Biosynthetic gene clusters with biotechnological applications in novel Antarctic isolates from Actinomycetota.

Actinomycetota have been widely described as valuable sources for the acquisition of secondary metabolites. Most microbial metabolites are produced via metabolic pathways encoded by biosynthetic gene clusters (BGCs). Although many secondary metabolites are not essential for the survival of bacteria, they play an important role in their adaptation and interactions within microbial communities. This is how bacteria isolated from extreme environments such as Antarctica could facilitate the discovery of new BGCs with biotechnological potential. This study aimed to isolate rare Actinomycetota strains from Antarctic soil and sediment samples and identify their metabolic potential based on genome mining and exploration of biosynthetic gene clusters. To this end, the strains were sequenced using Illumina and Oxford Nanopore Technologies platforms. The assemblies were annotated and subjected to phylogenetic analysis. Finally, the BGCs present in each genome were identified using the antiSMASH tool, and the biosynthetic diversity of the Micrococcaceae family was evaluated. Taxonomic annotation revealed that seven strains were new and two were previously reported in the NCBI database. Additionally, BGCs encoding type III polyketide synthases (T3PKS), beta-lactones, siderophores, and non-ribosomal peptide synthetases (NRPS) have been identified, among others. In addition, the sequence similarity network showed a predominant type of BGCs in the family Micrococcaceae, and some genera were distinctly grouped. The BGCs identified in the isolated strains could be associated with applications such as antimicrobials, anticancer agents, and plant growth promoters, among others, positioning them as excellent candidates for future biotechnological applications and innovations. KEY POINTS: • Novel Antarctic rare Actinomycetota strains were isolated from soil and sediments • Genome-based taxonomic affiliation revealed seven potentially novel species • Genome mining showed metabolic potential for novel natural products.

Extremophiles and their expanding biotechnological applications.

Microbial life is not restricted to any particular setting. Over the past several decades, it has been evident that microbial populations can exist in a wide range of environments, including those with extremes in temperature, pressure, salinity, and pH. Bacteria and Archaea are the two most reported types of microbes that can sustain in extreme environments, such as hot springs, ice caves, acid drainage, and salt marshes. Some can even grow in toxic waste, organic solvents, and heavy metals. These microbes are called extremophiles. There exist certain microorganisms that are found capable of thriving in two or more extreme physiological conditions simultaneously, and are regarded as polyextremophiles. Extremophiles possess several physiological and molecular adaptations including production of extremolytes, ice nucleating proteins, pigments, extremozymes and exopolysaccharides. These metabolites are used in many biotechnological industries for making biofuels, developing new medicines, food additives, cryoprotective agents etc. Further, the study of extremophiles holds great significance in astrobiology. The current review summarizes the diversity of microorganisms inhabiting challenging environments and the biotechnological and therapeutic applications of the active metabolites obtained as a response to stress conditions. Bioprospection of extremophiles provides a progressive direction with significant enhancement in economy. Moreover, the introduction to omics approach including whole genome sequencing, single cell genomics, proteomics, metagenomics etc., has made it possible to find many unique microbial communities that could be otherwise difficult to cultivate using traditional methods. These findings might be capable enough to state that discovery of extremophiles can bring evolution to biotechnology.

Microbial recovery of rare earth elements from various waste sources: a mini review with emphasis on microalgae.

Rare Earth Elements (REEs) are indispensable in contemporary technologies, influencing various aspects of our daily lives and environmental solutions. The escalating demand for REEs has led to increased exploitation, resulting in the generation of diverse REE-bearing solid and liquid wastes. Recognizing the potential of these wastes as secondary sources of REEs, researchers are exploring microbial solutions for their recovery. This mini review provides insights into the utilization of microorganisms, with a particular focus on microalgae, for recovering REEs from sources such as ores, electronic waste, and industrial effluents. The review outlines the principles and distinctions of bioleaching, biosorption, and bioaccumulation, offering a comparative analysis of their potential and limitations. Specific examples of microorganisms demonstrating efficacy in REE recovery are highlighted, accompanied by successful methods, including advanced techniques for enhancing microbial strains to achieve higher REE recovery. Moreover, the review explores the environmental implications of bio-recovery, discussing the potential of these methods to mitigate REE pollution. By emphasizing microalgae as promising biotechnological candidates for REE recovery, this mini review not only presents current advances but also illuminates prospects in sustainable REE resource management and environmental remediation.

