Applied Microbiology and Biotechnology最新文献

The biodegradation of organic aromatic compounds in subsurface environments is often hindered by limited dissolved oxygen. While oxygen supplementation can enhance in situ biodegradation, it poses financial and technical challenges. This study explores introducing low-oxygen concentrations in anaerobic environments for efficient contaminant removal, particularly in scenarios where coexisting pollutants are present. An innovative strategy of alternating nitrate-reducing and microaerobic conditions to stimulate biodegradation is proposed, utilizing nitrate initially to degrade easily-degradable compounds, and potentially reducing the need for additional oxygen. Batch experiments were conducted to assess the biodegradation of a BTEX, indene, indane, and naphthalene mixture using groundwater and sediments from an anaerobic contaminated aquifer. Two set-ups were incubated for 98 days to assess the redox transitions between microaerobic (oxygen concentrations < 0.5 mg O₂ L⁻¹) and nitrate-reducing conditions, aiming to minimize external electron acceptor usage while maximizing degradation. Comparative experiments under fully aerobic and fully anaerobic (nitrate-reducing) conditions were conducted, revealing that under microaerobic conditions, all compounds were completely degraded, achieving removal efficiencies comparable to fully aerobic conditions. A pre-treatment phase involving nitrate-reducing conditions followed by microaerobic conditions showed more effective utilization of oxygen specifically for contaminant degradation compared to fully aerobic conditions. Contrarily, under fully anaerobic conditions, without oxygen addition, partial degradation of ethylbenzene was observed after 400 days, while other compounds remained. The outcomes of this study can provide valuable insights for refining strategies involving oxygen and nitrate dosages, thereby enhancing the efficacy of in situ bioremediation approaches targeting complex hydrocarbon mixtures within anaerobic subsurface environments.

• BTEX, indene, indane, and naphthalene mix biodegraded under microaerobic conditions

• Subsurface microorganisms swiftly adapt from nitrate to microaerobic conditions

• More oxygen directed to hydrocarbon biodegradation via a pre-anaerobic treatment

Process intensification and simplification in biopharmaceutical manufacturing have driven the exploration of advanced feeding strategies to improve culture performance and process consistency. Conventional media design strategies, however, are often constrained by the stability and solubility challenges of amino acids, particularly in large-scale applications. As a result, dipeptides have emerged as promising alternatives. Despite extensive research on amino acids, dipeptide supplementation in Chinese hamster ovary (CHO) cell-based manufacturing has received comparatively less attention. In this review, we critically analyze challenges associated with amino acids prone to instability and poor solubility (e.g., glutamine, cysteine, and tyrosine), and explore the potential of dipeptides to address these limitations. We explore the intricate mechanisms of dipeptide transport and enzymatic cleavage, highlighting how chemical properties, stereoisomerism, and competitive metabolites influence their utilization. Notably, while most dipeptides exhibit enhanced solubility, their stabilization effects and culture performance remain variable, underlining the need for rational design. To guide future innovations, we propose tailored dipeptide strategies derived for specific biomanufacturing needs by integrating multi-omics analysis, metabolic flux modeling, and artificial intelligence (AI) modeling.

•Explored dipeptides as a solution to amino acid instability and poor solubility, enhancing cell culture performance.

•Discussed transporter kinetics and cleavage enzymes influencing dipeptide utilization in biomanufacturing.

•Suggested various design strategies for identifying appropriate dipeptide pairs to improve bioprocess efficiency.

