thermophilic thermoduric bacteria were 3.68 log and 1.81 log c.f.u. ml-1, respectively. Isolated psychrotrophic bacteria (26 genera and 50 species) and mesophilic thermoduric bacteria (20 genera and 32 species) showed high microbial diversity. Through metagenomic and proteomic analyses, significant disparities in the concentration and community structure of psychrotrophic and thermoduric bacteria were observed among different locations. A large number of peptidases were annotated by metagenomics, which may result in milk spoilage. They mainly come from some typical psychrotrophic and thermoduric bacteria, such as Chryseobacterium, Epilithonimonas, Pseudomonas, Psychrobacter, Acinetobacter, Lactococcus, Escherichia and Bacillus. However, the main proteins detected in fresh raw milk were associated with bacterial growth, reproduction and adaptation to cold environments. This investigation provides valuable insights into the microbial communities and protein profiles of raw milk, shedding light on the microbial factors contributing to milk deterioration.

A Thermostable Heme Protein Fold Adapted for Stereoselective C-H Bond Hydroxylation Using Peroxygenase Activity.

Thermostable protein folds of natural and synthetic origin are highly sought-after templates for biocatalyst generation due to their enhanced stability to elevated temperatures which overcomes one of the major limitations of applying enzymes for synthesis. Cytochrome P450 enzymes (CYPs) are a family of heme-thiolate monooxygenases that catalyse the oxidation of their substrates in a highly stereo- and regio-selective manner. The CYP enzyme (CYP107PQ1) from the thermophilic bacterium Meiothermus ruber binds the norisoprenoid β-ionone and was employed as a scaffold for catalyst design. The I-helix was modified to convert this enzyme from a monooxygenase into a peroxygenase (CYP107PQ1QE), enabling the enantioselective oxidation of β-ionone to (S)-4-hydroxy-β-ionone (94 % e.e.). The enzyme was resistant to 20 mM H2O2, 20 % (v/v) of organic solvent, supported over 1700 turnovers and was fully functional after incubation at 60 °C for 1 h and 30 °C for 365 days. The reaction was scaled-up to generate multi milligram quantities of the product for characterisation. Overall, we demonstrate that sourcing a CYP protein fold from an extremophile enabled the design of a highly stable enzyme for stereoselective C-H bond activation only using H2O2 as the oxidant, providing a viable strategy for future biocatalyst design.

Microbe-aided thermophilic composting accelerates manure fermentation.

Aerobic composting is a key strategy to the sustainable use of livestock manure, which is however constrained by the slow kinetics. Microbe-aided thermophilic composting provides an attractive solution to this problem. In this study, we identified key thermophilic bacteria capable of accelerating manure composting based on the deciphering of manure bacterial community evolution in a thermophilic system. High-throughput sequencing showed a significant evolution of manure bacterial community structure with the increasing heating temperature. Firmicutes were substantially enriched by the heating, particularly some known thermotolerant bacterial species, such as Novibacillus thermophiles, Bacillus thermolactis, and Ammoniibacillus agariperforans. Correspondingly, through function prediction, we found bacterial taxa with cellulolytic and xylanolytic activities were significantly higher in the thermophilic process relative to the initial stage. Subsequently, a total of 47 bacteria were isolated in situ and their phylogenetic affiliation and degradation capacity were determined. Three isolates were back inoculated to the manure, resulting in shortened composting process from 5 to 3 days with Germination Index increased up to 134%, and improved compost quality particularly in wheat growth promoting. Comparing to the mesophilic and thermophilic Bacillus, the genomes of the three isolates manifested some features similar to the thermophiles, including smaller genome size and mutation of specific genes that enhance heat tolerance. This study provide robust evidence that microbe-aided thermophilic composting is capable to accelerate manure composting and improve the quality of compost, which represents a new hope to the sustainable use of manure from the meat industry.

Structure and stability of an apo thermophilic esterase that hydrolyzes polyhydroxybutyrate.

