Microbial Biotechnology最新文献

In this study, we established a highly sensitive on-site detection technology for Bacillus anthracis. Firstly, we integrated Multiple Enzyme Isothermal Rapid Amplification (MIRA) with the clustered regularly interspaced short palindromic repeats (CRISPR) /associated protein 13a (CRISPR/Cas13a) detection system to develop a highly sensitive CRISPR/Cas13a assay. After testing crRNA selection, MIRA primers, reaction temperature, and CRISPR detection conditions, the CRISPR/Cas13a detection system employing dual crRNAs achieved a detection limit of 1000 copies/mL for B. anthracis. Quantitative analysis was additionally attempted. Compared with other common respiratory pathogens, the assay demonstrated high specificity. In clinically simulated samples, all 20 positive specimens were correctly identified, and all 13 negatives were unambiguously classified as negative. Based on these findings, we established a CRISPR point-of-care testing technology. By developing a CRISPR point-of-care testing device together with a tested lyophilised reagent system, the device achieved a detection limit of 250 copies/mL and delivered results within 30 min. All positive samples were accurately identified, and every negative sample was classified as negative. Consequently, this study presents a highly sensitive and portable technology for on-site detection of B. anthracis. It holds significant value for on-site detection of emerging infectious diseases.

This work describes the development of a CRISPR interference (CRISPRi) system for targeted gene repression in bifidobacteria. We first validated the CRISPRi-based approach using Bifidobacterium breve strains engineered to express nuclease-dead orthologs of Cas9 and demonstrated that the CRISPR-Cas system from Streptococcus thermophilus is efficient at targeting both reporter and endogenous genes through the use of single guide RNAs corresponding to the gene of interest. We also developed a one-plasmid system for targeted gene repression in bifidobacteria and demonstrated its utility by targeting genes involved in nucleotide metabolism and carbohydrate metabolism in several species of bifidobacteria. Efficient gene repression was achieved across all tested bifidobacterial species without the requirement for extensive optimization of transformation parameters or sequence optimization to avoid restriction modification systems thus removing the key barriers to genetic manipulation in this genus. This CRISPRi system provides a novel approach to functional genomics in bifidobacteria which facilitates future mechanistic studies in these commercially important microbes.

Surfactin, a lipopeptide antibiotic and quorum-sensing (QS) mediator from Bacillus subtilis, has dual functions in microbial ecology and plant disease suppression. This study engineered B. subtilis R31 to overproduce comK and phrC, key regulators of surfactin biosynthesis, increasing surfactin yield by 45% compared to the WT strain. While elevated surfactin enhanced antimicrobial potential, comK-mediated overproduction impaired biofilm formation and swarming motility, but rhizosphere colonisation was mostly unaffected. 16S rRNA sequencing of banana rhizospheres showed that surfactin selectively shaped the microbial community by enriching beneficial Bacillus species. Mechanistic studies confirmed surfactin's dual role as an antimicrobial and an intercellular signalling molecule for coordinated development in Bacillus populations. These results reveal the molecular mechanisms of R31-mediated suppression of banana Fusarium wilt and offer a strategy for engineering synthetic microbial consortia by manipulating metabolic signalling pathways.

Biodegradable compounds like rhamnolipids provide an environmentally friendly alternative to petroleum-based surfactants. Pseudomonas aeruginosa is the most proficient producer of rhamnolipids. The genetic plasticity and metabolic versatility of P. aeruginosa can be harnessed to leverage the production of rhamnolipids. However, P. aeruginosa carries virulence factors and can cause debilitating diseases in immunocompromised individuals. Bacteria other than P. aeruginosa have been used as heterologous hosts for rhamnolipid production through genetic engineering. Our lab had previously developed genetically attenuated strains of P. aeruginosa to produce polysaccharide alginate. These strains can also be used for rhamnolipid production. This review examines the unique genetic, biochemical, and physiological characteristics of P. aeruginosa amenable to be exploited via bioengineering and the existing models for rhamnolipid production. Further, we propose the attenuated strains of P. aeruginosa as a safe cell-factory for industrial production and biomedical use of rhamnolipids.

Pediococcus pentosaceus Li05, a strain of lactic acid bacteria isolated from the faeces of healthy volunteers, exhibited potential protective effects against various diseases. This study performed third-generation sequencing and detailed characterisation of its genome. The Li05 chromosome harboured conserved genes associated with acid resistance (atp), bile salt resistance (bsh), oxidative stress resistance (hsl, dltA, and et al.), and adhesion (nrd, gap, and et al.), whereas the plasmid did not contain antibiotic resistance or virulence genes. Following intervention with Li05 in loperamide-induced constipated mice, constipation symptoms improved. Meanwhile, alterations in gut microbiota, increased BSH activity in faeces, and modifications to the faecal bile acid profile were observed. Additionally, expression levels of TGR5 and TPH1 in the colon of the mice increased, leading to elevated 5-HT levels. When the TGR5 gene was knocked out or the TPH1 inhibitor LX1606 was administered to suppress 5-HT synthesis in constipated mice, the beneficial effects of Li05 on gastrointestinal motility and mucus secretion were reversed. Culturing intestinal organoids demonstrated that increased bile acids such as DCA, Iso-LCA, and EALCA could enhance 5-HT levels through the TGR5/TPH1 axis. Therefore, we concluded that Li05 regulated bile acid metabolism, subsequently increasing 5-HT levels through the TGR5/TPH1 axis, thus alleviating constipation.