Clocking out and letting go to unleash green biotech applications in a photosynthetic host.

Cyanobacteria are photosynthetic bacteria whose gene expression patterns are globally regulated by their circadian (daily) clocks. Due to their ability to use sunlight as their energy source, they are also attractive hosts for "green" production of pharmaceuticals, renewable fuels, and chemicals. However, despite the application of traditional genetic tools such as the identification of strong promoters to enhance the expression of heterologous genes, cyanobacteria have lagged behind other microorganisms such as Escherichia coli and yeast as economically efficient cell factories. The previous approaches have ignored large-scale constraints within cyanobacterial metabolic networks on transcription, predominantly the pervasive control of gene expression by the circadian (daily) clock. Here, we show that reprogramming gene expression by releasing circadian repressor elements in the transcriptional regulatory pathways coupled with inactivation of the central oscillating mechanism enables a dramatic enhancement of expression in cyanobacteria of heterologous genes encoding both catalytically active enzymes and polypeptides of biomedical significance.

Biochemical properties of a Flavobacterium johnsoniae dextranase and its biotechnological potential for Streptococcus mutans biofilm degradation.

Cariogenic biofilms have a matrix rich in exopolysaccharides (EPS), mutans and dextrans, that contribute to caries development. Although several physical and chemical treatments can be employed to remove oral biofilms, those are only partly efficient and use of biofilm-degrading enzymes represents an exciting opportunity to improve the performance of oral hygiene products. In the present study, a member of a glycosyl hydrolase family 66 from Flavobacterium johnsoniae (FjGH66) was heterologously expressed and biochemically characterized. The recombinant FjGH66 showed a hydrolytic activity against an early EPS-containing S. mutans biofilm, and, when associated with a α-(1,3)-glucosyl hydrolase (mutanase) from GH87 family, displayed outstanding performance, removing more than 80% of the plate-adhered biofilm. The mixture containing FjGH66 and Prevotella melaninogenica GH87 α-1,3-mutanase was added to a commercial mouthwash liquid to synergistically remove the biofilm. Dental floss and polyethylene disks coated with biofilm-degrading enzymes also degraded plate-adhered biofilm with a high efficiency. The results presented in this study might be valuable for future development of novel oral hygiene products.

Artificial photosynthesis: Promising approach for the efficient production of high-value bioproducts by microalgae.

Recently, microalgae had received extensive attention for carbon capture and utilization. But its overall efficiency still could not reach a satisfactory degree. Artificial photosynthesis showed better efficiency in the conversion of carbon dioxide. However, artificial photosynthesis could generally only produce C1-C3 organic matters at present. Some studies showed that heterotrophic microalgae can efficiently synthesize high value organic matters by using simple organic matter such as acetate. Therefore, the combination of artificial photosynthesis with heterotrophic microalgae culture showed great potential for efficient carbon capture and high-value organic matter production. This article systematically analyzed the characteristics and challenges of carbon dioxide conversion by microalgae and artificial photosynthesis. On this basis, the coupling mode and development trend of artificial photosynthesis combined with microalgae culture were discussed. In summary, the combination of artificial photosynthesis and microalgae culture has great potential in the field of carbon capture and utilization, and deserves further study.

Advancements in Nano-Enhanced microalgae bioprocessing.