The focus on microalgae for applications in several fields, e.g. resources for biofuel, the food industry, cosmetics, nutraceuticals, biotechnology, and healthcare, has gained increasing attention over the last decades. In this study, we investigate the microbiome of the cultured microalga Tetraselmis chui (T. chui) to highlight their potential for health benefits. In this context, biomolecules like antioxidants play a crucial role in the well-being of living organisms as they metabolise harmful reactive oxygen species (ROS) to reduce oxidative stress. Impaired processing of ROS leads to damaged cells and increases the risk of cancer, inflammatory diseases, and diabetes, among others. Here, we identify, characterise, and test bacterial antioxidants derived from the T. chui microbiome metagenome dataset. We identified 258 genes coding for proteins with potential antioxidant activity. Of those, four novel enzymes are expressed and identified as two superoxide dismutases (SOD), TcJM_SOD2 and TcIK_SOD3, and two catalases (CAT), TcJM_CAT2 and TcIK_CAT3. Extensive analyses characterised all implemented enzymes as active even in concentrations down to 25 ng*ml⁻¹ for the SODs and 15 ng*ml⁻¹ for the CATs. Furthermore, sequence-based analyses assign TcJM_SOD2 and TcIK_SOD3 to iron superoxide dismutases (Fe SODs) and TcJM_CAT2 and TcIK_CAT3 to heme-containing catalases. These candidates are phylogenetically classified within the phylum Pseudomonadota. Regarding the biotechnological potential, a toxicity assay did not indicate any harmful effects. The introduced enzymes may benefit medical applications and expand the potential of microalgae microbiomes.

• Omics-based discoveries of antioxidant enzymes from Tetraselmis chui microbiome

• Two superoxide dismutases and two catalases are identified and tested for activity

• Enzyme sensitivity highlights biotechnological potential of microalgae microbiomes

Two methods were compared for their ability to accurately identify Vibrio species of interest: whole genome sequencing as the reference method and MALDI-TOF MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) proteome fingerprinting. The accuracy of mass spectrometry–based identification method was evaluated for its ability to accurately identify isolates of Vibrio cholerae and Vibrio parahaemolyticus. Identification result of each isolate obtained by mass spectrometry was compared to identification by whole genome sequencing (WGS). The MALDI-TOF MS system had excellent performance for identification of V. cholerae and V. parahaemolyticus isolates grown on a non-selective solid agar media. Unlike the biochemical characterization performed by API20E. In this study, 161 isolates (V. cholerae, n = 33; V. parahaemolyticus, n = 102; V. spp., n = 23; other enteropathogens, Salmonella and E. coli, n = 3) were used to assess accuracy. The MALDI-TOF MS system was able to accurately identify 100% (33/33) of the V. cholerae isolates and 99.9% (101/102) of V. parahaemolyticus isolates, with 100% for both sensitivity and specificity for V. cholerae and 99% sensitivity and 98% specificity for V. parahaemolyticus. Thus, mass spectrometry for bacterial identification is comparable to the WGS. Furthermore, in comparison to a biochemical characterization, the use of MALDI-TOF MS system shortens the analysis time from over 72 h to less than 24 h.

• V. cholerae and V. parahaemolyticus were successfully ID-ed by MALDI-TOF

• MALDI-TOF sensitivity and specificity parallels the WGS method of identification

• MALDI-TOF is several days faster than the battery of culture-dependent methods

Detecting alterations in plasmid structures is often performed using conventional molecular biology. However, these methods are laborious and time-consuming for studying the conditions inducing these mutations, which prevent real-time access to cell heterogeneity during bioproduction. In this work, we propose combining both flow cytometry and fluorescence-activated cell sorting, integrated with mechanistic modelling to study conditions that lead to plasmid recombination using a limonene-producing microbial system as a case study. A gene encoding GFP was introduced downstream of the key enzymes involved in limonene biosynthesis to enable real-time kinetics monitoring and the identification of cell heterogeneity according to microscopic and flow cytometric analyses. Three different plasmid configurations (one correct and two incorrect) were identified through cell sorting based on subpopulations expressing different levels of GFP at 10 and 50 µM IPTG. Higher limonene production (530 mg/L) and lower subpopulation proportion carrying the incorrect plasmid (12%) were observed for 10 µM IPTG compared to 50 µM IPTG (96 mg/L limonene and more than 70% of cell population carrying the incorrect plasmid, respectively) in 100 mL production culture. We also managed to derive exploratory hypotheses regarding the plasmid recombination region using the model and successfully validated them experimentally. Additionally, the results also showed that limonene production was proportional to GFP fluorescence intensity. This correlation could serve as an alternative to using biosensors for a high-throughput screening process. The developed method enables rapid identification of plasmid recombination at single-cell level and correlates the heterogeneity with bioproduction performance.

• Strategy to study plasmid recombination during bioproduction.

• Different plasmid structures can be identified and monitored by flow cytometry.

• Mathematical modelling suggests specific alterations in plasmid structures.

