Microbial Biotechnology最新文献

Immunoassays represent sensitive, easy-to-use, and cost-effective tests useful for the detection of the SARS-CoV-2 virus. In this manuscript, we report on the binding specificity of a pair of novel monoclonal antibodies (MAbs) generated against the SARS-CoV-2 nucleocapsid protein (NP) and their development into sensitive sandwich enzyme-linked immunosorbent assays (sELISA) and a lateral flow immunoassay (LFIA). Binding of these MAbs to hCoVs is limited to variants of SARS-CoV-2 and SARS-CoV NP. Chemiluminescent and absorbance spectroscopy sELISAs report a limit of detection (LOD) for the SARS-CoV-2 B.1.1.529 NP variant at 15 pg/mL, and the LFIA using a red-dyed 200 nm particle at 10 ng/mL. The sELISA exhibits broad SARS-CoV-2 viral variant detection with assay LOD for SARS-CoV-2 B.1.1.529 virus at 1.4 × 10⁵ genome copies per mL (p ≤ 0.001). The availability of these MAbs should facilitate continued investment in the commercial development of immunoassays to increase global SARS-CoV-2 detection technologies.

To address the on-going need for chemical novelty and the limited information on Planctomycetota secondary metabolism, we focused on exploring the recently isolated marine planctomycetal strain Alienimonas chondri LzC2^T to uncover its potential production of novel compounds. This work contemplates the description of a large-scale cultivation study of strain LzC2^T, followed by metabolite extraction and compound isolation using chromatographic approaches, which resulted in the isolation of a novel molecule designated as alichondrichlorin. Structural elucidation of this new molecule was accomplished by a combination of high-resolution mass spectrometry and nuclear magnetic resonance. The molecule was additionally screened for anti-proliferative bioactivity against human tumoral and non-tumoral cell lines. These cytotoxicity assays revealed a targeted effect of alichondrichlorin in the growth of tumoral cell lines, especially human breast adenocarcinoma MCF-7 cell line (EC₅₀ = 4.06 μM) without effect on the human non-tumoral THLE-2 cell line (EC₅₀ > 50 μM).

This study investigated whether two exopolysaccharides could serve as exogenous carbon sources to enhance fermentation quality in oat silage, providing a theoretical foundation for their future application in silage. The oats were harvested at the heading stage and, following a period of wilting, were chopped into 2–3 cm lengths for the ensiling experiment. The treatments applied were as follows: (1) a control group (CK), which received only sterile water; (2) a group with added dextran (D); and (3) a group with added levan (L). The fermentation process was monitored at various intervals: 3, 7, 14, 30 and 60 days (d), respectively. Following 60 days of ensiling, the silage was subjected to a 5-day period of aerobic exposure (AE). EPS changed the fermentation quality of silage, altered the composition of the bacterial community, and had an impact on the feature dissimilarity between sample groups. Meanwhile, EPS showed different regulatory effects on carbohydrate metabolism at different fermentation times. EPS treatment increased the lactic acid content and decreased the pH of silage. After 60 days of fermentation, the treatment also increased the relative abundance of Lactobacillus. Dextran and levan increased the relative abundance of Hafnia–Obesumbacterium and Sediminibacterium, respectively. Under the treatment of dextran, silage retained more WSC content and achieved higher aerobic stability. Upon comparing the bacterial correlation networks, it became evident that the fermentation time altered the composition of inter-bacterial correlations. In conclusion, EPS can effectively enhance the fermentation quality of oat silage, with dextran yielding the most pronounced positive effects.

Accurate strain identification is essential for economically significant fungi, as it aids in understanding their diverse agronomic traits, pathogenicity, and other important characteristics. However, traditional methods often face challenges related to limited accuracy, high workloads, and reproducibility issues. Recently, multiple nucleotide polymorphism (MNP) markers have been employed in mushroom strain identification, demonstrating significantly improved accuracy and reproducibility. Nevertheless, the identification of strains across different species still heavily depends on specific and often overly complex MNP markers. In this study, we address these challenges by developing a novel method for constructing high-resolution phylogenomic topologies using core gene-associated multiple nucleotide polymorphism (cgMNP) markers, focusing on Agaricus bisporus (button mushroom). Utilising resequencing data from 213 cultivated and wild strains of A. bisporus, we identified 84 cgMNP markers within 83 core genes from 1011 MNP markers. Phylogenetic analysis based on cgMNP sequences and the genetic distance between strain pairs allowed for precise identification of all strains. Moreover, the successful transferability of these cgMNP markers to an additional 385 A. bisporus strains and other fungal species, including Flammulina filiformis (enoki mushroom) and Saccharomyces cerevisiae (yeast), highlights their cross-species applicability. The high resolution and strong congruence of cgMNP markers with whole-genome data provide a robust and reliable method for strain-level discrimination in fungi. The success of this approach in A. bisporus sets a promising precedent for its application to a broader range of fungal taxa.