Microalgae are promising sources of valuable compounds: carotenoids, polyunsaturated fatty acids, lipids, etc. To overcome the feasibility challenge due to low yield and attain commercial potential, researchers merge technologies to enhance algal bioprocess. In this context, nanomaterials are attractive for enhancing microalgal bioprocessing, from cultivation to downstream extraction. Nanomaterials enhance biomass and product yields (mainly lipid and carotenoids) through improved nutrient uptake and stress tolerance during cultivation. They also provide mechanistic insights from recent studies. They also revolutionize harvesting via nano-induced sedimentation, flocculation, and flotation. Downstream processing benefits from nanomaterials, improving extraction and purification. Special attention is given to cost-effective extraction, showcasing nanomaterial integration, and providing a comparative account. The review also profiles nanomaterial types, including metallic nanoparticles, magnetic nanomaterials, carbon-based nanomaterials, silica nanoparticles, polymers, and functionalized nanomaterials. Challenges and future trends are discussed, emphasizing nanomaterials' role in advancing sustainable and efficient microalgal bioprocessing, unlocking their potential for bio-based industries.

From biomass-derived fructose to γ-valerolactone: Process design and techno-economic assessment.

This work proposes a process design and techno-economic assessment for the production of γ-valerolactone from lignocellulosic derived fructose at industrial scale, with the aim of exploring its feasibility, identifying potential obstacles, and suggesting improvements in the context of France. First, the conceptual process design is developed, the process modelled and optimized. Second, different potential scenarios for the energy supply to the process are analyzed by means of a set of economic key performance indicators, aimed at highlighting the best potential profitability scenario for the sustainable exploitation of waste biomass in the context analyzed. The lowest Minimum Selling Price for GVL is obtained at 10 kt/y plant fueled by biomass, i.e. 1.89 €/kg, along with the highest end-of-live revenue, i.e. 113 M€. Finally, a sensitivity and uncertainties analysis, based on Monte Carlo simulations, are carried out on the results in order to test their robustness with respect to key input parameters.

Kinetic, electrochemical and spectral characterization of bacterial and archaeal rusticyanins; unexpected stability issues and consequences for applications in biotechnology.

Motivated by the ambition to establish an enzyme-driven bioleaching pathway for copper extraction, properties of the Type-1 copper protein rusticyanin from Acidithiobacillus ferrooxidans (AfR) were compared with those from an ancestral form of this enzyme (N0) and an archaeal enzyme identified in Ferroplasma acidiphilum (FaR). While both N0 and FaR show redox potentials similar to that of AfR their electron transport rates were significantly slower. The lack of a correlation between the redox potentials and electron transfer rates indicates that AfR and its associated electron transfer chain evolved to specifically facilitate the efficient conversion of the energy of iron oxidation to ATP formation. In F. acidiphilum this pathway is not as efficient unless it is up-regulated by an as of yet unknown mechanism. In addition, while the electrochemical properties of AfR were consistent with previous data, previously unreported behavior was found leading to a form that is associated with a partially unfolded form of the protein. The cyclic voltammetry (CV) response of AfR immobilized onto an electrode showed limited stability, which may be connected to the presence of the partially unfolded state of this protein. Insights gained in this study may thus inform the engineering of optimized rusticyanin variants for bioleaching processes as well as enzyme-catalyzed solubilization of copper-containing ores such as chalcopyrite.

Novel CRISPR/SpRY system for rapid detection of CRISPR/Cas-mediated gene editing in rice.

