Microbial Biotechnology最新文献

The cytochrome P450 monooxygenase enzymes (CYPs) of the CYP102 family are versatile, self-sufficient biocatalysts. The archetypal example is CYP102A1 (P450_BM3) from the bacterium Bacillus (Priestia) megaterium, and variants of this enzyme can oxidise many substrates with high activity and selectivity. However, this enzyme has relatively low thermal stability. Here, we identify and characterise a CYP102 family enzyme from the moderately thermophilic bacterium Thermosporothrix hazakensis. We were able to produce this enzyme using Escherichia coli and demonstrate the in vivo oxidation of fatty acids. However, the activity of the isolated holoenzyme was low, so we generated a peroxygenase variant by introducing the E278Q and T279E mutations into the heme domain (‘HazakQE’). This isolated variant was able to catalyse the oxidation of a range of substrates using hydrogen peroxide as the oxidant. The product distributions arising from fatty acid oxidation using the holoprotein monooxygenase and heme domain peroxygenase variants of this enzyme were broadly similar to those obtained with P450_BM3. For fatty acids, the oxidation occurred predominantly at the ω-1 through to ω-3 positions. Styrene was epoxidised and tetralone hydroxylated at the benzylic carbon. The oxidation of 1-methoxynaphthalene generated the dimeric Russig's blue, enabling colorimetric assays of the enzyme activity. Although the HazakQE heme peroxygenase was more thermostable than the mesophilic CYP199A4 enzyme from Rhodopseudomonas palustris, it was not more resistant to heating than the heme domain of P450_BM3. These peroxygenase variants offer a simple platform for metabolite identification and biocatalysts for oxidation reactions, which could be enhanced through protein engineering.

Microbial and biochemical research has historically focused on pathogenic agents due to their clear association with disease. This is a perspective that has saved countless lives but encourages a skewed, threat-centered view of microbes and biogenic compounds. Emerging evidence shows that exposure to diverse environmental microbiomes and natural biochemical products is also salutogenic—promoting health and resilience. Here we introduce the ‘Database of Salutogenic Potential’, a prototype relational repository cataloguing environmental microbes and biochemical compounds linked to health benefits. Drawing from more than 200 articles, we identified 124 potentially salutogenic microbial taxa, 14 biochemical compounds and 63 associated benefits. By creating a structured and open platform, we aim to shift the balance between pathogen-centric and salutogenic perspectives, potentially enabling future applications in public health, urban planning and ecosystem restoration. While the current iteration of the database primarily centers on human health outcomes, it is designed to expand into ecosystem health domains, embedding salutogenic thinking into One Health frameworks. We present this as a first step, not a ready-to-use tool, and invite collaborative refinement from the scientific community.

Microbial and biochemical research has historically focused on pathogenic agents due to their clear association with disease. This is a perspective that has saved countless lives but encourages a skewed, threat-centered view of microbes and biogenic compounds. Emerging evidence shows that exposure to diverse environmental microbiomes and natural biochemical products is also salutogenic—promoting health and resilience. Here we introduce the ‘Database of Salutogenic Potential’, a prototype relational repository cataloguing environmental microbes and biochemical compounds linked to health benefits. Drawing from more than 200 articles, we identified 124 potentially salutogenic microbial taxa, 14 biochemical compounds and 63 associated benefits. By creating a structured and open platform, we aim to shift the balance between pathogen-centric and salutogenic perspectives, potentially enabling future applications in public health, urban planning and ecosystem restoration. While the current iteration of the database primarily centers on human health outcomes, it is designed to expand into ecosystem health domains, embedding salutogenic thinking into One Health frameworks. We present this as a first step, not a ready-to-use tool, and invite collaborative refinement from the scientific community.

