Microbial Biotechnology最新文献

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.

Neurotropic viral infections continue to pose significant global health challenges, with pathogens such as herpes simplex virus (HSV), varicella-zoster virus, human immunodeficiency virus, poliovirus, enteroviruses, parechovirus, West Nile virus and Japanese encephalitis virus driving the search for more effective therapeutic interventions. Current antiviral strategies, including small molecules and monoclonal antibodies, often face limitations such as drug resistance, narrow spectrum activity and adverse side effects, underscoring the need for alternative approaches. Antiviral peptides are emerging as potential therapeutic agents against these viral infections as entry and fusion inhibitors. However, their clinical development is limited by poor stability, low bioavailability and insufficient cellular penetration. To address these limitations, peptide stapling, a chemical modification that stabilises peptide α-helices through covalent linkage, has emerged as a transformative technique to enhance the therapeutic potential of peptides, especially in antiviral drug development. Stapling techniques, including hydrocarbon staples, lactam bridges and metal-coordination bonds, are explored for their ability to improve peptide stability, bioavailability and target binding affinity. This review examines the application of stapling in the development of antiviral peptides with a focus on stapled peptides targeting viral fusion and entry mechanisms, highlighting their potential against neurotropic viruses such as HSV and influenza. By integrating the structural rigidity conferred by stapling, these constructs promise to overcome delivery barriers and achieve superior antiviral efficacy. This paper underscores the pivotal role of peptide stapling by highlighting recent advancements in antiviral therapeutics and presents a roadmap for future research into multifunctional stapled peptides.

Autoinducer-2 (AI-2) is a key signalling molecule that in many bacteria facilitates interspecies communication by regulating gene expression in response to population density through a process known as quorum sensing. While this signalling mechanism has been extensively studied in Gram-negative bacteria, its role in the genus Bifidobacterium remains poorly understood. In this study, an in silico analysis was conducted to examine the distribution of the luxS gene, which encodes the enzyme that synthesises the AI-2 precursor, across Bifidobacterium genomes. Our analysis revealed that luxS is ubiquitously present in all publicly available bifidobacterial genomes. To explore the functional implications of luxS, gene expression profiling was performed on the model strain B. bifidum PRL2010 and its isogenic luxS insertion mutant, both grown in a medium simulating the human gut environment. RNA sequencing results indicated that disruption of luxS impairs the mutant strain's ability to (i) interact and communicate with the host, (ii) transport sugars, (iii) internalise potassium and iron, and (iv) cope with stress conditions. Collectively, these findings highlight the crucial role of AI-2 in promoting colonisation and ensuring the persistence of PRL2010 within the competitive ecosystem of the human gut.

This study reports the efficacy of a rationally designed Pseudomonas putida strain to bring about the specific removal of S atoms from dibenzothiophene (DBT), the model heterocyclic sulfur-containing component of raw petroleum. The emphasis on DBT as a model compound stems from its prevalence in fossil fuels and its resistance to hydrodesulfurization, which positions it as a critical target for improving biodesulfurization technologies. To this end, we explored the combinatorial space of the dsz operon of the naturally occurring strain Rhodococcus qingshengii IGTS8—known to achieve dibenzothiophene degradation—by re-engineering the native regulation of the operon, generating permutations of the order of the cognate genes and their ribosomal-binding sites, testing the effects of multicopy versus monocopy doses and introducing the resulting constructs in the tailor-made host. The combination that emerged as best in terms of catalytic efficacy, moderate physiological burden, and durability was one in which the original dsz operon was refactored by [i] reordering its native gene order to dszBCA, [ii] decompressing their naturally occurring translational coupling with optimised ribosomal-binding sites, [iii] engineering its constitutive expression with a heterologous promoter and [iv] inserting the thereby refactored pathway in the Tn7 site of the genome-edited strain P. putida EM384, which is optimised for greater stability and hosting harsh redox reactions. The resulting P. putida DS006 exhibited exceptional DBT desulfurization activity as well as efficiency in model biphasic biodesulfurization systems.

The seed microbiota, a still underexplored component of plant–microbe interactions, plays a pivotal role in plant development and holds significant promise for advancing sustainable agriculture. By influencing essential processes such as germination, stress tolerance, nutrient acquisition and defence, seed-associated microbes offer unique advantages beyond those of soil- or rhizosphere-associated microbiomes. Notably, they are transmitted both vertically and horizontally; however, fundamental questions remain regarding their origin, ecological dynamics and functional roles across environments. This article explores the diversification of seed microbiota as a consequence of crop domestication, emerging insights into functional microbial genes and key challenges that must be addressed to fully unlock its potential.

The Brucella abortus A19 attenuated live vaccine poses potential infection risks during practical applications and interferes with serological diagnostics, thereby affecting quarantine measures and the establishment of disease-free zones. Consequently, this study aimed to reduce its potential virulence, enhance its protective efficacy and differentiate it from wild-type strains by knocking out the immunosuppressive virulence gene btpB in the A19 strain. Using homologous recombination, we successfully obtained the A19ΔBtpB deletion strain. In a mouse model, the A19ΔBtpB strain demonstrated improved safety and significantly increased TLR2 and TLR4 expression levels in splenic lymphocytes, suggesting attenuated immune suppression. The A19ΔBtpB strain induced Brucella-specific IgG antibodies comparable to those of the A19 strain but exhibited superior performance in promoting cellular immunity. It effectively induced Th1-type cytokine (IL-6 and TNF-α) production, reduced Th2-type cytokine (IL-4 and IL-10) expression and promoted IFN-γ expression in T lymphocytes. Notably, the A19ΔBtpB deletion strain provided better protection against the virulent M28 strain in mice than did the A19 strain. In addition, an indirect ELISA diagnostic method based on BtpB protein has been developed, effectively distinguishing vaccine and wild-type strains in the infection of Brucella. The A19ΔBtpB strain thus represents a promising candidate for a Brucella gene-deletion vaccine, offering both theoretical and empirical support for future vaccine development.

The Brucella abortus A19 attenuated live vaccine poses potential infection risks during practical applications and interferes with serological diagnostics, thereby affecting quarantine measures and the establishment of disease-free zones. Consequently, this study aimed to reduce its potential virulence, enhance its protective efficacy and differentiate it from wild-type strains by knocking out the immunosuppressive virulence gene btpB in the A19 strain. Using homologous recombination, we successfully obtained the A19ΔBtpB deletion strain. In a mouse model, the A19ΔBtpB strain demonstrated improved safety and significantly increased TLR2 and TLR4 expression levels in splenic lymphocytes, suggesting attenuated immune suppression. The A19ΔBtpB strain induced Brucella-specific IgG antibodies comparable to those of the A19 strain but exhibited superior performance in promoting cellular immunity. It effectively induced Th1-type cytokine (IL-6 and TNF-α) production, reduced Th2-type cytokine (IL-4 and IL-10) expression and promoted IFN-γ expression in T lymphocytes. Notably, the A19ΔBtpB deletion strain provided better protection against the virulent M28 strain in mice than did the A19 strain. In addition, an indirect ELISA diagnostic method based on BtpB protein has been developed, effectively distinguishing vaccine and wild-type strains in the infection of Brucella. The A19ΔBtpB strain thus represents a promising candidate for a Brucella gene-deletion vaccine, offering both theoretical and empirical support for future vaccine development.