Microbial Cell Factories最新文献

Background: During the last decades, the advancements in synthetic biology opened the doors for a profusion of cost-effective, fast, and ecologically friendly medical applications priorly unimaginable. Following the trend, the genetic engineering of the baker's yeast, Saccharomyces cerevisiae, propelled its status from an instrumental ally in the food industry to a therapy and prophylaxis aid.

Main text: In this review, we scrutinize the main applications of engineered S. cerevisiae in the medical field focusing on its use as a cell factory for pharmaceuticals and vaccines, a biosensor for diagnostic and biomimetic assays, and as a live biotherapeutic product for the smart in situ treatment of intestinal ailments. An extensive view of these fields' academic and commercial developments as well as main hindrances is presented.

Conclusion: Although the field still faces challenges, the development of yeast-based medical applications is often considered a success story. The rapid advances in synthetic biology strongly support the case for a future where engineered yeasts play an important role in medicine.

Background: Ecotoxicology is essential for the evaluation and comprehension of the effects of emergency pollutants (EP) such as heavy metal ions on the natural environment. EPs pose a substantial threat to the health of humans and the proper functioning of the global ecosystem. The primary concern is the exposure of humans and animals to heavy metal ions through contaminated water. The presence of heavy metal ions in drinking water ought to be monitored in accordance with World Health Organization regulations. Among the numerous harmful metal ions, copper ions are responsible for a variety of human diseases.

Results: This study investigates the application of phage display as a screening method for heavy metal toxicological targets, with copper served as the main focus. To identify a variety of Cu-binding M13 phage clones with unique peptides and to assess their affinity for metal ions, the study utilized Escherichia coli as a factories producing recombinant bacteriophages, modified biopanning procedure and an ELISA assay. The research highlights the increasing importance of phage display as a screening tool in ecotoxicology. We synthesized and modified the selected peptide to enable the rapid optical detection of Cu(II) ions in aqueous solutions. By incorporating the dansyl group into a designated peptide sequence, we implemented fluorescence detection assays for real-time measurements. The Cu²⁺- binding peptide's efficacy was confirmed through spectroscopic measurements, which allowed for real-time detection with rapid response times with high selectivity.

Conclusions: The phage display technique was successfully applied to develop the fluorescent peptide-based chemosensor that exhibited high selectivity and sensitivity for Cu²⁺.

Background: In the soy sauce fermentation industry, Aspergillus oryzae (A. oryzae) plays an essential role and is frequently subjected to high salinity levels, which pose a significant osmotic stress. This environmental challenge necessitates the activation of stress response mechanisms within the fungus. The Zn(II)₂Cys₆ family of transcription factors, known for their zinc binuclear cluster-containing proteins, are key regulators in fungi, modulating various cellular functions such as stress adaptation and metabolic pathways.

Results: Overexpression of AozC decreased growth rates in the presence of salt, while its knockdown enhanced growth, the number of spores, and biomass, particularly under conditions of 15% salt concentration, doubling these metrics compared to the wild type. Conversely, the knockdown of AozC via RNA interference significantly enhanced spore density and dry biomass, particularly under 15% salt stress, where these parameters were markedly improved over the wild type strain. Moreover, the overexpression of AozC led to a downregulation of the FAD2 gene, a pivotal enzyme in the biosynthesis of unsaturated fatty acids (UFAs), which are essential for preserving cell membrane fluidity and integrity under saline conditions. Transcriptome profiling further exposed the influence of AozC on the regulation of UFA biosynthesis and the modulation of critical stress response pathways. Notably, the regulatory role of AozC in the mitogen-activated protein kinase (MAPK) signaling and ABC transporters pathways was highlighted, underscoring its significance in cellular osmotic balance and endoplasmic reticulum homeostasis. These findings collectively indicate that AozC functions as a negative regulator of salt tolerance in A. oryzae.

Conclusion: This research suggest that AozC acts as a negative regulator in salt tolerance and modulates fatty acid biosynthesis in response to osmotic stress. These results provide insights into the regulatory mechanisms of stress adaptation in A. oryzae.