Pollution from plastics is a global problem that threatens the biosphere for a host of reasons, including the time scale that it takes for most plastics to degrade. Biodegradation is an ideal solution for remediating bioplastic waste as it does not require the high temperatures necessary for thermal degradation and does not introduce additional pollutants into the environment. Numerous organisms can scavenge for bioplastics, such as polylactic acid (PLA) or poly-(R)-hydroxybutyrate (PHB), which they can use as an energy source. Recently, a promiscuous PHBase from the thermophilic soil bacterium Lihuaxuella thermophila (LtPHBase) was identified. LtPHBase can accommodate many substrates, including PHB granules and films and PHB block copolymers, as well as the unrelated polymers polylactic acid (PLA) and polycaprolactone (PCL). LtPHBase uses the expected Ser-His-Asp catalytic triad for hydrolysis at an optimal enzyme activity near 70°C. Here, the 1.75 Å resolution crystal structure of apo LtPHBase is presented and its chemical stability is profiled. Knowledge of its substrate preferences was extended to different-sized PHB granules. It is shown that LtPHBase is highly resistant to unfolding, with barriers typical for thermophilic enzymes, and shows a preference for low-molecular-mass PHB granules. These insights have implications for the long-term potential of LtPHBase as an industrial PHB hydrolase and shed light on the evolutionary role that this enzyme plays in bacterial metabolism.

A lab-scale frugal solar bioreactor design for cleaner production of bioproducts from thermophilic bacteria in outdoor conditions

Our study introduces an economical solar-powered bioreactor design for producing a model bioproduct, elucidated as a variant of poly-gamma-glutamic acid, using a thermophilic Bacillus licheniformis in a sterile-compromised growth environment. This frugal solar bioreactor (SB) setup repurposes an out-of-service jacketed stirred tank reactor with a working volume of 5 L and incorporates solar photovoltaic (PV) modules and a solar thermal unit to supply the necessary electrical load (1680 Wh) and heat for carrying out 24-hour batch fermentation at 50 °C. By repurposing the condemned equipments, the cost of SB fabrication was brought down by 59 %. In a batch mode of operation, the solar bioreactor yielded a maximum biopolymer concentration of 19.8 ± 0.9 g/L, which was 14.5 % less than the biopolymer titre obtained from a commercial bioreactor under similar production conditions. The molecular weight (MW) of the biopolymer was found to be dependent on the temperature of fermentation, with a maximum MW of 865.7 kDa achieved at 50 °C. It is envisaged that the proposed solar bioreactor design can be used for the effective production of thermophilic bacterial products suited for various low-cost applications.

Study of biotechnological potential of hydrocarbon-oxidizing bacteria from oil-contaminated soils of the Uzen oil field

Background: Oil-contaminated soils are one of the significant environmental concerns in Western Kazakhstan. Cleaning soils from oil contamination is becoming extremely important. Biological soil treatment, which uses hydrocarbon-oxiding bacteria for microbiological remediation, is a more environmentally friendly and delicate method of soil treatment than any other currently used technology (physical, chemical, biological). One of the important aspects of microbiological remediation is the use of bacteria endomimic to the treated soil. This guarantees the most effective soil purification, as the bacteria perform in relatively optimal conditions for themselves. The soil of the Mangistau region, especially the coastal regions, is distinctive due to its high mineralisation and low moisture content, which have helped to form a specific microflora that is adapted to these conditions. Aim: The study aims to search and isolate highly effective hydrocarbon-oxidizing bacteria that are native to highly mineralized soils of the Uzen oil field. Materials and methods: The research employs a variety of analytical techniques, including water chemistry analysis and infrared spectrometry, as well as the microbiological culture of aerobic bacteria on liquid and dense media in environments (temperature and salinity) that closely resemble their native ecotopes. Results: Four enrichment cultures of hydrocarbon-oxidizing bacteria were obtained, and three pure cultures of oil-oxidizing bacteria were isolated. Their hydrocarbon-oxidizing efficiency has been studied. On the basis of heavy, extremely viscous paraffinic oil from the Uzen field, their hydrocarbon-oxidizing efficiency was examined. Conclusion: In this study, active enrichment cultures of halophilic hydrocarbon-oxidizing bacteria as well as active accumulative cultures of halophilic and moderately thermophilic aerobic bacteria were obtained. These bacteria are capable of oxidising a wide range of hydrocarbons, including high-molecular polycyclic and sulfur-containing compounds. Their high biotechnological potential will be studied in further studies.

