Microbial Biotechnology最新文献

Microbial volatile organic compounds (VOCs) are integral to microbial ecological communication. Their potential as tools for sustainable crop protection is increasingly recognised, yet practical implementation remains limited. There are numerous in vitro lab-based studies focussed on screening single strains of soil or plant-associated microbes for their ability to produce VOCs and demonstrate their potential to inhibit plant pathogens or pests. Most of these, however, lack any validation in planta or in the field after petri dish experiments. This extends to a lack of understanding on whether the same VOCs are produced in vitro as in planta. How do we shift this focus and move from exciting lab-based discoveries to practical, scalable crop protection solutions for farmers? This opinion piece explores the current state of research on microbial VOCs for crop protection, translational challenges in deploying them on-farm, and highlights areas where learnings from the ecological roles of microbial VOCs can be leveraged towards field application.

Bacterial microcompartments (BMCs) are protein-based organelles that spatially organise metabolic pathways in prokaryotes, playing critical roles in enhancing metabolic processes and microbe fitness. Notably, many bacterial species possess multiple types of BMCs. While recent studies have advanced our knowledge about the assembly and function of individual BMC types, the mechanisms governing the coexistence and interplay of distinct BMC families within a single bacterial cell remain poorly understood. Here, we engineered Salmonella enterica serovar Typhimurium LT2 to co-express native 1,2-propanediol utilisation (Pdu) BMCs and synthetic α-carboxysomes (α-CBs), providing a unique platform for dissecting their assembly dynamics and functional crosstalk. By exploiting super-resolution fluorescence imaging, electron microscopy, biochemical and enzymatic assays, our studies demonstrate the formation of hybrid BMCs through the exchange of shell proteins between Pdu BMCs and α-CBs, whereas cargo proteins exhibit only limited compatibility, highlighting the specificity of encapsulation mechanisms. Furthermore, the generated hybrid BMCs display altered mobility and enzymatic activities, revealing emergent properties arising from shell protein interchangeability. Our findings provide insights into the inherent structural plasticity and modular architecture of BMCs. More broadly, this study has implications for deciphering how bacterial cells modulate the construction and functions of diverse metabolic modules within a single cellular context and could inform the rational design and engineering of synthetic organelles and bio-factories with tailored metabolic functions for biotechnological applications.

Komagataella pastoris is extensively used as a microbial cell factory for the production of recombinant proteins and high-value compounds. However, tightly controlled promoter systems responsive to safe and economical inducers are required for precise metabolic and pathway engineering in this yeast species. Cumate-inducible promoters are an ideal choice due to the safety and low cost of cumate. In this study, we systematically optimised the insertion sites of the CuO operator sequence within the strong promoter P_GCW14 to isolate a high-activity variant that we designated as P_GCWCuO03. To fine-tune the expression of the repressor protein CymR, we developed a truncated promoter of P_GAP, designated as P_GAP200. Based on the optimal promoter P_GCWCuO03 and the CymR expression unit, we constructed a robust CymR/CuO-mediated cumate-inducible promoter, designated as P_gc, in K. pastoris. P_gc demonstrated outstanding induction properties, resulting in an approximately 11-fold increase in target protein production following induction. Promoter substitution assays validated the effectiveness of P_gc in temporal gene expression control, highlighting the significant potential of this promoter for both basic research and industrial bioprocessing applications in synthetic biology and biotechnology in K. pastoris.

Crops depend on microbial partners for their growth, development, and overall resilience. A pivotal understanding has emerged showing the direct involvement of the root microbiota in regulating the tiller number of rice, a crucial architecture that influences yield. Novel frontiers in microbiological applications for agriculture highlight the profound role of the root microbiota in shaping crop architecture to boost productivity. We propose that improvements in crop production are moving from a genetic perspective on “architecture” to embracing “holobiont architecture.” As such, microbial orchestration provides a dynamic fine-tune function for breeding “architecture-smart crops” characterised by phenotypic plasticity under environmental uncertainty.

