Microbial Biotechnology最新文献

We here explore the potential of the fungal genus Aureobasidium as a prototype for a microbial chassis for industrial biotechnology in the context of a developing circular bioeconomy. The study emphasizes the physiological advantages of Aureobasidium, including its polyextremotolerance, broad substrate spectrum, and diverse product range, making it a promising candidate for cost-effective and sustainable industrial processes. In the second part, recent advances in genetic tool development, as well as approaches for up-scaled fermentation, are described. This review adds to the growing body of scientific literature on this remarkable fungus and reveals its potential for future use in the biotechnological industry.

Over time, humanity has addressed microbial water contamination in various ways. Historically, individuals resorted to producing beer to combat the issue. Fast forward to the 19th century, and we witnessed a scientific approach by Robert Koch. His groundbreaking gelatine plating method aimed to identify and quantify bacteria, with a proposed limit of 100 colony-forming units per millilitre (CFU/mL) to avoid Cholera outbreaks. Despite considerable advancements in plating techniques through experimentation with media compositions and growth temperatures, the reliance on a century-old method for water safety remains the state-of-the-art. Even though most countries succeed in producing qualitative water at the end of the production centres, it is difficult to control, and guarantee, the same quality during distribution. Rather than focusing solely on specific sampling points, we propose a holistic examination of the entire water network to ensure comprehensive safety. Current practices leave room for uncertainties, especially given the low concentrations of pathogens. Innovative methods like flow cytometry and flow cytometric fingerprinting offer the ability to detect changes in the microbiome of drinking water. Additionally, molecular techniques and emerging sequencing technologies, such as third-generation sequencing (MinION), mark a significant leap forward, enhancing detection limits and emphasizing the identification of unwanted genes rather than the unwanted bacteria/microorganisms itself. Over the last decades, there has been the realization that the drinking water distribution networks are complex ecosystems that, beside bacteria, comprise of viruses, protozoans and even isopods. Sequencing techniques to find eukaryotic DNA are necessary to monitor the entire microbiome of the drinking water distribution network. Or will artificial intelligence, big data and machine learning prove to be the way to go for (microbial) drinking water monitoring? In essence, it is time to transcend century-old practices and embrace modern technologies to ensure the safety of our drinking water from production to consumption.

Built environments (BEs) currently represent the areas in which human beings spend most of their life. Consistently, microbes populating BEs mostly derive from human occupants and can be easily transferred from BE to occupants. The hospital microbiome is a paradigmatic example, representing a reservoir for harmful pathogens that can be transmitted to susceptible patients, causing the healthcare-associated infections (HAIs). Environmental cleaning is a crucial pillar in controlling BE pathogens and preventing related infections, and chemical disinfectants have been largely used so far towards this aim. However, despite their immediate effect, chemical-based disinfection is unable to prevent recontamination, has a high environmental impact, and can select/increase antimicrobial resistance (AMR) in treated microbes. To overcome these limitations, probiotic-based sanitation (PBS) strategies were recently proposed, built on the use of detergents added with selected probiotics able to displace surrounding pathogens by competitive exclusion. PBS was reported as an effective and low-impact alternative to chemical disinfection, providing stable rebalance of the BE microbiome and significantly reducing pathogens and HAIs compared to disinfectants, without exacerbating AMR and pollution concerns. This minireview summarizes the most significant results obtained by applying PBS in sanitary and non-sanitary settings, which overall suggest that PBS may effectively tackle the infectious risk meanwhile preventing the further spread of pathogenic and resistant microbes.

Antibiotic resistance, one of the major medical threats worldwide, can be selected and induced by metals through multiple mechanisms such as co-resistance, cross-resistance, and co-regulation. Compared with co-resistance and cross-resistance which are attributed to the physically or functionally linked metal and antibiotic resistance genes, co-regulation of antibiotic resistance genes by metal-responsive regulators and pathways is much more complex and elusive. Here, we discussed the main mechanisms by which antibiotic resistance is regulated in response to metals and showed recent attempts to combat antibiotic resistance by interfering with metal-based signalling pathways. Further efforts to depict the intricate metal-based regulatory network of antibiotic resistance will provide tremendous opportunities for the discovery of novel anti-resistance targets, and blocking or rewiring the metal-based signalling pathways is emerging as a promising stratagem to reverse bacterial resistance to antibiotics and rejuvenate the efficacy of conventional antibiotics.