Iron oxide nanoparticles, recognized for their superparamagnetic properties, are promising for future healthcare therapies. However, their extensive use in medicine and electronics contributes to their discharge into our environments, highlighting the need for further research on their cellular damage effects on aquatic organisms. While the detrimental properties of other compounds have been stated in the early-life stages of fish, the cytotoxic consequences of superparamagnetic iron oxide nanoparticles (SPIONs) in these stages are still unexplored. Therefore, using the red tilapia (Oreochromis sp.) as a model organism, this study is the first to talk about the subtle cellular alterations caused by biologically induced biomineralized Fe₃O₄-SPIONs by Bacillus sp. in the early-life stages. Once the red tilapia eggs were fertilized, they were challenged to different doses of SPIONs (0, 5, 10, 15, and 30 mg/l), and their tenfold increases (50, 100, 150, and 300 mg/l) for 72 h. The hatching rate, malformation rate, body length, and deformities of the larvae were all studied. Our research showed that iron oxide nanoparticles were harmful to the early stages of life in red tilapia embryos and larvae. They slowed hatching delay, a decrease in survival rate, an increase in heart rate, bleeding, arrested development, and membrane damage and changed the axis’s physiological structure. Additionally, results indicated numerous deformities of red tilapia larvae, with lordosis, kyphosis, and scoliosis once subjected to 50 and 150 mg/l of SPIONs concentrations, respectively. This study could assist us in recognizing the risk and evaluating the disrupting potential of nanoparticles. The key objective of this inquiry is to describe the existing features of the produced magnetite SPIONs (29.44 g/l) including their morphological, chemical, and magnetic characteristics. Illustrate their current role in medicinal applications and aquatic organisms by studying in vivo cytotoxic effects to motivate the development of enhanced SPIONs systems. As a recommendation, more research is needed to completely understand how various exposure endpoints of SPIONs disturb the bodies of red tilapia in the early stages.

• Biogenic SPIONs: a material of the future.

• Characterization is essential to assess the functional properties of the produced SPIONs.

• Fe₃O₄-SPIONs’ impact on the red tilapia ontogeny.

Bacteria-based tumor therapy, which releases therapeutic payloads or remodels the tumor’s immune-suppressive microenvironment and directly kills tumor cells or initiates an anti-tumor immune response, is recently recognized as a promising strategy. Bacteria could be endowed with the capacities of tumor targeting, tumor cell killing, and anti-tumor immune activating by established gene engineering. Furthermore, the integration of synthetic biology and nanomedicine into these engineered bacteria could further enhance their efficacy and controllability. This comprehensive review systematically elucidates the classification and mechanisms of bacterial gene expression induction systems, as well as strategies for constructing bacterial-nanomaterial nanobiohybrids. The review concludes by highlighting the challenges associated with quality control and regulation of bacteria-based tumor therapy while also providing insights into the future prospects of this therapeutic technology.

• A comprehensive overview of the current status of research on bacteria-based tumor therapy.

• The classification and mechanisms of bacterial gene expression induction systems are summarized.

• The challenges and perspectives in clinical translation.

Several strategies have been developed in recent years to improve virus-like particle (VLP)-based vaccine production processes. Among these, the metabolic engineering of cell lines has been one of the most promising approaches. Based on previous work and a proteomic analysis of HEK293 cells producing Human Immunodeficiency Virus-1 (HIV-1) Gag VLPs under transient transfection, four proteins susceptible of enhancing VLP production were identified: ataxia telangiectasia mutated (ATM), ataxia telangiectasia and rad3-related (ATR), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit delta (PDEδ). The knockdown of ATM, ATR, and PDEδ in HEK293 cells increased HIV-1 VLP titers in the supernatant by 3.4-, 2.1-, and 2.2-fold, respectively. Also, possible metabolic synergies between plasmids were investigated by statistical design of experiments (DoE), enabling us to identify the optimal production strategy, that was further demonstrated at lab-scale stirred tank bioreactor operated in perfusion, significantly increasing both VLPs specific and volumetric productivities to 8.3 × 10³ VLPs/cellxday and 7.5 × 10¹² VLPs/Lxday, respectively.

• ATM, ATR, and PDEδ knockdowns increased VLP production in HEK293 cells.

