Microbial Biotechnology最新文献

Root-knot nematodes (Meloidogyne spp.) represent a major threat to global crop production, and current chemical nematicides pose serious environmental and health risks. RNA interference (RNAi) offers a promising gene-specific strategy for nematode control. However, the efficient and sustainable delivery of RNA molecules into nematodes remains a significant challenge. In this study, we developed an innovative RNA delivery platform using extracellular vesicles (EVs) derived from the nematode-trapping fungus Arthrobotrys oligospora. EVs were either exogenously loaded with synthetic siRNAs targeting the Mi-flp-18 gene of M. incognita or harvested from engineered fungal strains expressing short hairpin RNAs (shRNAs) or double-stranded RNAs (dsRNAs) against multiple nematode neuropeptide genes (flp and nlp families). The engineered EVs efficiently delivered RNA cargos into nematodes, leading to significant downregulation of target gene expression. Functional assays and greenhouse experiments revealed the biocontrol potential of the engineered fungal strains, with reductions in nematode motility, root invasion and infectivity. This is the first demonstration in a nematophagous fungus that EVs can serve as effective RNA delivery vehicles for the control of root-knot nematodes. The use of engineered A. oligospora strains provides a scalable, eco-friendly alternative to synthetic delivery systems and transgenic crops. Our findings establish fungal EVs as a powerful tool in cross-kingdom RNAi applications and open new avenues for sustainable pest management in agriculture.

Despite the availability of vaccines, foot-and-mouth disease (FMD) remains a significant concern in many developing countries, causing severe economic losses and affecting local farming communities. Virus-like particle (VLP) vaccines are highly regarded for their safety and efficacy. N-glycosylation for stabilisation and recognition by antigen-presenting cells has been a widely adopted strategy, particularly in enveloped viruses. Here, FMD virus (FMDV) VLPs were employed as a model for artificial glycosylation. N-glycosylation was introduced by mutating the potential glycosylation site of VP1 and then N-glycosylated FMDV VLPs were successfully produced in Pichia pastoris. Glycan profiling revealed that the majority of associated glycans (72.93%) were of the high-mannose type, with additional hybrid type (4.16%) and complex type (22.92%) detected. Functional analyses demonstrated that glycosylation significantly enhanced the stability of VLPs and facilitated the uptake by antigen-presenting cells. Animal experiments further revealed that glycosylation could induce a higher cellular immune response compared to WT VLPs, offering a reference for the glycosylation design of VLP vaccines.

Despite its unusual structure and detrimental role as a chaotropic guanidinium ion, guanidine [HNC(NH₂)₂] exists as a genuine metabolite in many microbes, and its negative effects are mitigated by specific exporters. The metabolic origin of this molecule remains unknown, except in a few cases. We propose here that it results from the deep oxidation of guanine-containing nucleotides derived from 8-oxoguanine in the presence of molecular oxygen. Analysis of the co-evolutionary patterns of guanidine exporters in distant bacteria, together with the analysis of operons involved in purine catabolism, revealed that although purines are generally broken down to urea, guanidine can be produced instead in the presence of molecular oxygen. We investigated how this process could enable guanidine to play a distinct regulatory role in directing metabolism in the presence of molecular oxygen. We propose that it is used as a signal meant to control the generation of reactive oxygen species at an optimal level for the cell.

Over the past decades, biodiesel production has sharply increased worldwide and has led to an overproduction of glycerol, as by-product. Therefore, glycerol is not only produced at low cost with a wide availability but is also a versatile precursor of useful value-added chemicals such as1,3-propanediol. At an industrial scale, glycerol conversion into 1,3-propanediol is almost entirely carried out by fermentation processes as they have shown the best economic and environmental performances. The aim of this article is to provide an up-to-date state of the art on the fundamentals and fermentation process strategies for the microbial conversion of glycerol into 1,3-propanediol. Glycerol fermentation metabolism is detailed and strategies concerning microbial inoculum (i.e., pure cultures of natural or genetically modified strains vs. mixed cultures or artificial consortia), process configuration (i.e., batch, fed-batch and continuous reactors, biomass immobilisation) and related operational parameters (i.e., temperature, pH, oxido-reduction potential) are discussed for the optimisation of 1,3-propanediol production by fermentation.