Formaldehyde is a key metabolite in natural and synthetic one-carbon metabolism. To facilitate the engineering of formaldehyde-producing enzymes, the development of sensitive, user-friendly, and cost-effective detection methods is required. In this study, we engineered Escherichia coli to serve as a cellular biosensor capable of detecting a broad range of formaldehyde concentrations. Using both natural and promiscuous formaldehyde assimilation enzymes, we designed three distinct E. coli growth biosensor strains that depend on formaldehyde for cell growth. These strains were engineered to be auxotrophic for one or several essential metabolites that could be produced through formaldehyde assimilation. The respective assimilating enzyme was expressed from the genome to compensate the auxotrophy in the presence of formaldehyde. We first predicted the formaldehyde dependency of the biosensors by flux balance analysis and then analysed it experimentally. Subsequent to strain engineering, we enhanced the formaldehyde sensitivity of two biosensors either through adaptive laboratory evolution or modifications at metabolic branch points. The final set of biosensors demonstrated the ability to detect formaldehyde concentrations ranging approximately from 30 μM to 13 mM. We demonstrated the application of the biosensors by assaying the in vivo activity of different methanol dehydrogenases in the most sensitive strain. The fully genomic nature of the biosensors allows them to be deployed as “plug-and-play” devices for high-throughput screenings of extensive enzyme libraries. The formaldehyde growth biosensors developed in this study hold significant promise for advancing the field of enzyme engineering, thereby supporting the establishment of a sustainable one-carbon bioeconomy.

Pseudomonas putida has become an increasingly important chassis for producing valuable bioproducts. This development is not least due to the ever-improving genetic toolbox, including gene and genome editing techniques. Here, we present a novel, one-plasmid design of a critical genetic tool, the pEMG/pSW system, guaranteeing one engineering cycle to be finalized in 3 days. The pEMG/pSW system proved in the last decade to be valuable for targeted genome engineering in Pseudomonas, as it enables the deletion of large regions of the genome, the integration of heterologous gene clusters or the targeted generation of point mutations. Here, to expedite genetic engineering, two alternative plasmids were constructed: (1) The sacB gene from Bacillus subtilis was integrated into the I-SceI expressing plasmid pSW-2 as a counterselection marker to accelerated plasmid curing; (2) double-strand break introducing gene I-sceI and sacB counterselection marker were integrated into the backbone of the original pEMG vector, named pEMG-RIS. The single plasmid of pEMG-RIS allows rapid genome editing despite the low transcriptional activity of a single copy of the I-SceI encoding gene. Here, the usability of the pEMG-RIS is shown in P. putida KT2440 by integrating an expression cassette including an msfGFP gene in 3 days. In addition, a large fragment of 12.1 kb was also integrated. In summary, we present an updated pEMG/pSW genome editing system that allows efficient and rapid genome editing in P. putida. All plasmids designed in this study will be available via the Addgene platform.

The capture and reduction of atmospheric dinitrogen gas to ammonium can be accomplished through the enzyme nitrogenase in a process known as biological nitrogen fixation (BNF), by a class of microbes known as diazotrophs. The diazotroph Azotobacter vinelandii is a model organism for the study of aerobic nitrogen fixation, and in recent years has been promoted as a potential producer of biofertilizers. Prior reports have demonstrated the potential to partially deregulate BNF in A. vinelandii, resulting in accumulation and extracellular release of ammonium. In many cases, deregulation requires the introduction of transgenic genes or elements to yield the desired phenotype, and the long-term stability of these strains has been reported to be somewhat problematic. In this work, we constructed two strains of A. vinelandii where regulation can be precisely controlled without the addition of any foreign genes or genetic markers. Regulation is maintained through native promoters found in A. vinelandii that can be induced through the addition of extraneous galactose. These strains result in varied degrees of regulation of BNF, and as a result, the release of extracellular ammonium is controlled in a precise, and galactose concentration-dependent manner. In addition, these strains yield high biomass levels, similar to the wild-type A. vinelandii strain and are further able to produce high percentages of the bioplastic polyhydroxybutyrate.