Historically, art and science have often been viewed as distinct disciplines, each with its own methodologies and modes of expression. However, a closer examination reveals a rich and complex web of interplay between the two, where scientific inquiry and artistic creativity converge to explore and interpret the natural world. In this article, we dig into the flourishing field of microbial art, with a particular focus on the utilisation of extremophilic microorganisms – organisms that thrive in conditions once deemed uninhabitable – as both objects and subjects in contemporary artistic practices. Tracing the lineage from early intersections between these two fields to modern pioneers, we highlight how microorganisms have transitioned from subjects of scientific study to integral components of artistic creation. Through case studies, we illustrated how the unique properties of extremophiles – such as their pigmentation, resilience, and metabolic capabilities – offer novel avenues for artistic exploration. Furthermore, we emphasised the reciprocal benefits of interdisciplinary collaborations between artists and scientists. In an era marked by environmental challenges and the proliferation of misinformation, the fusion of art and science emerges as a compelling strategy to promote public understanding and appreciation of complex scientific phenomena, serving also as potent tools for science communication and outreach.

Biogas inocula with distinct taxonomic compositions often converge to similar communities when fed the same substrate, indicating strong substrate-driven deterministic assembly. Nevertheless, stochastic processes have also been suggested as a critical element for microbial assembly in biogas systems. To date, assembly processes have mainly been investigated with undefined, non-sterile substrates, making it difficult to exclude the influence of external microorganisms. The aim of the present study was to investigate whether three taxonomically distinct anaerobic digestion (AD) communities would converge when exposed to uniform growth conditions during semi-continuous operation with a sterilised defined medium. The inocula originated from mesophilic processes using different substrates (food waste, sludge, and manure) and total ammonia levels (0.5–7.2 g/L). The medium was formulated to support all four main metabolic steps of AD: hydrolysis, fermentation, anaerobic oxidation, and methanogenesis. Taxonomic, phylogenetic, and functional analyses conducted via 16S and metagenomic sequencing showed that the substrate had no deterministic effect on microbial community taxonomic composition. Instead, the final community structure was dictated primarily by the initial inoculum, regardless of changes in substrate composition or ammonia levels. Despite taxonomic divergence, broad-level functionality and operational performance remained similar between communities.

Host defence peptides (HDPs) represent a valuable class of antimicrobial agents with the potential to address the growing threat of antimicrobial resistance (AMR). Here, we have studied recombinant constructs based on a combination of HDPs fused to the GFP protein and multidomain proteins combining three or four HDPs in a single polypeptide, referred to as first and second generation antimicrobials, respectively. These recombinant peptides were tested against Gram-positive and Gram-negative bacteria associated with healthcare infections. In addition, in silico studies provided insight into the antimicrobial structure–activity relationships of these biomolecules. For the first generation of antimicrobials, amphipathicity mainly explains the average antimicrobial activity against the Gram-positive strains. In the case of the Gram-negative bacteria, it depends on the quantity and the exposed area of the Ser and Thr amino acids. For the second generation of antimicrobials, the order of domains is crucial to act against Gram-positive strains, preferably by positioning the most bioactive domain against the Gram-positive pathogen at the ends.

The mechanisms of antioxidant action of lactic acid bacteria (LAB) have not been fully explained. This review aimed to characterise the antioxidant properties that can be presented by LAB strains isolated from food. The review presents a definition and classification of the antioxidants, mechanisms of antioxidant action of LAB, discusses the most popular antioxidant assays, taking into account the mechanisms underlying each test and the practice of assessing antioxidant capacity, and presents examples of studies of food-derived LAB and fermented food with antioxidant properties. LAB are an important part of the human microbiota, and their role in antioxidant processes is extremely important. They can respond quickly and effectively to free radicals by enhancing antioxidant activity, chelating metal ions, producing antioxidant enzymes and other metabolites, and thus mitigating the damage caused by oxidative stress. This review also presents methods for testing antioxidant properties that can be used for LAB screening. The most commonly used methods are the classical methods of testing antioxidant activity, such as DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), or FRAP (Ferric Reducing Antioxidant Power) assays. We recommend using at least three different assays. It is important to consider whether to test live or inactivated cells, post-culture supernatant, cell lysates, protein fractions or purified exopolysaccharides. In conclusion, due to their properties, lactic acid bacteria strains may prove to be an interesting and natural alternative to synthetic antioxidants used in food production. Lactic acid bacteria have been shown to be not only useful as microorganisms that support the proper functioning of the digestive tract or as probiotics, but also allow their antioxidant properties to be noticed and strengthen the defence against oxidative stress.