Over the past decades, biodiesel production has sharply increased worldwide and has led to an overproduction of glycerol, as by-product. Therefore, glycerol is not only produced at low cost with a wide availability but is also a versatile precursor of useful value-added chemicals such as1,3-propanediol. At an industrial scale, glycerol conversion into 1,3-propanediol is almost entirely carried out by fermentation processes as they have shown the best economic and environmental performances. The aim of this article is to provide an up-to-date state of the art on the fundamentals and fermentation process strategies for the microbial conversion of glycerol into 1,3-propanediol. Glycerol fermentation metabolism is detailed and strategies concerning microbial inoculum (i.e., pure cultures of natural or genetically modified strains vs. mixed cultures or artificial consortia), process configuration (i.e., batch, fed-batch and continuous reactors, biomass immobilisation) and related operational parameters (i.e., temperature, pH, oxido-reduction potential) are discussed for the optimisation of 1,3-propanediol production by fermentation.

Centromeres are the chromosomal sites at which the kinetochore forms and attaches to spindle microtubules, directing chromosome segregation. Plasmids based on centromeres can maintain stability and distribute accurately during cell division, supporting them as effective genetic tools for research and biotechnological applications. Here, the centromeric regions on seven chromosomes of Ogataea polymorpha, a methylotrophic non-conventional yeast with great potential in biotechnology, were located by ChIP-seq with the native Cse4. The actual centromeric sequences of chromosomes 1, 2 and 5 were characterised, which are very different from those of other eukaryotes and each unique. Although long terminal repeats (LTR) were found in these centromeres, they are ‘solo LTR elements’ and not important for the function of centromeres. Hence, the O. polymorpha centromeres were categorised into small regional centromeres. O. polymorpha centromeric plasmids were constructed for the first time, which exhibit high genetic stability and compatibility. Application potential for multigene or multicopy expression was validated by the production of uricase and triterpene squalene. This research elucidates the structural features of O. polymorpha centromeres and constructs new, stable centromeric plasmids, expanding the phenomenal diversity of centromeres and providing a powerful genetic toolbox for multi-node and multiple-layered engineering of cell metabolism and physiological functions.

Sweet sorghum (Sorghum bicolor L.) silage is highly prone to aerobic spoilage due to its high sugar content, leading to significant nutritional losses. This study applied absolute microbial quantification, providing novel insights into how Lactobacillus buchneri and Lactobacillus hilgardii, alone or in combination, influence microbial succession and improve the aerobic stability of sweet sorghum silage. The treatments included: (1) control (CK, sterilised water); (2) Lactobacillus buchneri NX205 (LB); (3) Lactobacillus hilgardii M1814 (LH); and (4) a combination of LB and LH (LBLH). After 60 days of ensiling, lactic acid bacteria (LAB)-inoculated groups exhibited significantly lower pH, butyric acid and ammonia-N (except for the LB group), along with higher acetic acid compared with the CK group (p < 0.05), whereas lactic acid and propionic acid contents did not differ significantly among treatments (p > 0.05). LAB inoculation significantly improved aerobic stability, with the LBLH group exhibiting the longest stability period compared to CK, LB and LH groups (462 h; p < 0.005). During aerobic exposure, the LBLH group delayed nutritional and fermentation losses by maintaining lower pH and ammonia-N levels while sustaining higher lactic and acetic contents compared to CK, LB and LH groups. Microbial analysis showed that LBLH reshaped bacterial and fungal communities, with Gluconobacter oxydans prevailing among bacteria and Zygosaccharomyces bailii and Penicillium paneum dominating fungi. Functional pathway prediction further revealed enrichment in carbohydrate degradation, xenobiotic metabolism and energy utilisation in LAB-inoculated silages. Collectively, these results demonstrate that heterofermentative LAB, particularly the LBLH combination, enhances sweet sorghum silage quality by improving aerobic stability and regulating microbial succession.

