Microbial Biotechnology最新文献

Bacterial protein toxins constitute a vast, largely untapped reservoir of potent bioactive molecules for antimicrobial discovery and biotechnology. However, their intrinsic toxicity often prevents recombinant expression in microbial hosts, limiting biochemical and functional characterization. Here, we present a modular platform for the recombinant production of highly toxic bacterial proteins based on covalent fusion of polymorphic toxins (PTs) to their cognate immunity proteins (PIMs), enabling transient toxin neutralization during expression. This strategy enabled recombinant production and purification of three previously refractory toxin families, PT7, PT8 and PT9. We further streamlined the workflow by introducing intracellular protease-mediated cleavage of the toxin-immunity fusion, reducing purification from four steps to two and increasing the recovered yield of PT9 from approximately 1–2 mg/L to ~4 mg/L. Extending the approach to PT37 further showed that computational redesign of the immunity protein and fusion architecture can enable production when the native fusion is insufficient. Purified PT7 and PT8 retained nuclease activity following denaturation and refolding, while refolded PT9 was structurally validated by X-ray crystallography, demonstrating recovery of functionally and structurally competent proteins. Together, these findings establish a modular strategy for recombinant production of highly toxic bacterial proteins and facilitate their biochemical and functional characterization.

Bacterial protein toxins constitute a vast, largely untapped reservoir of potent bioactive molecules for antimicrobial discovery and biotechnology. However, their intrinsic toxicity often prevents recombinant expression in microbial hosts, limiting biochemical and functional characterization. Here, we present a modular platform for the recombinant production of highly toxic bacterial proteins based on covalent fusion of polymorphic toxins (PTs) to their cognate immunity proteins (PIMs), enabling transient toxin neutralization during expression. This strategy enabled recombinant production and purification of three previously refractory toxin families, PT7, PT8 and PT9. We further streamlined the workflow by introducing intracellular protease-mediated cleavage of the toxin-immunity fusion, reducing purification from four steps to two and increasing the recovered yield of PT9 from approximately 1–2 mg/L to ~4 mg/L. Extending the approach to PT37 further showed that computational redesign of the immunity protein and fusion architecture can enable production when the native fusion is insufficient. Purified PT7 and PT8 retained nuclease activity following denaturation and refolding, while refolded PT9 was structurally validated by X-ray crystallography, demonstrating recovery of functionally and structurally competent proteins. Together, these findings establish a modular strategy for recombinant production of highly toxic bacterial proteins and facilitate their biochemical and functional characterization.

IgA nephropathy (IgAN), the most common primary glomerular disease, involves mucosa–kidney axis dysregulation, with gut dysbiosis increasingly implicated in its pathogenesis. Certain Escherichia coli (E. coli) strains harbour virulence-associated features that promote intestinal colonization, barrier disruption and immune activation. Recent microbiome and metagenomic studies have reported an enrichment of E. coli in patients with IgAN, suggesting a potential link to the mucosa–kidney axis. Nevertheless, E. coli may contribute to IgAN through two major pathways. First, sustained LPS exposure may induce aberrant production of galactose-deficient IgA1 (Gd-IgA1) by stimulating B cells and activate the NLR family pyrin domain containing 3 (NLRP3) inflammasome in macrophages, thereby amplifying systemic and renal inflammation. Second, metabolic reprogramming is associated with increased circulating sphingosine-1-phosphate (S1P) and altered production of indole and trimethylamine (TMA); S1P may promote mesangial proliferation and fibrosis, whereas indole- and TMA-related pathways may contribute to tubulointerstitial injury and chronic kidney disease (CKD) progression. Beyond conventional gut microbiota-targeted interventions, emerging precision therapeutic strategies offer promising new directions for the etiological treatment of IgAN. This review summarizes E. coli virulence factors, pathogenesis, and host, environmental, and ecological factors driving its gut expansion in IgAN patients and outlines corresponding therapeutic strategies.

The filamentous fungus Penicillium rubens is well known as an industrial host for producing penicillin and also used as a promising chassis for secondary metabolite biosynthesis. Chromosomal position effect significantly influences the functional implementation of exogenous genetic modules, but it remains underexplored in filamentous fungi such as P. rubens. In this study, a PiggyBac (PB) transposon-mediated mutagenesis library was constructed in P. rubens. High-throughput sequencing confirmed a broadly distributed with moderate local hopping of PB insertions across this fungal genome. To characterize the genome position effect, two genomic loci libraries including low-expression (P4) and high-expression (P7) positions were obtained respectively by flow cytometry. Heterologous biosynthesis of the plant-derived anthraquinone physcion in P. rubens, via targeted integration of its biosynthetic gene cluster (BGC) into the P7 genomic loci, substantially boosted the titre to a level significantly higher than that achieved via integration at the P4 loci, demonstrating a direct correlation between the integration locus and the secondary metabolite output. To our knowledge, this is the first systematic elucidation of chromosomal position effect on heterologous biosynthesis of secondary metabolites in P. rubens and it also provides an insight for rational selection of integration sites in filamentous fungi.