• Knockdown of ATM increased budding efficiency and extracellular vesicle concentration.

• ATM knockdown could be intensified to bioreactor scale operated in perfusion.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted genome editing has been applied to several major edible agaricomycetes, enabling efficient gene targeting. This method is promising for rapid and efficient breeding to isolate high-value cultivars and overcome cultivation challenges. However, the integration of foreign DNA fragments during this process raises concerns regarding genetically modified organisms (GMOs) and their regulatory restrictions. In this study, we developed a foreign-DNA-free genome editing method in Pleurotus ostreatus by transferring the Cas9/guide RNA (gRNA) complex between nuclei in the dikaryotic state. We isolated a donor monokaryotic P. ostreatus strain expressing Cas9 and gRNA targeting pyrG by introducing a recombinant plasmid, which exhibited uracil auxotrophy and 5-fluoroorotic acid (5-FOA) resistance. This strain was then crossed with a pyrG⁺ recipient monokaryon, resulting in dikaryotic strains exhibiting 5-FOA resistance after mycelial growth. When these strains were de-dikaryonized into monokaryons through protoplasting, we obtained monokaryotic isolates harboring the recipient nucleus with small indels at the pyrG target site. Importantly, these isolates were confirmed to be free of foreign DNA through genomic PCR, Southern blotting, and whole-genome resequencing analyses. This is the first report of an efficient genome editing protocol in agaricomycetes that ensures no integration of exogenous DNA. This approach is expected to be applicable to other fungi with a dikaryotic life cycle, opening new possibilities for molecular breeding without the concerns associated with GMOs.

• Successful genome editing via CRISPR/Cas9 trans-nuclei manner in P. ostreatus.

• Recipient monokaryons from gene-edited dikaryons showed no exogenous DNA sequences.

• Efficient genome editing protocol for safer molecular breeding in mushroom fungus.

Transcriptomics is a powerful approach for functional genomics and systems biology, yet it can also be used for genetic part discovery. Here, we derive constitutive and light-regulated promoters directly from transcriptomics data of the basidiomycete red yeast Xanthophyllomyces dendrorhous CBS 6938 (anamorph Phaffia rhodozyma) and use these promoters with other genetic elements to create a modular synthetic biology parts collection for this organism. X. dendrorhous is currently the sole biotechnologically relevant yeast in the Tremellomycete class—it produces large amounts of astaxanthin, especially under oxidative stress and exposure to light. Thus, we performed transcriptomics on X. dendrorhous under different wavelengths of light (red, green, blue, and ultraviolet) and oxidative stress. Differential gene expression analysis (DGE) revealed that terpenoid biosynthesis was primarily upregulated by light through crtI, while oxidative stress upregulated several genes in the pathway. Further gene ontology (GO) analysis revealed a complex survival response to ultraviolet (UV) where X. dendrorhous upregulates aromatic amino acid and tetraterpenoid biosynthesis and downregulates central carbon metabolism and respiration. The DGE data was also used to identify 26 constitutive and regulated genes, and then, putative promoters for each of the 26 genes were derived from the genome. Simultaneously, a modular cloning system for X. dendrorhous was developed, including integration sites, terminators, selection markers, and reporters. Each of the 26 putative promoters were integrated into the genome and characterized by luciferase assay in the dark and under UV light. The putative constitutive promoters were constitutive in the synthetic genetic context, but so were many of the putative regulated promoters. Notably, one putative promoter, derived from a hypothetical gene, showed ninefold activation upon UV exposure. Thus, this study reveals metabolic pathway regulation and develops a genetic parts collection for X. dendrorhous from transcriptomic data. Therefore, this study demonstrates that combining systems biology and synthetic biology into an omics-to-parts workflow can simultaneously provide useful biological insight and genetic tools for nonconventional microbes, particularly those without a related model organism. This approach can enhance current efforts to engineer diverse microbes.

• Transcriptomics revealed further insights into the photobiology of X. dendrorhous, specifically metabolic nodes that are transcriptionally regulated by light.

• A modular genetic part collection was developed, including 26 constitutive and regulated promoters derived from the transcriptomics of X. dendrorhous.

• Omics-to-parts can be applied to nonconventional microbes for rapid “onboarding”.