In Komagataella phaffii, the use of formate as an AOX1 promoter (P_AOX1) inducer in combination with sorbitol, a non-repressive carbon source, has emerged as a promising alternative to methanol-based expression systems. Recently, we demonstrated that formate derived from the tetrahydrofolate-mediated one-carbon (THF-C1) metabolism accumulates in K. phaffii cells deficient in formate dehydrogenase (FdhKO) when grown in sorbitol-based methanol-free medium. Using the lipase CalB from Candida antarctica as a model protein, we observed that recombinant protein (rProt) productivity in an FdhKO strain grown on sorbitol was comparable to that of an Fdh-proficient strain grown on methanol. However, sorbitol is inefficiently metabolised in K. phaffii, leading to a low growth rate and potentially limiting rProt productivity due to insufficient energy and carbon supply. Here, we increased the sorbitol uptake rate, and thus improved sorbitol metabolism, by overexpressing the gene encoding sorbitol dehydrogenase (SOR1) in an FdhKO strain. Our results demonstrate that while increased sorbitol metabolism promotes biomass formation, it reduces P_AOX1 induction, as evidenced by lower formate accumulation and decreased rProt productivity, both for intracellular eGFP and secreted proteins namely CalB lipase and glucose oxidase (Gox) from Aspergillus niger in SOR1-overexpressing strains. Additionally, oxygen availability for cells influences these dynamics, with lower oxygen transfer favouring higher P_AOX1 induction due to increased formate accumulation in an FdhKO strain. Our data also suggest that at low oxygen transfer and low sorbitol uptake rate, the proportion of cells in an induced state increased significantly. This work provides valuable insights into the interplay between sorbitol metabolism and oxygen transfer conditions, contributing to the development of improved recombinant protein production strategies in K. phaffii.

In Komagataella phaffii, the use of formate as an AOX1 promoter (P_AOX1) inducer in combination with sorbitol, a non-repressive carbon source, has emerged as a promising alternative to methanol-based expression systems. Recently, we demonstrated that formate derived from the tetrahydrofolate-mediated one-carbon (THF-C1) metabolism accumulates in K. phaffii cells deficient in formate dehydrogenase (FdhKO) when grown in sorbitol-based methanol-free medium. Using the lipase CalB from Candida antarctica as a model protein, we observed that recombinant protein (rProt) productivity in an FdhKO strain grown on sorbitol was comparable to that of an Fdh-proficient strain grown on methanol. However, sorbitol is inefficiently metabolised in K. phaffii, leading to a low growth rate and potentially limiting rProt productivity due to insufficient energy and carbon supply. Here, we increased the sorbitol uptake rate, and thus improved sorbitol metabolism, by overexpressing the gene encoding sorbitol dehydrogenase (SOR1) in an FdhKO strain. Our results demonstrate that while increased sorbitol metabolism promotes biomass formation, it reduces P_AOX1 induction, as evidenced by lower formate accumulation and decreased rProt productivity, both for intracellular eGFP and secreted proteins namely CalB lipase and glucose oxidase (Gox) from Aspergillus niger in SOR1-overexpressing strains. Additionally, oxygen availability for cells influences these dynamics, with lower oxygen transfer favouring higher P_AOX1 induction due to increased formate accumulation in an FdhKO strain. Our data also suggest that at low oxygen transfer and low sorbitol uptake rate, the proportion of cells in an induced state increased significantly. This work provides valuable insights into the interplay between sorbitol metabolism and oxygen transfer conditions, contributing to the development of improved recombinant protein production strategies in K. phaffii.

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.