The gene editing technology represented by clustered rule-interspersed short palindromic repeats (CRISPR)/Cas9 has developed as a common tool in the field of biotechnology. Many gene-edited products in plant varieties have recently been commercialized. However, the rapid on-site visual detection of gene-edited products without instrumentation remains challenging. This study aimed to develop a novel and efficient method, termed the CRISPR/SpRY detection platform, for the rapid screening of CRISPR/Cas9-induced mutants based on CRISPR/SpRY-mediated in vitro cleavage using rice (Oryza sativa L.) samples genetically edited at the TGW locus as an example. We designed the workflow of the CRISPR/SpRY detection platform and conducted a feasibility assessment. Subsequently, we optimized the reaction system of CRISPR/SpRY, and developed a one-pot CRISPR/SpRY assay by integrating recombinase polymerase amplification (RPA). The sensitivity of the method was further verified using recombinant plasmids. The proposed method successfully identified various types of mutations, including insertions, deletions (indels), and nucleotide substitutions, with excellent sensitivity. Finally, the applicability of this method was validated using different rice samples. The entire process was completed in less than an hour, with a limit of detection as low as 1%. Compared with previous methods, our approach is simple to operate, instrumentation-free, cost-effective, and time-efficient. The primary significance lies in the liberation of our developed system from the limitations imposed using protospacer adjacent motif sequences. This expands the scope and versatility of the CRISPR-based detection platform, making it a promising and groundbreaking platform for detecting mutations induced by gene editing.

Extremophiles in a changing world.

Extremophiles and their products have been a major focus of research interest for over 40 years. Through this period, studies of these organisms have contributed hugely to many aspects of the fundamental and applied sciences, and to wider and more philosophical issues such as the origins of life and astrobiology. Our understanding of the cellular adaptations to extreme conditions (such as acid, temperature, pressure and more), of the mechanisms underpinning the stability of macromolecules, and of the subtleties, complexities and limits of fundamental biochemical processes has been informed by research on extremophiles. Extremophiles have also contributed numerous products and processes to the many fields of biotechnology, from diagnostics to bioremediation. Yet, after 40 years of dedicated research, there remains much to be discovered in this field. Fortunately, extremophiles remain an active and vibrant area of research. In the third decade of the twenty-first century, with decreasing global resources and a steadily increasing human population, the world's attention has turned with increasing urgency to issues of sustainability. These global concerns were encapsulated and formalized by the United Nations with the adoption of the 2030 Agenda for Sustainable Development and the presentation of the seventeen Sustainable Development Goals (SDGs) in 2015. In the run-up to 2030, we consider the contributions that extremophiles have made, and will in the future make, to the SDGs.

This is the Age of Microbial Technology: Crucial roles of learned societies and academies.

Microbial technologies constitute a huge and unique potential for confronting major humanitarian and biosphere challenges, especially in the realms of sustainability and providing basic goods and services where they are needed and particularly in low-resource settings. These technologies are evolving rapidly. Powerful approaches are being developed to create novel products, processes, and circular economies, including new prophylactics and therapies in healthcare, bioelectric systems, and whole-cell understanding of metabolism that provides novel insights into mechanisms and how they can be utilised for applications. The modulation of microbiomes promises to create important applications and mitigate problems in a number of spheres. Collectively, microbial technologies save millions of lives each year and have the potential, through increased deployment, to save many more. They help restore environmental health, improve soil fertility, enable regenerative agriculture, reduce biodiversity losses, reduce pollution, and mitigate polluted environments. Many microbial technologies may be considered to be 'healing' technologies - healing of humans, of other members of the biosphere, and of the environment. This is the Age of Microbial Technology. However, the current exploitation of microbial technologies in the service of humanity and planetary health is woefully inadequate and this failing unnecessarily costs many lives and biosphere deterioration. Microbiologists - the practitioners of these healing technologies - have a special, preordained responsibility to promote and increase their deployment for the good of humanity and the planet. To do this effectively - to actually make a difference - microbiologists will need to partner with key enablers and gatekeepers, players such as other scientists with essential complementary skills like bioengineering and bioinformatics, politicians, financiers, and captains of industry, international organisations, and the general public. Orchestration and coordination of the establishment and functioning of effective partnerships will best be accomplished by learned societies, their academies, and the international umbrella organisations of learned societies. Effective dedication of players to the tasks at hand will require unstinting support from employers, particularly the heads of institutes of higher education and of research establishments. Humanity and the biosphere are currently facing challenges to their survival not experienced for millennia. Effectively confronting these challenges is existential, and microbiologists and their learned societies have pivotal roles to play: they must step up and act now.

