Microbial Cell Factories最新文献

Background: Aminoglycoside antibiotics continue to play an indispensable role in clinical antibacterial agents. However, the protection and deprotection procedures in the chemical pathways of semi-synthetic antibiotics are long, atom- and step-inefficient, which severely hampers the development of novel AGs.

Results: Here, GenB3 and GenB4 are employed to synthesize sisomicin, Oxo-verdamicin, Oxo-gentamicin C1a, and Oxo-gentamicin C2a. Subsequently, a semi-rational strategy is applied to enhance the activities of GenB3 and GenB4. The activity of GenB3^M1 (Q270N) towards JI-20A-P is 1.74 times higher than that of GenB3^WT. Similarly, the activity of GenB3^M2 (L361C/A412T/Q270N) towards JI-20Ba-P is 1.34 times higher than that of GenB3^WT. The activity of GenB4^M1 (L356C) towards sisomicin is 1.51 times higher than that of GenB4^WT, while GenB4^M2 (L356C/A407T/Q265N) towards verdamicin C2a is 1.34 times higher than that of GenB4^WT. Furthermore, the beneficial effects of these mutants have been validated in engineered strains. Molecular dynamics simulations indicate that GenB3^M1 establishes a hydrogen bond network in the active center, while GenB4^M1 reduces the distance between K238 and the reaction center. It is also noted that the GenB3^M2 exhibits a synergistic effect specifically on JI-20Ba-P, as the C6'-CH₃ group stabilization restricts the movement of the substrate, which contrasts with JI-20A-P.

Conclusion: Our results not only lay the foundation for the mild and efficient synthesis of C6'-modified AGs analogues but also serve as a reference for synthesizing additional single components in M. echinospora by further enhancing the dideoxygenation process.

Biocatalysis using whole cell biotransformation presents an alternative approach to producing complex molecules when compared to traditional synthetic chemical processes. This method offers several advantages, including scalability, self-contained co-factor recycling systems, the use of cost-effective raw materials, and reduced purification costs. Notably, biotransformation using microbial consortia provides benefits over monocultures by enhancing biosynthesis efficiency and productivity through division of labor and a reduction in metabolic burden. However, reliably controlling microbial cell populations within a consortium remains a significant challenge. In this work, we address this challenge through mechanical constraints. We describe the encapsulation and immobilization of cells in a hyper-porous hydrogel block, using methods and materials that are designed to be amenable to industrial scale-up. The porosity of the block provides ample nutrient access to ensure good cell viability, while the mechanical properties of the hydrogel matrix were optimized for Escherichia coli encapsulation, effectively limiting their proliferation while sustaining recombinant protein production. We also demonstrated the potential of this method for achieving stable co-cultivation of microbes by maintaining two different microbial strains spatially in a single porous hydrogel block. Finally, we successfully applied encapsulation to enable biotransformation in a mixed culture. Unlike its non-encapsulated counterpart, encapsulated E. coli expressing RadH halogenase achieved halogenation of the genistein substrate in a co-culture with genistein-producing Streptomyces. Overall, our strategy of controlling microbial cell populations through physical constraints offers a promising approach for engineering synthetic microbial consortia for biotransformation at an industrial scale.

Background: Metabolic engineering to increase the supply of precursors, such as 2,3-oxidosqualene (OSQ), and manipulate heterologous biosynthetic pathways through the strategic overexpression of multiple genes is promising for increasing the microbial production of triterpenoid saponins. However, the multiple use of constitutive promoters, typically derived from glycolytic or ribosomal protein promoters, can cause transcription factor competition, reducing the expression of each gene. To avoid this issue, we overexpressed transcriptional factor repressor activator protein 1 (Rap1), known to upregulate glycolytic gene expression and be involved in various metabolic pathways, including pyruvate dehydrogenase (PDH) bypass, the mevalonate (MVA) pathway, and sterol synthesis.

Results: Transcriptome analysis of a wild-type yeast strain revealed that Rap1 overexpression significantly upregulated several central carbon metabolism (CCM)-related genes for OSQ production, including glycolytic genes, particularly after the diauxic shift phase. To validate the effect on triterpenoid saponin production, we engineered a Saccharomyces cerevisiae strain capable of producing ginsenoside compound K (CK). Notably, compared with the control strain, the CK-producing strain with Rap1 overexpression showed upregulation of heterologous genes controlled by TDH3 promoter, and a continuous supply of precursors to the CK synthesis pathway, resulting in a 4.5-fold increase in CK production.

Conclusion: These results highlight Rap1 overexpression as a robust strategy to increase triterpenoid production in yeast cell factories. Additionally, this approach provides a versatile framework for enhancing both precursor supply and heterologous gene expression.

Background: Leuconostoc mesenteroides (L. mesenteroides) has known as an electrogenic probiotic bacterium. However, metabolites related to electro-fermentation in ferments of L. mesenteroides are not unveiled.