The contemporary home remains trapped in a mechanistic paradigm of twentieth-century resource consumption, where metabolic biological presence is considered irrelevant to the functioning of a building. Presenting the home as a conceptual vehicle for metabolically optimised living spaces in dense urban environments, this paper develops a conceptual framework of metabolic design patterns that enables the staged retrofit of bioreactors, from composters and fermenters to bioelectrical systems, into human-occupied living spaces. By bringing circular resource processing into the heart of domestic life the principles for regenerative homes are established. Bioelectrical systems are given particular attention because they provide real-time, connectable data through electricity as a medium for information, power, and chemical transformation simultaneously, forming a connective backbone for generating the metabolic design patterns. Three patterns are developed: Toilet as Hearth, Kitchen Garden, and Greywater Wetland, each integrating bioelectrical systems into the habitation programme to provide the respective architectural affordances. The framework proposes a staged retrofit pathway to implementation from passive sensing to neighbourhood metabolic exchange, supported by a conceptual anatomical atlas for diagnosing metabolic failures in established metabolic systems. This revaluation positions metabolic presence as a generative resource rather than a cost, directly addressing SDGs 6, 7, 9, 11 and 12.

Cytochrome P450 enzymes form a large superfamily of monooxygenases capable of oxidising diverse substrates. Among them, members of the CYP108 family are recognised for their role in monoterpenoid oxidation. In this study, we report the identification and characterisation of a new CYP108 family member, CYP108D18, from a metagenome-assembled genome derived from microbial communities in a geothermal hot spring located in northern Queensland, Australia. Taxonomic binning associated CYP108D18 with a Novosphingobium species, a genus well-known for its bioremediation potential. CYP108D18 was heterologously expressed in Escherichia coli, yielding 640 nmol L⁻¹ of culture, and purified to homogeneity. The enzyme exhibited moderate thermostability, with a ¹⁵T₅₀ value of 47°C ± 1°C. Spectroscopic substrate binding assays revealed that while CYP108D18 bound monoterpenoids similarly to other CYP108 enzymes, it showed higher affinity and pronounced larger heme spin-state shifts with aromatic hydrocarbons such as phenylcyclohexane and phenanthrene. Subsequent in silico docking and spectral-binding studies also suggested that CYP108D18 may accommodate larger plant-derived sesquiterpenes, indicating a potential role in their metabolism. Reconstitution of CYP108D18 with spinach ferredoxin and ferredoxin reductase resulted in the oxidation of phenylcyclohexane to trans-4-phenylcyclohexanol. Molecular dynamics simulations and ONIOM (QM/MM) calculations supported selective hydrogen abstraction at the cyclohexane C4 position, rationalising trans-4-phenylcyclohexanol as the predominant product. The high-resolution crystal structure of substrate-free CYP108D18 revealed a bound dioxygen ligand positioned above the heme iron, an unusual feature among P450 structures. Both substrate-free and phenylcyclohexane-bound structures revealed a persistent open conformation, suggesting the absence of a canonical open-to-closed transition upon ligand binding. Spectroelectrochemical analysis showed a heme redox potential of −451 mV versus NHE for the substrate-free CYP108D18, shifting subtly to −409 mV upon phenylcyclohexane binding, which contributes to reduced activity with non-native [2Fe-2S] ferredoxins.

According to the United Nations, up to 40% of the world‘s terrestrial ecosystems are degraded and in need of ecological restoration. Here, we argue that ecosystem restoration requires not only the reintroduction of plant species but also the recovery of the biological interactions that sustain ecosystem functioning. Plants and their associated microbiomes form integrated functional units in which microorganisms contribute to nutrient cycling, stress tolerance, contaminant degradation and ecosystem resilience. These plant–microbe partnerships therefore represent a new opportunity for environmental biotechnology. Advances in microbiome engineering, multi-omics and artificial intelligence are enabling the identification, design and management of microbial communities with complementary functions adapted to specific restoration contexts. However, the complexity of environmental microbiomes, the technical limitations of multi-omics in contaminated environments and the gap between computational predictions and ecological performance highlight the need for experimental and field-scale validation. In this context, we propose Holobiont Engineering for Ecosystem Restoration (HEER) as a conceptual framework that treats plants and their microbiomes as functional units and makes ecological interactions explicit targets of restoration. This perspective positions microbial biotechnology at the interface between ecological restoration and environmental engineering, supporting a shift from species-focused approaches towards the recovery of ecosystem functions.