Background: Sporobolomyces pararoseus is a well-studied oleaginous red yeast that can synthesize a variety of high value-added bioactive compounds. Biofilm is one of the important biological barriers for microbial cells to resist environmental stresses and maintain stable fermentation process. Here, the effect of acidic conditions on the biosynthesis of biofilms in S. pararoseus NGR was investigated through the combination of morphology, biochemistry, and multi-omics approaches.

Results: The results showed that the acidic environment was the key factor to trigger the biofilm formation of S. pararoseus NGR. When S. pararoseus NGR was cultured under pH 4.7, the colony morphology was wrinkled, the cells were wrapped by a large amount of extracellular matrix, and the hydrophobicity and anti-oxidative stress ability were significantly improved, and the yield of intracellular carotenoids was significantly increased. Transcriptome and metabolome profiling indicated that carbohydrate metabolism, amino acid metabolism, lipid metabolism, and nucleic acid metabolism in S. pararoseus NGR cells were significantly enriched in biofilm cells under pH 4.7 culture conditions, including 56 differentially expressed genes and 341 differential metabolites.

Conclusions: These differential genes and metabolites may play an important role in the formation of biofilms by S. pararoseus NGR in response to acidic stress. The results will provide strategies for the development and utilization of beneficial microbial biofilms, and provide theoretical support for the industrial fermentation production of microorganisms to improve their resistance and maintain stable growth.

Shiga toxin-producing Escherichia coli (STEC) is one of the major pathogens responsible for severe foodborne infections, and the common serotypes include E. coli O157, O26, O45, O103, O111, O121, and O145. Vaccination has the potential to prevent STEC infections, but no licensed vaccines are available to provide protection against multiple STEC infections. In this study, we constructed an engineered S. Typhimurium to rapidly produce the outer membrane vesicle (OMV) with low endotoxic activity to deliver the O-antigen of E. coli. S. Typhimurium OMV (STmOMV), which displays mixed heterologous O-antigens, was systematically investigated in mice for immunogenicity and the ability to prevent wild-type STEC infection. Animal experiments demonstrated that STmOMV displaying both E. coli O111 and O157 O-antigens by intraperitoneal injection not only induced robust humoral immunity but also provided effective protection against wild-type E. coli O111 and O157 infection in mice, as well as long-lasting immunity. Meanwhile, the O-antigen polysaccharides of E. coli O26 and O45, and O145 and O103 were also mixedly exhibited on STmOMV as O-antigens of the O111 and O157 did. Three mixed STmOMVs were inoculated intraperitoneally to mice, and confer effective protection against six E. coli infections. The STmOMV developed in this study to display mixed heterologous O-antigens provides an innovative and improved strategy for the prevention of multiple STEC infections.

Carbapenem-resistant Klebsiella pneumoniae poses a severe risk to global public health, necessitating the immediate development of novel therapeutic strategies. The current study aimed to investigate the effectiveness of the green algae Arthrospira maxima (commercially known as Spirulina) both in vitro and in vivo against carbapenem-resistant K. pneumoniae. In this study, thirty carbapenem-resistant K. pneumoniae isolates were collected, identified, and then screened for their susceptibility to several antibiotics and carbapenemase production genes using PCR. Both bla_KPC and bla_OXA-48 genes were the most predominant detected carbapenemase genes in the tested isolates. The phytochemical profiling of A. maxima algal extract was conducted using LC-MS/MS in a positive mode technique. The minimum inhibitory concentrations (MIC) of the algal extract ranged from 500 to 1000 µg/mL. The algal extract also resulted in decreasing the membrane integrity and distortion in the bacterial cells as revealed by scanning electron microscope. The bioactive compounds that were responsible for the antibacterial action were fatty acids, including PUFAs, polysaccharides, glycosides, peptides, flavonoids, phycocyanin, minerals, essential amino acids, and vitamins. Moreover, A. maxima algal extract revealed an antibiofilm activity by crystal violet assay and qRT-PCR. A murine pneumonia model was employed for the in vivo assessment of the antibacterial action of the algal extract. A. maxima showed a promising antibacterial action which was comparable to the action of colistin (standard drug). This was manifested by improving the pulmonary architecture, decreasing the inflammatory cell infiltration, and fibrosis after staining with hematoxylin and eosin and Masson's trichrome stain. Using immunohistochemical investigations, the percentage of the immunoreactive cells significantly decreased after using monoclonal antibodies of the tumor necrosis factor-alpha and interleukin six. So, A. maxima may be considered a new candidate for the development of new antibacterial medications.