LPPerfectus001 (LPP; GDMCC 62334) is a strain isolated from traditional pickled vegetables and identified as Lactiplantibacillus plantarum by 16s rRNA sequencing and biochemical analysis. Whole-genome sequencing and functional prediction revealed that it is rich in carbohydrate metabolism and lipid metabolism genes, as well as short-chain fatty acid synthesis genes. Preliminary experiments on high-sugar and high-fat zebrafish showed that LPP has the potential to improve glucose and lipid metabolism. Subsequently, in a high-fat/high-fructose obese rat experiment, a 7-week intervention with LPP was conducted. Without altering energy intake, the high-dose LPP (LPPH) significantly reduced body weight and fat mass (p < 0.05), improved insulin sensitivity (reduced HOMA-IR and fasting blood glucose, p < 0.01) and normalised lipid profiles, thereby enhancing glucose homeostasis. This intervention reversed gut microbial dysbiosis by enriching beneficial bacteria (Akkermansia and Lactobacillus) and suppressing pathogens (Klebsiella pneumoniae and Ruminococcus), while also increasing short-chain fatty acids (butyrate/acetate) associated with improved metabolism. Transcriptome analysis revealed that LPPH remodelled adipose tissue through the PPAR signalling pathway (downregulating Pparg and upregulating Cpt1a) and activated the hepatic PI3K-Akt signalling pathway to enhance insulin sensitivity. These results suggest that LPPH alleviates metabolic disorders by synergistically regulating the gut microbiota-fat-liver axis (independent of appetite suppression), making it a promising probiotic therapy for the treatment of obesity-related metabolic disorders.

Clostridium perfringens is a multi-host opportunistic pathogen whose plasmid-encoded toxins cause gas gangrene, necrotic enteritis and enterotoxemia, resulting in substantial economic losses in animal husbandry. In light of antibiotic bans and the need for alternatives, we employed reverse network pharmacology to screen and in vitro validate artemisinin (ART), then assessed its efficacy in murine and rabbit infection models challenged with C. perfringens type F. ART treatment did not significantly affect body weight change or intestinal histopathological damage. However, it significantly modulated inflammatory cytokines and antioxidant parameters in a tissue- and species-dependent manner. Specifically, ART increased serum TNF-α in mice, decreased IL-1β in rabbits and elevated IL-10 in multiple tissues. In addition, ART enhanced hepatic SOD and T-AOC in mice and reduced hepatic MDA in rabbits. Microbiota analysis revealed limited and subtle shifts in community structure following ART intervention. Transcriptomic analysis further indicated that ART treatment induced marked changes in hepatic gene expression, particularly involving detoxification, lipid metabolism and stress response pathways, with notable species-specific differences in enrichment profiles. While correlation analysis suggested associations of Anaerotruncus with hepatic detoxification genes and Bacteroides with inflammation-regulatory genes, these genus-level findings are based on correlation only and should be interpreted with caution given the lack of significant changes in overall microbial community structure. Collectively, these results indicate that ART can modulate host inflammatory and antioxidant responses, but its impact on gut microbiota composition in C. perfringens infection models appears limited, and the biological significance of observed genus-level associations remains to be elucidated.

The COVID-19 pandemic has underscored the urgent need for sustainability-driven biotechnology that strikes a balance between economic development, environmental stewardship and social impact, all while upholding fundamental human rights.

The COVID-19 pandemic has underscored the urgent need for sustainability-driven biotechnology that strikes a balance between economic development, environmental stewardship and social impact, all while upholding fundamental human rights.

The gut microbiota composition in Crohn's disease (CD) patients may influence their response to ustekinumab (UST) therapy. A total of 51 patients with active CD undergoing UST therapy were prospectively enrolled. Clinical activity was evaluated using the Crohn's Disease Activity Index (CDAI), and faecal microbiota were characterised by 16S rRNA sequencing at baseline and week 24. Microbial compositional and functional alterations were assessed, and their correlations with clinical outcomes were examined. At week 24, 46.7% of patients achieved clinical remission and 82.2% achieved clinical response. At baseline, Megamonas (p = 0.009) and Erysipelatoclostridium (p = 0.030) were enriched in the remission group, whereas Fusobacterium (p = 0.016) was more abundant in the non-remission group and correlated positively with C-reactive protein (CRP) but negatively with body mass index (BMI) and serum albumin (ALB). Longitudinal analysis showed that CR patients exhibited increased Clostridium sensu stricto 1 (p = 0.028) and decreased Granulicatella (p = 0.043) after 24 weeks. This study provides real-world evidence supporting the clinical efficacy of UST in Asian patients with active CD. The observed association between elevated baseline Fusobacterium abundance and poorer treatment response suggests a potential microbial influence on therapeutic outcomes. These findings highlight the potential of Fusobacterium as a predictive biomarker for UST response and could provide a rationale for integrating microbiota-modulating strategies to enhance the efficacy of biologics in the future.