The limited number of well-characterised model bacteria cannot address all the challenges in a circular bioeconomy. Therefore, there is a growing demand for new production strains with enhanced resistance to extreme conditions, versatile metabolic capabilities and the ability to utilise cost-effective renewable resources while efficiently generating attractive biobased products. Particular thermophilic microorganisms fulfil these requirements. Non-virulent Gram-negative Caldimonas thermodepolymerans DSM15344 is one such attractive thermophile that efficiently converts a spectrum of plant biomass sugars into high quantities of polyhydroxyalkanoates (PHA)—a fully biodegradable substitutes for synthetic plastics. However, to enhance its biotechnological potential, the bacterium needs to be ‘domesticated’. In this study, we established effective homologous recombination and transposon-based genome editing systems for C. thermodepolymerans. By optimising the electroporation protocol and refining counterselection methods, we achieved significant improvements in genetic manipulation and constructed the AI01 chassis strain with improved transformation efficiency and a ΔphaC mutant that will be used to study the importance of PHA synthesis in Caldimonas. The advances described herein highlight the need for tailored approaches when working with thermophilic bacteria and provide a springboard for further genetic and metabolic engineering of C. thermodepolymerans, which can be considered the first model of thermophilic PHA producer.

Microbes constitute a ubiquitous warp, a highly sensitive skin of the biosphere that can be scratched and damaged by all human activities. However, the existence of life in general and the human species, in particular, depends on the intelligent preservation of such a biological microbiological cement linking our health with the health of Earth. We are responsible for maintaining sustainable health by managing our damaging individual and social behaviour, and we are also charged with the duty of correcting the microbial disequilibrium we are provoking. The harmful secondary effects resulting from the nature of the species Homo sapiens are frequently neglected. However, sustainable health by microbial causes depends on our individual and social psychology. The role of individual psychology, social behaviour (including the ‘tragedy of the commons’), based on collective psychology, culture, values and social norms, and the influence on sustainable health of the methodology of research and management of interventions are briefly analysed. As a general antidote to our unavoidable natural stultified behaviour, education in science is the only possibility to counteract mistakes and restore human dignity.

Imazalil (IMZ), a major surface water contaminant characterised by high environmental recalcitrance and toxicity, is used in fruit-packaging plants to control fungal infestations during storage. This leads to the production of wastewaters which should be treated on site before their environmental release. We previously isolated a Cladosporium herbarum strain, the first microorganism that could degrade IMZ. Here we describe the genetic network utilised by the fungus to degrade IMZ and its detailed transformation. Genomic and transcriptomic analysis of C. herbarum pointed to the involvement of strongly upregulated CYP450s in IMZ degradation, as further verified by cessation of its biodegradation by CYP450 inhibitors. LC-QTOF-HRMS analysis and suspect/non-target screening identified nine transformation products (TPs) of IMZ. IMZ biotransformation mainly proceeded through O-dealkylation, while other less important paths, most probably controlled by upregulated oxidases, were operative involving successive hydroxylation reactions. These lead to the formation of TPs like IMZ_313 and IMZ_331, with the former being further transformed through imidazole ring scission to IMZ_288, a TP reported for the first time. We provide first evidence for the transformation mechanism of IMZ by C. herbarum and the potential genes/enzymes involved, paving the way for the use of C. herbarum in the biodepuration of agro-industrial effluents.