Diverse and abundant phages exploit conjugative plasmids.

Phages exert profound evolutionary pressure on bacteria by interacting with receptors on the cell surface to initiate infection. While the majority of phages use chromosomally encoded cell surface structures as receptors, plasmid-dependent phages exploit plasmid-encoded conjugation proteins, making their host range dependent on horizontal transfer of the plasmid. Despite their unique biology and biotechnological significance, only a small number of plasmid-dependent phages have been characterized. Here we systematically search for new plasmid-dependent phages targeting IncP and IncF plasmids using a targeted discovery platform, and find that they are common and abundant in wastewater, and largely unexplored in terms of their genetic diversity. Plasmid-dependent phages are enriched in non-canonical types of phages, and all but one of the 65 phages we isolated were non-tailed, and members of the lipid-containing tectiviruses, ssDNA filamentous phages or ssRNA phages. We show that plasmid-dependent tectiviruses exhibit profound differences in their host range which is associated with variation in the phage holin protein. Despite their relatively high abundance in wastewater, plasmid-dependent tectiviruses are missed by metaviromic analyses, underscoring the continued importance of culture-based phage discovery. Finally, we identify a tailed phage dependent on the IncF plasmid, and find related structural genes in phages that use the orthogonal type 4 pilus as a receptor, highlighting the evolutionarily promiscuous use of these distinct contractile structures by multiple groups of phages. Taken together, these results indicate plasmid-dependent phages play an under-appreciated evolutionary role in constraining horizontal gene transfer via conjugative plasmids.

Use of the Phylobone database for the annotation of bone extracellular matrix proteins in reindeer (Rangifer tarandus).

Bone extracellular matrix (ECM) proteins play a key role in bone formation and regeneration, including structural and regulatory functions. The Phylobone database consists of 255 ECM protein groups from 39 species and can be used to support bone research. Here, we gathered bone ECM proteins from reindeer (Rangifer tarandus), a member of the Cervidae family. The importance of reindeer lies in their ability to regenerate their antlers, in both male and female individuals. Protein sequences were extracted from the National Center for Biotechnology Information's repository and selected by homology searches. We identified 215 proteins and their corresponding functional domains, which are putatively present in the bone ECM of reindeer. Protein sequence alignments have shown a high degree of conservation between R. tarandus and other members of the Cervidae family. This update expands the Phylobone database and shows that it is a useful resource for the preliminary annotation of bone ECM proteins in novel proteomes.

Identification of transporters involved in aromatic compounds tolerance through screening of transporter deletion libraries.

Aromatic compounds are used in pharmaceutical, food, textile and other industries. Increased demand has sparked interest in exploring biotechnological approaches for their sustainable production as an alternative to chemical synthesis from petrochemicals or plant extraction. These aromatic products may be toxic to microorganisms, which complicates their production in cell factories. In this study, we analysed the toxicity of multiple aromatic compounds in common production hosts. Next, we screened a subset of toxic aromatics, namely 2-phenylethanol, 4-tyrosol, benzyl alcohol, berberine and vanillin, against transporter deletion libraries in Escherichia coli and Saccharomyces cerevisiae. We identified multiple transporter deletions that modulate the tolerance of the cells towards these compounds. Lastly, we engineered transporters responsible for 2-phenylethanol tolerance in yeast and showed improved 2-phenylethanol bioconversion from L-phenylalanine, with deletions of YIA6, PTR2 or MCH4 genes improving titre by 8-12% and specific yield by 38-57%. Our findings provide insights into transporters as targets for improving the production of aromatic compounds in microbial cell factories.

Bacterial extracellular vesicles: biotechnological perspective for enhanced productivity.