Background: The healthcare sector faces a growing threat from the rise of highly resistant microorganisms, particularly Methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDR P. aeruginosa). Facing the challenge of antibiotic resistance, nanoparticles have surfaced as promising substitutes for antimicrobial therapy. Recent studies showcase the effectiveness of various fungi species in nanoparticle synthesis. Mycosynthesized silver nanoparticles (AgNPs) and selenium nanoparticles (SeNPs) using Aspergillus carneus MAK 259 has been investigated and demonstrate antibacterial, antibiofilm and synergistic activities against (MRSA) and (MDR P. aeruginosa).

Results: In the current research, silver nanoparticles (AgNPs) and selenium nanoparticles (SeNPs) were produced extracellularly using A. carneus MAK 259 culture supernatants. Colour change, an initial evaluation of the production of AgNPs and SeNPs. Then, UV absorption peaks at 410 nm and 260 nm confirmed the production of AgNPs and SeNPs, respectively. AgNPs and SeNPs were dispersed consistently between 5‒26 nm and 20-77 nm in size, respectively using TEM. FT-IR analysis was used for assessing proteins bound to the produced nanoparticles. The crystallinity and stability of AgNPs and SeNPs was confirmed using X-ray diffraction analysis and zeta potential measurements, respectively. Antibacterial, antibiofilm and synergistic effects of both (NPs) with antibiotics against MRSA and MDR P. aeruginosa were tested by Agar well diffusion, tissue culture plate and disc diffusion method respectively. Both (NPs) inhibited the growth of P. aeruginosa more than S. aureus. But, SeNPs was stronger. AgNPs had stronger antibiofilm effect especially on biofilms producing S. aureus. as regard synergestic effects, Both (NPs) had higher synergestic effects in combination with cell wall inhibiting antibiotics against P. aeuroginosa While, on S. aureus with antibiotics that inhibit protein synthesis and affect metabolic pathways.

Conclusions: Our study demonstrated that the mycosynthesized SeNPs had remarkable antibacterial effect while, mycosynthesized AgNPs exhibited a considerable antibiofilm effect. Both NPs exhibited higher synergistic effect with antibiotics with different modes of action. This approach could potentially enhance the efficacy of existing antibiotics, providing a new weapon against drug-resistant bacteria where the described silver and selenium nanoparticles play a pivotal role in revolutionizing healthcare practices, offering innovative solutions to combat antibiotic resistance, and contributing to the development of advanced medical technologies.

Background: In response to iron deficiency and other environmental stressors, cyanobacteria producing siderophores can help in ameliorating plant stress and enhancing growth physiological and biochemical processes. The objective of this work was to screen the potential of Arthrospira platensis, Pseudanabaena limnetica, Nostoc carneum, and Synechococcus mundulus for siderophore production to select the most promising isolate, then to examine the potentiality of the isolated siderophore in promoting Zea mays seedling growth in an iron-limited environment.

Results: Data of the screening experiment illustrated that Synechococcus mundulus significantly recorded the maximum highest siderophore production (78 ± 2%) while the minimum production was recorded by Nostoc carneum (24.67 ± 0.58%). Therefore, Synechococcus mundulus was chosen for the beneficiary study and the intended agricultural application. Siderophore-type identification tests proved that Synechococcus mundulus produced hydroxamate-type. The response surface approach was successful in optimizing the conditions of siderophore production in Synechococcus mundulus with actual values for maximum biomass (387.11 mg L^- 1) and siderophore production (91.84%) higher than the predicted values. The proton nuclear magnetic resonance (¹H NMR) analysis data and the Fourier transformer-infrared spectrum analysis (FT-IR) signify the hydroxamate nature of Synechococcus mundulus isolated siderophore. Zea mays seedlings' growth response in the hydroponic system was significantly stimulated in response to supplementation with Synechococcus mundulus siderophore in the absence of iron compared to plants grown without iron and the positive controls. Additionally, the contents of chlorophyll a, chlorophyll b, carotenoids, total carbohydrates, and total protein were all surpassed in siderophore-treated plants, which is expected due to the increased iron content.