Antimicrobial resistance (AMR) represents a significant global health problem which challenges Sustainable Development Goal 3 of the United Nations, with growing concerns about the possibility of AMR transmission through the food chain. The indiscriminate use of antimicrobials for the treatment of food production animals and for agricultural crop improvement, in addition to the direct discharge of livestock farm residues to sewage and the use of animal manure in agriculture, are among the factors that can facilitate the selection and transmission of AMR throughout the food chain. The study of food microbiomes has been boosted by the advent of next-generation sequencing techniques, which have enabled gaining in-depth understanding of the diversity of antimicrobial resistance genes present in food and associated environments (the so-called resistome). The aim of this review is to provide an accurate and comprehensive overview of the knowledge currently available on the resistome of the most frequently consumed foods worldwide, from a One Health perspective. To this end, the different metagenomic studies which have been conducted to characterize the resistome of foods are compiled and critically discussed.

As a Gram-negative anaerobic bacterium, Akkermansia muciniphila (AKK) participates in the immune response in many cancers. Our study focused on the factors and molecular mechanisms of AKK affecting immune escape in lung adenocarcinoma (LUAD). We cultured AKK bacteria, prepared AKK outer membrane protein Amuc_1100 and constructed a subcutaneous graft tumour mouse model. A549, NCI-H1395 cells and mice were respectively treated with inactivated AKK, Amuc_1100, Ruxolitinib (JAK inhibitor) and RO8191 (JAK activator). CD8⁺ T cells that penetrated the membrane were counted in the Transwell assay. The toxicity of CD8⁺ T cells was evaluated by lactate dehydrogenase assay. Western blot was applied to determine JAK/STAT-related protein and PD-L1 expression, whilst CCL5, granzyme B and INF-γ expression were assessed through enzyme-linked immunosorbent assay (ELISA). The proportion of tumour-infiltrating CD8⁺ T cells and the levels of granzyme B and INF-γ were determined by flow cytometry. AKK markedly accelerated A549 and NCI-H1395 recruiting CD8⁺ T cells and enhanced CD8⁺ T cell toxicity. Amuc_1100 purified from AKK exerted the same promoting effects. Besides, Amuc_1100 dramatically suppressed PD-L1, p-STAT and p-JAK expression and enhanced CCL5, granzyme B and INF-γ expression. Treatment with Ruxolitinib accelerated A549 and NCI-H1395 cells recruiting CD8⁺ T cells, enhanced CD8⁺ T cell toxicity, CCL5, granzyme B and INF-γ expression, and inhibited PD-L1 expression. In contrast, the RO8191 treatment slowed down the changes induced by Amuc_1100. Animal experiments showed that Amuc_1100 was found to increase the number of tumour-infiltrating CD8⁺ T cells, increase the levels of granzyme B and INF-γ and significantly inhibit the expression of PD-L1, p-STAT and p-JAK, which exerted an antitumour effect in vivo. In conclusion, through inhibiting the JAK/STAT signalling pathway, AKK outer membrane protein facilitated the recruitment of CD8⁺ T cells in LUAD and suppressed the immune escape of cells.

Different international agencies recognize that antibiotic resistance is one of the most severe human health problems that humankind is facing. Traditionally, the introduction of new antibiotics solved this problem but various scientific and economic reasons have led to a shortage of novel antibiotics at the pipeline. This situation makes mandatory the implementation of approaches to preserve the efficacy of current antibiotics. The concept is not novel, but the only action taken for such preservation had been the ‘prudent’ use of antibiotics, trying to reduce the selection pressure by reducing the amount of antibiotics. However, even if antibiotics are used only when needed, this will be insufficient because resistance is the inescapable outcome of antibiotics' use. A deeper understanding of the alterations in the bacterial physiology upon acquisition of resistance and during infection will help to design improved strategies to treat bacterial infections. In this article, we discuss the interconnection between antibiotic resistance (and antibiotic activity) and bacterial metabolism, particularly in vivo, when bacteria are causing infection. We discuss as well how understanding evolutionary trade-offs, as collateral sensitivity, associated with the acquisition of resistance may help to define evolution-based therapeutic strategies to fight antibiotic resistance and to preserve currently used antibiotics.