Beyond low lignin: Identifying the primary barrier to plant biomass conversion by fermentative bacteria.

Renewable alternatives for nonelectrifiable fossil-derived chemicals are needed and plant matter, the most abundant biomass on Earth, provide an ideal feedstock. However, the heterogeneous polymeric composition of lignocellulose makes conversion difficult. Lignin presents a formidable barrier to fermentation of nonpretreated biomass. Extensive chemical and enzymatic treatments can liberate fermentable carbohydrates from plant biomass, but microbial routes offer many advantages, including concomitant conversion to industrial chemicals. Here, testing of lignin content of nonpretreated biomass using the cellulolytic thermophilic bacterium, Anaerocellum bescii, revealed that the primary microbial degradation barrier relates to methoxy substitutions in lignin. This contrasts with optimal lignin composition for chemical pretreatment that favors high S/G ratio and low H lignin. Genetically modified poplar trees with diverse lignin compositions confirm these findings. In addition, poplar trees with low methoxy content achieve industrially relevant levels of microbial solubilization without any pretreatments and with no impact on tree fitness in greenhouse.

Cooperation of rhamnolipid and thermophilic bacteria modifies proteinic structure, microbial community, and metabolic traits for efficient solubilization and acidogenesis of mariculture solid wastes

Anaerobic fermentation combined with thermophilic bacteria (TB) pretreatment is a promising method to realize effective waste management and carbon resource recovery. However, undesirable properties of high-strength mariculture solid wastes (MSW) such as high solids concentration, excessive salinity and poor bioavailability limited the overall solubilization and acidogenic efficiency. This study innovatively introduced rhamnolipid (RL) to alleviate this adverse effect, and unveiled its cooperation with TB on enhancing organic matter dissolution and volatile fatty acids (VFAs) production. The results showed that VFAs yield from pretreated MSW was improved by 9.4-15.1 folds with enriched acetate (81.4%-94.4%) in the TB+RL groups. The co-pretreatment of RL and TB disintegrated substrate structure for efficient release of electron shuttles and biodegradable organics. This was because introducing RL reconstructed solid-liquid interfacial charge and molecular arrangement, improved thermophilic enzyme activity, and reduced apoptosis and necrosis cells of TB. Substrate bioavailability was further improved with proteinic structure shifted from α-helix and β-sheet to random coil and aggregated strands, and amide II and carboxyl groups interacted with RL molecules. These changes induced the selective enrichment of hydrolytic and acidogenic bacteria, and the upregulated expression of encoding genes responsible for transmembrane transport, protein hydrolysis, carbohydrate metabolism and acetate biosynthesis. This study provides a new strategy to overcome the bottlenecks of acidogenesis from high-strengthen organic wastes and deciphers the underlying mechanism.

Draft genome of Thermoanaerobacter thermohydrosulfuricus strain AK152, a novel thermophilic and anaerobic bacterium isolated from a hot spring in Iceland.

We present the draft genome of the bacterium Thermoanaerobacter thermohydrosulfuricus strain AK152, a thermophilic, endospore-spore-forming, anaerobe isolated from a hot spring in Grensdalur, in Southwestern Iceland. This assembled genome will lay the foundation for identifying the carboxylic and amino acid fermentation pathways, suggesting biotechnological applications for this strain.