Conclusions: The results introduced in this study highlighted the significant potential of Synechococcus mundulus-derived siderophore in stimulating Zea mays physicochemical growth parameters and iron uptake. Findings of this study present novel visions of cyanobacteria producing siderophores as an ecofriendly alternative candidate to synthetic iron chelators and their role in plant stress management.

Background: 2'-Fucosyllactose (2'-FL) is a predominant human milk oligosaccharide that significantly enhances infant nutrition and immune health. This study addresses the need for a safe and economical production of 2'-FL by employing Generally Recognized As Safe (GRAS) microbial strain, Priestia megaterium ATCC 14581. This strain was chosen for its robust growth and established safety profile and attributing suitable for industrial-scale production.

Results: The engineering targets included the deletion of the lacZ gene to prevent lactose metabolism interference, introduction of α-1,2-fucosyltransferase derived from the non-pathogenic strain, and optimization of the GDP-L-fucose biosynthesis pathway through the overexpression of manA and manC. These changes, coupled with improvements in lactose uptake and utilization through random mutagenesis, led to a high 2'-FL yield of 28.6 g/L in fed-batch fermentation, highlighting the potential of our metabolic engineering strategies on P. megaterium.

Conclusions: The GRAS strain P. megaterium ATCC 14581 was successfully engineered to overproduce 2'-FL, a valuable human milk oligosaccharide, through a series of genetic modifications and metabolic pathway optimizations. This work underscores the feasibility of using GRAS strains for the production of oligosaccharides, paving the way for safer and more efficient methods in biotechnological applications. Future studies could explore additional genetic modifications and optimization of fermentation conditions of the strain to further enhance 2'-FL yield and scalability.

Background: Because the process is cost-effective, microbial pectinase is used in juice clearing. The isolation, immobilization, and characterization of pectinase from Aspergillus nidulans (Eidam) G. Winter (AUMC No. 7147) were therefore the focus of the current investigation.

Results: Ammonium sulphate (85%), DEAE-cellulose, and Sephadex G-200 were used to purify the enzyme. With a yield of 30.4%, the final specific activity was 400 units mg^-1 protein and 125-fold purification. Using SDS-PAGE to validate the purification of the pectinase, a single band showing the homogeneity of the purified pectinase with a molecular weight of 50 kD was found. Chitosan and calcium alginate both effectively immobilized pectinase, with immobilization efficiencies of 85.7 and 69.4%, respectively. At 50, 55, 60, and 65 °C, the thermostability of both free and chitosan-immobilized pectinase was examined. The free and chitosan-immobilized enzymes had half-lives (t_1/2) of 23.83 and 28.64 min at 65 °C, and their K_d values were 0.0291 and 0.0242 min^-1, respectively. In addition, the Z values were 44.6 and 31.54 °C, while the D values were 79.2 and 95.1 min. Compared to the untreated one, the orange, mango, and pineapple juices treated with immobilized pure pectinase showed greater clarity. Following treatment with pure pectinase, the fruit juice's 1, 1-diphenyl-2-picrylhydrazyl and 2, 2'-azino-bis 3-ethylbenzothiazoline-6-sulfonate scavenging activities increased. Following treatment with pure pectinase, the amounts of total phenolics and total flavonoids increased.

Conclusion: The procedure is deemed cost-effective in the food industry because the strong affinity of fungal pectinase for pectin. The investigated pectinase supported its usage in the food industry by being able to clear orange, mango, and pineapple juices.