Pseudomonas syringae is a plant pathogen complex responsible for bacterial canker in cherry. In the absence of any control measures, bacteriophages (phages) have the potential for biocontrol. However, it is crucial to first evaluate the role of bacterial antiviral defence systems (ADS) in phage infection dynamics for careful design of a phage cocktail (mixture). Investigating 250 Pseudomonas strains revealed the Ps complex possessed diverse ADS with defence profiles being influenced by phylogeny. Phage host range assays revealed five MR phages with distinct genotypes possessed strong lytic activity against several bacterial canker-causing Ps pathovars, including syringae and morsprunorum race 1 and 2. Phage susceptibility and resistance appeared to be associated with individual ADS rather than defence profiles as a whole. Multisequence alignment of lipopolysaccharide biosynthesis genes glucose-1-phosphate thymidylyltransferase (gpt), glycosyltransferase family 1 (gst1) and lipopolysaccharide kinase (lpk) found these potential receptor genes to be highly conserved within Ps phylogroups. However, gpt alone appeared to influence phage infectivity. Our findings indicate that the gpt gene is a potential primary predictor of MR phage susceptibility, hypothesised to influence phage absorption, while individual ADS only have a secondary role in phage resistance. This study highlights that understanding the genetic mechanisms underlying phage-bacterial interactions is crucial for designing more effective phage cocktails capable of targeting a broader range of pathogenic strains, but phage screening still is a powerful tool to select phages for biocontrol treatments.

The demand for strong and easily inducible promoters to produce heterologous proteins in Saccharomyces cerevisiae has attracted considerable attention in the last years. In this organism, alkalinisation triggers a wide and well-characterised transcriptional response that includes activation of the calcium–dependent calcineurin-Crz1 and the phosphate-responsive PHO pathways. Here, we present the construction of random libraries containing multiple combinations of Crz1- and Pho4-binding sequences, and we show that these elements are able to promote efficient expression of GFP by simple addition of KOH to the medium. The expression in Crz1 or Pho4-deficient cells allowed us to define the relative contribution of these elements to GFP production. We also show that the addition of a single copy of a 60-bp fragment of the ENA1 promoter containing an Stp1/2 site further enhances expression. Finally, we demonstrate that these constructs drive strong expression of secretable laccase, an enzyme of industrial interest in processing lignin biopolymers, and that the level of expression can be adjusted by modifying the pH of the medium. In conclusion, our work presents a novel expression system whose induction is simple, cheap, and easy to monitor, and that could be an attractive alternative to current expression platforms for both research and industrial protein production purposes.

Bacterial Rieske non-heme iron oxygenases catalyse the transformation of a wide range of aromatic compounds to vicinal cis-dihydrodiols. Such compounds have been successfully applied in chemoenzymatic synthetic routes for, for example, pharmaceuticals, natural products and polymers. In the case of benzoate, only (1S,2R)-cis-1,2-dihydroxy-2-hydrobenzoate is readily accessible via enzymatic transformation, but not the regioisomeric cis-2,3-dihydroxy-2,3-dihydrobenzoate (2,3-DD) or cis-3,4-dihydroxy-3,4-dihydrobenzoate. While trace amounts of putative cis-2,3-DD have been obtained before by using p-cumate 2,3-dioxygenase (PCDO) or a combination of chlorobenzene dioxygenase and nitrilase, none of these approaches enabled its production and isolation at a greater scale for potential use as a chiral building block in organic synthesis. We here provide a protocol for biotransformation of benzoate yielding (2R,3S)-2,3-dihydroxy-2,3-dihydrobenzoate using the PCDO of Pseudomonas citronellolis strain EB200 with negligible formation of side products. An isolation procedure suitable for production of the 2,3-DD sodium salt monohydrate at high purity (> 95%) at a gram scale, and a comprehensive characterisation of this novel metabolite is given.