According to the United Nations, up to 40% of the world‘s terrestrial ecosystems are degraded and in need of ecological restoration. Here, we argue that ecosystem restoration requires not only the reintroduction of plant species but also the recovery of the biological interactions that sustain ecosystem functioning. Plants and their associated microbiomes form integrated functional units in which microorganisms contribute to nutrient cycling, stress tolerance, contaminant degradation and ecosystem resilience. These plant–microbe partnerships therefore represent a new opportunity for environmental biotechnology. Advances in microbiome engineering, multi-omics and artificial intelligence are enabling the identification, design and management of microbial communities with complementary functions adapted to specific restoration contexts. However, the complexity of environmental microbiomes, the technical limitations of multi-omics in contaminated environments and the gap between computational predictions and ecological performance highlight the need for experimental and field-scale validation. In this context, we propose Holobiont Engineering for Ecosystem Restoration (HEER) as a conceptual framework that treats plants and their microbiomes as functional units and makes ecological interactions explicit targets of restoration. This perspective positions microbial biotechnology at the interface between ecological restoration and environmental engineering, supporting a shift from species-focused approaches towards the recovery of ecosystem functions.

Adaptive laboratory evolution (ALE) is a powerful strategy for exploring functional solution spaces, yet its pace is fundamentally constrained by low spontaneous mutation rates. Here, we present a broadly applicable genetic device that accelerates genome diversification in Gram-negative bacteria through the transient action of a dual base editor (ACBE3). This construct combines cytidine and adenosine deaminase activities with a single-stranded DNA-binding module, enabling efficient genome-wide mutagenesis without reliance on host-specific replication or repair systems. The expression of ACBE3 increased mutation rates by up to four orders of magnitude in Escherichia coli and Pseudomonas putida, outperforming classical random mutagenesis approaches such as UV irradiation and chemical treatment. Whole-genome sequencing revealed extensive and uniformly distributed mutations, predominantly consistent with deamination signatures, as well as a broader spectrum of nucleotide changes and small indels. The system displayed strong orthogonality, functioning across diverse bacterial taxa. As a proof of concept, coupling ACBE3-mediated diversification with selective growth conditions enabled the rapid evolution of Acinetobacter calcoaceticus P320, yielding mutant strains with enhanced growth on n-dodecane within only a few induction cycles. This approach decouples diversification from selection, enabling the controlled exploration of adaptive landscapes at unprecedented speeds. While high mutational loads impose cellular burdens and require tight regulatory control, the platform offers a versatile route to accelerate microbial evolution at both single-strain and community levels. Our results thus document dual base editing as a powerful framework for fostering evolution in microbial biotechnology.

Adaptive laboratory evolution (ALE) is a powerful strategy for exploring functional solution spaces, yet its pace is fundamentally constrained by low spontaneous mutation rates. Here, we present a broadly applicable genetic device that accelerates genome diversification in Gram-negative bacteria through the transient action of a dual base editor (ACBE3). This construct combines cytidine and adenosine deaminase activities with a single-stranded DNA-binding module, enabling efficient genome-wide mutagenesis without reliance on host-specific replication or repair systems. The expression of ACBE3 increased mutation rates by up to four orders of magnitude in Escherichia coli and Pseudomonas putida, outperforming classical random mutagenesis approaches such as UV irradiation and chemical treatment. Whole-genome sequencing revealed extensive and uniformly distributed mutations, predominantly consistent with deamination signatures, as well as a broader spectrum of nucleotide changes and small indels. The system displayed strong orthogonality, functioning across diverse bacterial taxa. As a proof of concept, coupling ACBE3-mediated diversification with selective growth conditions enabled the rapid evolution of Acinetobacter calcoaceticus P320, yielding mutant strains with enhanced growth on n-dodecane within only a few induction cycles. This approach decouples diversification from selection, enabling the controlled exploration of adaptive landscapes at unprecedented speeds. While high mutational loads impose cellular burdens and require tight regulatory control, the platform offers a versatile route to accelerate microbial evolution at both single-strain and community levels. Our results thus document dual base editing as a powerful framework for fostering evolution in microbial biotechnology.