Cabbage head rot, caused by Sclerotinia sclerotiorum, threatens crop yield and quality. Among the 21 mycoparasitic fungi isolated from sclerotia, dormant structure and primary sources of inoculum for the pathogen, the strongest antagonism (78.51% mycelial growth inhibition) was observed in Paraphaeosphaeria minitans strain TNAU-CM 1. Scanning electron microscopy (SEM) revealed its destructive colonisation, including pycnidia and pycnidiospore formation, with visible shrinkage and deformation of sclerotia. Gas chromatography–mass spectrometry (GC–MS) analysis identified 24 bioactive metabolites at the point of interactions between P. minitans TNAU-CM 1 and S. sclerotiorum TNAU-SS-5 strains in dual-culture assays. Further, crude metabolites from P. minitans TNAU-CM 1 cultures inhibited the pathogen's mycelial growth by 54.4% at 100 ppm. In the molecular docking of 14 key compounds, linoleic acid and butyl octyl phthalate, well-known antifungal compounds, displayed the highest binding affinity of −7.6 and −6.2 kcal/mol, respectively, against Saccharomyces cerevisiae cupin protein (1ZNP) YML079w, a homologue of SsYCP1, a YML079-like cupin protein (YCP) and a virulence molecule from S. sclerotiorum. Field trials demonstrated that foliar application of P. minitans TNAU-CM 1 stock solution (8–10 × 10⁸ spores per mL) at 5 mL/L dilutions significantly reduced disease incidence and the crops produced a yield of 41.37 tons/ha, comparable to chemical fungicide treatment (43.51 tons/ha). Thus, molecular interaction studies and field evaluations suggest that P. minitans TNAU-CM 1 is a promising eco-friendly alternative to synthetic fungicides for the management of cabbage head rot. Furthermore, our findings indicate that linoleic acid and butyl octyl phthalate are the key antifungal metabolites of P. minitans, active against S. sclerotiorum and will serve as potential candidates for developing bio-fungicide formulations to control head rot in cabbage.

Coastal regions support approximately 60% of the global population and face escalating anthropogenic pollution, which disrupts the dynamics of marine and coastal ecosystems. The discharge of over 80% of sewage without adequate treatment introduces human pathogenic microorganisms into coastal waters, posing significant risks to ecological integrity and public health. This challenge is exacerbated by cross-resistance between antibiotics and biocides, whereby biocide use for biofilm control in coastal industries may inadvertently select for resistant pathogens of terrestrial origin. While microbial biofilms are known to promote macrofouling by facilitating invertebrate larval settlement, a major operational challenge for marine industries, the role of anthropogenic microbial contamination in influencing macrofouling dynamics remains poorly understood. Here, we provide evidence linking anthropogenic microbial contamination to marine biofouling. We isolated an antibiotic and biocide-resistant Enterobacter cloacae strain from a marine cooling water circuit at an operational power plant and identified it as a potent inducer of barnacle (Amphibalanus reticulatus) larval settlement. Salt-tolerance assays combined with Multi-Locus Sequence Typing (MLST) and AAI/ANI-based comparative genomics against reference strains indicated a likely terrestrial origin for this isolate. Larval settlement and choice assays demonstrated that E. cloacae biofilms increased barnacle settlement by > 70% relative to controls. To develop sustainable mitigation strategies against this biocide-resistant organism, we isolated natural bacteriophages targeting E. cloacae from the same water sample. Phage-mediated selective elimination of E. cloacae from biofilms reduced larval settlement by 80% in plate-based assays, providing proof of concept for bacteriophage-based targeted elimination of biofouling-promoting bacteria. Our findings reveal a previously unrecognised connection between anthropogenic bacterial contamination and biofouling dynamics, establishing bacteriophages as an environmentally sustainable strategy for controlling biofilm-mediated larval settlement in marine industries.