Expanded genome and proteome reallocation in a novel, robust Bacillus coagulans strain capable of utilizing pentose and hexose sugars.

Bacillus coagulans, a Gram-positive thermophilic bacterium, is recognized for its probiotic properties and recent development as a microbial cell factory. Despite its importance for biotechnological applications, the current understanding of B. coagulans' robustness is limited, especially for undomesticated strains. To fill this knowledge gap, we characterized the metabolic capability and performed functional genomics and systems analysis of a novel, robust strain, B. coagulans B-768. Genome sequencing revealed that B-768 has the largest B. coagulans genome known to date (3.94 Mbp), about 0.63 Mbp larger than the average genome of sequenced B. coagulans strains, with expanded carbohydrate metabolism and mobilome. Functional genomics identified a well-equipped genetic portfolio for utilizing a wide range of C5 (xylose, arabinose), C6 (glucose, mannose, galactose), and C12 (cellobiose) sugars present in biomass hydrolysates, which was validated experimentally. For growth on individual xylose and glucose, the dominant sugars in biomass hydrolysates, B-768 exhibited distinct phenotypes and proteome profiles. Faster growth and glucose uptake rates resulted in lactate overflow metabolism, which makes B. coagulans a lactate overproducer; however, slower growth and xylose uptake diminished overflow metabolism due to the high energy demand for sugar assimilation. Carbohydrate Transport and Metabolism (COG-G), Translation (COG-J), and Energy Conversion and Production (COG-C) made up 60%-65% of the measured proteomes but were allocated differently when growing on xylose and glucose. The trade-off in proteome reallocation, with high investment in COG-C over COG-G, explains the xylose growth phenotype with significant upregulation of xylose metabolism, pyruvate metabolism, and tricarboxylic acid (TCA) cycle. Strain B-768 tolerates and effectively utilizes inhibitory biomass hydrolysates containing mixed sugars and exhibits hierarchical sugar utilization with glucose as the preferential substrate.IMPORTANCEThe robustness of B. coagulans makes it a valuable microorganism for biotechnology applications; yet, this phenotype is not well understood at the cellular level. Through phenotypic characterization and systems analysis, this study elucidates the functional genomics and robustness of a novel, undomesticated strain, B. coagulans B-768, capable of utilizing inhibitory switchgrass biomass hydrolysates. The genome of B-768, enriched with carbohydrate metabolism genes, demonstrates high regulatory capacity. The coordination of proteome reallocation in Carbohydrate Transport and Metabolism (COG-G), Translation (COG-J), and Energy Conversion and Production (COG-C) is critical for effective cell growth, sugar utilization, and lactate production via overflow metabolism. Overall, B-768 is a novel, robust, and promising B. coagulans strain that can be harnessed as a microbial biomanufacturing platform to produce chemicals and fuels from biomass hydrolysates.

Characterization of the intracellular polyphosphate granules of the phototrophic green sulfur bacterium Chlorobaculum tepidum

The ability to generate polyphosphate (polyP) granules is important for survival for bacteria during resistance to diverse environmental stresses, however the genesis of polyP granules is poorly understood. Chlorobaculum tepidum (Cba tepidum) is a thermophilic green sulfur anoxygenic phototrophic bacterium which uses reduced sulfur compounds as electron donors. The presence of electron rich granules inside the Cba tepidum was reported, but no further information was provided. In this work we used cell thin sections at three different time points of cultivation to observe the biogenesis of the inclusions over time, and the in cell total phosphate concentration was monitored over time as well. Furthermore, the elemental analysis (EDS) of the electron rich inclusions showed the presence of phosphorus and oxygen. The existence of polyphosphate was demonstrated by 31P NMR spectroscopy of cell lysates. Finally, we show that the biogenesis of the phosphorus granules correlates with an abundance of proteins that are closely related to polyphosphate metabolism.