Mesophotic coral ecosystems (MCEs) host a diverse array of sponge species, which represent a promising source of bioactive compounds. Increasing evidence suggests that sponge-associated bacteria may be the primary producers of these compounds. However, cultivating these bacteria under laboratory conditions remains a significant challenge. To investigate the rich resource of bioactive compounds synthesised by mesophotic sponge-associated bacteria, we retrieved 429 metagenome-assembled genomes (MAGs) from 15 mesophotic sponges, revealing a strong correlation between bacterial diversity and sponge species. Furthermore, we identified 1637 secondary metabolite biosynthetic gene clusters (BGCs) within these MAGs. Among the identified BGCs, terpenes were the most abundant (495), followed by 369 polyketide synthases (PKSs), 293 ribosomally synthesised and post-translationally modified peptides (RiPPs) and 135 nonribosomal peptide synthetases (NRPSs). The BGCs were classified into 1086 gene cluster families (GCFs) based on sequence similarity. Notably, only five GCFs included experimentally validated reference BGCs from the Minimum Information about a Biosynthetic Gene cluster database (MIBiG). Additionally, an unusual abundance of BGCs was detected in Entotheonella sp. (s191209.Bin93) from the Tectomicrobia phylum. In contrast, members of Proteobacteria and Acidobacteriota harboured fewer BGCs (6–7 on average), yet their high abundance in MCE sponges suggests a potentially rich reservoir of BGCs. Analysis of the BGC distribution patterns revealed that a subset of BGCs, including terpene GCFs (FAM_00447 and FAM_01046), PKS GCF (FAM_00235), and RiPPs GCF (FAM_01143), were widespread across mesophotic sponges. Furthermore, 32 GCFs were consistently present in the same MAGs across different sponges, highlighting their potential key biological roles and capacity to yield novel bioactive compounds. This study not only underscores the untapped potential of mesophotic sponge-associated bacteria as a source of bioactive compounds but also provides valuable insights into the intricate interactions between sponges and their symbiotic microbial communities.

The yeast Komagataella phaffii (syn. Pichia pastoris) is a highly effective and well-established host for the production of recombinant proteins. The redox balance of its secretory pathway, which is multi-organelle dependent, is of high importance for producing secretory proteins. Redox imbalance and oxidative stress can significantly influence protein folding and secretion. Glutathione serves as the main redox buffer of the cell and cellular redox conditions can be assessed through the status of the glutathione redox couple (GSH-GSSG). Previous research often focused on the redox potential of the endoplasmic reticulum (ER), where oxidative protein folding and disulphide bond formation occur. In this study, in vivo measurements of the glutathione redox potential were extended to different subcellular compartments by targeting genetically encoded redox sensitive fluorescent proteins (roGFPs) to the cytosol, ER, mitochondria and peroxisomes. Using these biosensors, the impact of oxygen availability on the redox potentials of the different organelles was investigated in non-producing and producing K. phaffii strains in glucose-limited chemostat cultures. It was found that the transition from normoxic to hypoxic conditions affected the redox potential of all investigated organelles, while the exposure to hyperoxic conditions did not impact them. Also, as reported previously, hypoxic conditions led to increased recombinant protein secretion. Finally, transcriptome and proteome analyses provided novel insights into the short-term response of the cells from normoxic to hypoxic conditions.

The isoprenoid bisabolene, one of the simplest monocyclic sesquiterpenes, is a natural plant product that, in addition to its biological function, serves as a precursor for many industrial products. Due to the low concentration of bisabolene and the long harvest cycle, industrial production of this isoprenoid in plants is economically challenging. Chemical synthesis of bisabolene also suffers from significant disadvantages, such as low yields, toxic side products and high costs. Archaea appear suitable producers of isoprenoids, as their membrane lipids consist of isoprenoid ethers, which are synthesised via a variant of the mevalonate (MVA) pathway. Archaeal model species have versatile metabolic capacities, which makes them potential candidates for biotechnological applications. Here, we engineered Methanosarcina acetivorans for production of α-bisabolene from one-carbon substrates by introducing a bisabolene synthase from Abies grandis. Expression of a codon-optimised bisabolene synthase gene in M. acetivorans resulted in 10.6 mg bisabolene/L of culture. Overexpressing genes of the MVA pathway only slightly increased bisabolene yields, which, however, were reached much earlier during incubations than in the corresponding parent strain. The data presented argue for the suitability of M. acetivorans for the biotechnical production of certain isoprenoids.