Bacterial extracellular vesicles (BEVs) are non-replicative nanostructures released by Gram-negative and Gram-positive bacteria as a survival mechanism and inter- and intraspecific communication mechanism. Due to BEVs physical, biochemical, and biofunctional characteristics, there is interest in producing and using them in developing new therapeutics, vaccines, or delivery systems. However, BEV release is typically low, limiting their application. Here, we provide a biotechnological perspective to enhance BEV production, highlighting current strategies. The strategies include the production of hypervesiculating strains through gene modification, bacteria culture under stress conditions, and artificial vesicles production. We discussed the effect of these production strategies on BEVs types, morphology, composition, and activity. Furthermore, we summarized general aspects of BEV biogenesis, functional capabilities, and applications, framing their current importance and the need to produce them in abundance. This review will expand the knowledge about the range of strategies associated with BEV bioprocesses to increase their productivity and extend their application possibilities.

Advances in biotin biosynthesis and biotechnological production in microorganisms.

Biotin, also known as vitamin H or B7, acts as a crucial cofactor in the central metabolism processes of fatty acids, amino acids, and carbohydrates. Biotin has important applications in food additives, biomedicine, and other fields. While the ability to synthesize biotin de novo is confined to microorganisms and plants, humans and animals require substantial daily intake, primarily through dietary sources and intestinal microflora. Currently, chemical synthesis stands as the primary method for commercial biotin production, although microbial biotin production offers an environmentally sustainable alternative with promising prospects. This review presents a comprehensive overview of the pathways involved in de novo biotin synthesis in various species of microbes and insights into its regulatory and transport systems. Furthermore, diverse strategies are discussed to improve the biotin production here, including mutation breeding, rational metabolic engineering design, artificial genetic modification, and process optimization. The review also presents the potential strategies for addressing current challenges for industrial-scale bioproduction of biotin in the future. This review is very helpful for exploring efficient and sustainable strategies for large-scale biotin production.

Role of oxalic acid in fungal and bacterial metabolism and its biotechnological potential.

Oxalic acid and oxalates are secondary metabolites secreted to the surrounding environment by fungi, bacteria, and plants. Oxalates are linked to a variety of processes in soil, e.g. nutrient availability, weathering of minerals, or precipitation of metal oxalates. Oxalates are also mentioned among low-molecular weight compounds involved indirectly in the degradation of the lignocellulose complex by fungi, which are considered to be the most effective degraders of wood. The active regulation of the oxalic acid concentration is linked with enzymatic activities; hence, the biochemistry of microbial biosynthesis and degradation of oxalic acid has also been presented. The potential of microorganisms for oxalotrophy and the ability of microbial enzymes to degrade oxalates are important factors that can be used in the prevention of kidney stone, as a diagnostic tool for determination of oxalic acid content, as an antifungal factor against plant pathogenic fungi, or even in efforts to improve the quality of edible plants. The potential role of fungi and their interaction with bacteria in the oxalate-carbonate pathway are regarded as an effective way for the transfer of atmospheric carbon dioxide into calcium carbonate as a carbon reservoir.

Size-Selective Capturing of Exosomes Using DNA Tripods.

Fractionating and characterizing target samples are fundamental to the analysis of biomolecules. Extracellular vesicles (EVs), containing information regarding the cellular birthplace, are promising targets for biology and medicine. However, the requirement for multiple-step purification in conventional methods hinders analysis of small samples. Here, we apply a DNA origami tripod with a defined aperture of binders (e.g., antibodies against EV biomarkers), which allows us to capture the target molecule. Using exosomes as a model, we show that our tripod nanodevice can capture a specific size range of EVs with cognate biomarkers from a broad distribution of crude EV mixtures. We further demonstrate that the size of captured EVs can be controlled by changing the aperture of the tripods. This simultaneous selection with the size and biomarker approach should simplify the EV purification process and contribute to the precise analysis of target biomolecules from small samples.