Large-scale production of polyhydroxybutyrate (PHB), a biodegradable bioplastic, using genetically engineered cyanobacteria offers a sustainable alternative to petrochemical-derived plastics. However, monoculture-based phototrophic systems face major limitations, such as poor resilience in large-scale reactors, hindering industrial upscaling. To address these challenges, we replaced the native cyanobacterium of a natural microbial consortium with a genetically engineered Synechocystis strain optimised for PHB production, establishing what we define a hybrid photosynthetic microbiome. This new community preserved the ecological structure and stability of the original microbiome while gaining synthetic production capacity. Compared to the axenic strain, the hybrid system exhibited enhanced robustness under abiotic stress, including light and temperature fluctuations, and improved tolerance to operational instability. These features made it suitable for upscaling and application in non-sterile environments. The hybrid microbiome sustained PHB production in scaled photobioreactors, reaching up to 32% PHB per cell dry weight (CDW) equal to ~230 mg L⁻¹ under fully photoautotrophic conditions. Production was also achieved under dark conditions with acetate supplementation, highlighting the system's metabolic flexibility. This work demonstrates the successful integration of an engineered phototroph into a stable native microbiome, positioning hybrid communities as powerful platform for industrial biotechnology.

Lacticaseibacillus rhamnosus GG (LGG) is one of the most extensively studied probiotic strains, widely used in food and health applications. However, the absence of efficient, precise genome editing methods has limited its broader potential and functional versatility. Here, we present an endogenous type II-A CRISPR-Cas genome editing workflow for LGG designed for functional strain construction. Using a plasmid interference assay together with single-nucleotide substitutions, we confirm the precise PAM requirement as 5′-NGAAA-3′. We pair a synthetic sgRNA cassette with homology-directed repair donors to enable targeted deletions and insertions across multiple loci, achieving modest but practically relevant editing efficiencies (11.1-25.0% of recovered transformants) that support routine strain construction. Using this optimised and precise genome engineering method, we generated a β-glucuronidase (GUS)-expressing LGG strain for robust strain tracking within complex microbial communities. This work removes barriers to LGG engineering, expands the probiotic CRISPR toolkit, and provides broadly applicable strategies for designing next-generation probiotics with applications in food biotechnology and microbial therapeutics.

Secretory protein production by microbial hosts simplifies product recovery and is therefore preferred over intracellular production. Efficient secretion of heterologous proteins by bacteria requires the identification of optimal signal peptides (SPs), a step that often limits process development. Using Corynebacterium glutamicum as a model host, we established a modular cloning system enabling rapid assembly of expression plasmids for secretory protein production. Screening a library of 30 individually cloned endogenous SPs with a fungal cutinase as target protein demonstrated that several native SPs achieved substantially higher secretion levels than the widely used Bacillus subtilis NprE reference SP. To accelerate SP discovery, we developed a one-pot approach in which C. glutamicum was directly transformed with a single modular cloning mixture containing all 30 SPs. Combined with the AutoBioTech high-throughput platform for cultivation, harvesting, and protein quantification, this strategy enabled screening of several hundred clones in parallel. Superior SPs were rapidly identified not only for cutinase but also for four polyethylene terephthalate hydrolases (PETases). This streamlined workflow significantly reduces time and cost for selecting effective SPs and provides a versatile platform for advancing secretory protein production in C. glutamicum.

Acute pancreatitis (AP) pathogenesis involves gut microbiota dysbiosis. Although Lactobacillus salivarius Li01 (Li01) is a well-characterised probiotic strain, its specific role in AP via the ‘gut-pancreas axis’ remains unclear. Li01 pretreatment via oral gavage was assessed in an L-arginine-induced AP mouse model. The gut microbiota composition and abundance were analysed via 16S rRNA sequencing, complemented by untargeted faecal metabolomics and pancreatic transcriptomics analyses. Li01 pretreatment significantly alleviated histopathological damage to the pancreas and reduced serum amylase activity in AP model mice. Pancreatic transcriptomic analysis revealed that Li01 modulated the expression of 89 differentially expressed genes (DEGs), thereby impacting key immune-related signalling pathways, including the TNF-α signalling pathway. Furthermore, Li01 mitigated gut microbiota dysbiosis in AP mice, notably by increasing the relative abundance of bacteria such as Paramuribaculum. Faecal metabolomics analysis indicated that Li01 intervention significantly increased the levels of metabolites involved in steroid hormone biosynthesis, including 17α-estradiol. Li01 may alleviate AP by modulating the gut microbiota composition, increasing the relative abundance of bacteria such as Paramuribaculum, and regulating faecal metabolite profiles, particularly those involved in the steroid hormone biosynthesis pathway. These modulations, in turn, appear to influence pancreatic inflammation-related signalling pathways, including the TNF signalling pathway.