Transcriptomic profiling under poly (L-lactide) polymer degradation of the thermophilic filamentous bacterium Laceyella sacchari LP175 and its potential for polymer film hydrolysis

Polylactide or polylactic acid (PLA) is a hydrolysable polymer used in many applications including medical devices, agricultural films, and packaging materials. Biological methods that completely decompose biodegradable polymers are environmentally beneficial. Laceyella sacchari LP175, a thermophilic filamentous bacterium, had a high potential producing poly (L-lactide) (PLLA)-degrading enzyme which is characterized as a serine protease. This study demonstrated that this strain is an effective degrader of PLLA at a high temperature of 50 °C, which resulted in an increase in enzyme activity within the initial 24 h of incubation. The alterations in the physical and structural characteristics of the PLLA polymer film were verified to occur when incubated with a culture supernatant of this strain. Transcriptomic profiling revealed the differentially expressed genes of Laceyella sacchari LP175 under PLLA degradation, showing 289 and 296 up-regulated and down-regulated genes, respectively. Findings indicated that serine protease and transport-related genes played a key role in the biodegradation of PLLA in Laceyella sacchari LP175. The functions and metabolic pathway annotation of the enriched expression genes showed that propanoate metabolism, protein secretion, and membrane transportation were the main pathways induced when cells were grown in the presence of PLLA powder. Therefore, transcriptome data will be helpful in the development and manipulation of microbial systems to improve the degradation of biodegradable polymers.

Structural studies of β-glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus

β-Glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus (Bgl1) has been denoted as having an attractive catalytic profile for various industrial applications. Bgl1 catalyses the final step of in the decomposition of cellulose, an unbranched glucose polymer that has attracted the attention of researchers in recent years as it is the most abundant renewable source of reduced carbon in the biosphere. With the aim of enhancing the thermostability of Bgl1 for a broad spectrum of biotechnological processes, it has been subjected to structural studies. Crystal structures of Bgl1 and its complex with glucose were determined at 1.47 and 1.95 Å resolution, respectively. Bgl1 is a member of glycosyl hydrolase family 1 (GH1 superfamily, EC 3.2.1.21) and the results showed that the 3D structure of Bgl1 follows the overall architecture of the GH1 family, with a classical (β/α)8 TIM-barrel fold. Comparisons of Bgl1 with sequence or structural homologues of β-glucosidase reveal quite similar structures but also unique structural features in Bgl1 with plausible functional roles.

Secondary metabolites with antimicrobial activity produced by thermophilic bacteria from a high-altitude hydrothermal system.

Thermophilic microorganisms possess several adaptations to thrive in high temperature, which is reflected as biosynthesis of proteins and thermostable molecules, isolation and culture represent a great methodological challenge, therefore High throughput sequencing enables screening of the whole bacterial genome for functional potential, providing rapid and cost-effective information to guide targeted cultures for the identification and characterization of novel natural products. In this study, we isolated two thermophilic bacterial strains corresponding to Bacillus LB7 and Streptomyces LB8, from the microbial mats in the Atacama Desert. By combining genome mining, targeted cultures and biochemical characterization, we aimed to identify their capacity to synthesize bioactive compounds with antimicrobial properties. Additionally, we determined the capability to produce bioactive compounds under controlled in vitro assays and detected by determining their masses by Thin-Layer Chromatography/Mass Spectrometry (TLC/MS). Overall, both isolates can produce antimicrobial (e.g., Myxalamide C by-product) and antioxidants (e.g. Dihydroxymandelic Acid, Amide biotine and Flavone by-products) compounds. Bacillus LB7 strain possesses a more diverse repertoire with 51.95% of total metabolites unmatched, while Streptomyces LB8 favors mainly antioxidants, but has over 70% of unclassified compounds, highlighting the necessity to study and elucidate the structure of novel compounds. Based on these results, we postulate that the uncultured or rare cultured thermophiles inhabiting high-altitude hydrothermal ecosystems in the Atacama Desert offer a promising opportunity to the study of novel microbial bioactive compounds.