Result: Electrogenic L. mesenteroides fermentatively metabolized bovine milk to dense ferments with homogeneous particle-size distribution. A non-targeted metabolomics approach was performed on non-fermented and L. mesenteroides-fermented milk. A total of 917 metabolites were identified and quantified by ultra-high performance liquid chromatography (UHPLC)-tandem mass spectrometry (MS-MS). Thirteen prokaryotic metabolic pathways associated with differentially expressed metabolites (DEMs) were revealed through Koto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Anthranilic acid (AA) and 3-hydroxyanthranilin acid (3-HAA), potentially as electron donors, and quinolinic acid, an electron donor precursor, in the tryptophan kynurenine pathway were significantly increased in the fermented milk. Histidine, arginine, and riboflavin involved in bacterial survival or bioelectricity production were elevated after fermentation.

Conclusions: Results indicate that electrogenic L. mesenteroides can mediate electro-fermentation to transform milk to a new nutritional source which is rich in electron donors reportedly acting as antioxidants.

Background: Clostridium perfringens (C. perfringens) is an important zoonotic pathogen. The diseases such as necrotic enteritis (NE), enterotoxemia, gas gangrene and food poisoning caused by its infection seriously threaten the lives of both humans and animals. However, under the severe situation of antibiotic resistance, the development of new antibacterial strategies or drugs deserves great attention.

Results: In this study, we selected the virulence factor Type IV pili (TFP) of C. perfringens as the target for drug screening. The gliding motility, biofilm formation, cell adhesion and antibacterial activity of the natural compound isoxanthohumol (IXN) against C. perfringens were determined. Transmission electron microscopy (TEM), TFP gene transcription analysis and Western blot were used to detect the expression of PilA pilin. The therapeutic effect of IXN on C. perfringens infection was demonstrated through a mouse gas gangrene model. It was confirmed that IXN inhibits the function of TFP by down-regulating TFP-encoding genes and two-component regulatory genes.

Conclusions: In conclusion, our study shows that IXN has the potential to inhibit the function of TFP in C. perfringens and for anti-infection applications.

Sustainable biosynthesis of metal oxide nanoparticles using an eco-friendly approach is a growing research area owing to their promising environmental and biomedical applications. This work aims to biosynthesize and characterize magnesium oxide nanoparticles (MgONPS@Aj) for possible application in dye biosorption and antibacterial activity. For the first time, MgONPS@Aj was successfully synthesized by harnessing exometabolites of Aspergillus japonicus. Various parameters were statistically optimized to maximize the production of MgONPS@Aj using Plackett Burman's design and central composite design. The analysis of variance (ANOVA) revealed that pH was the most significant variable, affecting the bioproduction process followed by biomass quantity and Mg²⁺ precursor concentration. The suggested model (quadratic) was greatly significant and acceptable due to the nonsignificant lack of fit (15.10), and P-value (0.001). The optimized nanoparticles were characterized using X-ray powder diffraction, Fourier-transform infrared (FTIR) spectroscopy, transmission electron microscope (TEM), and Scanning electron microscopy. A high biosorption capacity (204.08 mg/g) of reactive black 5 dye was achieved within 40 min using a 5 mg biosorbent dose (MgONPS@Aj), 100 mg/l initial dye concentration, and pH 6.0. The biosorption process followed a pseudo-second-order (R² of 0.9842) and Langmuir isotherm (R² of 0.9422) models with a dimensionless separation factor (R_L) of 8 × 10⁴, hinting favorable and effective biosorption of dye molecules. A biosorption capacity of 81.97 mg/g after five successive cycles hints that the nanomaterial is suitable for several time utilization. Biogenic MgONPS@Aj displayed dramatic concentration-dependent antibacterial activity with the largest inhibition zones for P. aeruginosa (24.1 ± 0.8 mm, MIC: 3.125 µg/ml), followed by E. coli (22.3 ± 0.7 mm, MIC 6.25), B. subtilis (14.7 ± 0.4 mm, MIC: 12.5 µg/ml) and S. aureus (19.2 ± 0.6 mm, MIC: 6.25 µg/ml). The antibacterial activity was further interpreted using molecular simulation analysis. The lowest binding affinity was determined between MgONPS@Aj and target bacterial proteins (chloramphenicol acetyltransferase E. coli, and S. aureus MurE). The ligand (MgONPS@Aj) can bind to the active site's residues (Tyr¹⁷² and SER²²⁴), indicating a possible antibacterial mechanism. This study recommends MgONPS@Aj as an eco-friendly, and reusable alternative to traditional anionic dye sorbents and a uniquely promising candidate for antimicrobial applications.

During the past decades, the importance of developing sustainable, carbon dioxide (CO₂)-neutral and biodegradable alternatives to conventional plastic has become evident in the context of global pollution issues. Therefore, heterotrophic bacteria such as Cupriavidus sp. have been intensively explored for the synthesis of the biodegradable polymer polyhydroxybutyrate (PHB). PHB is also naturally produced by a variety of phototrophic cyanobacteria, which only need sunlight and CO_2, thereby allowing a CO₂ negative, eco-friendly synthesis of this polymer. However, a major drawback of the use of cyanobacteria is the need of a two-stage production process, since relevant amount of PHB synthesis only occurs after transferring the cultures to conditions of nitrogen starvation, which hinders continuous, large-scale production.This study aimed at generating, by means of genetic engineering, a cyanobacterium that continuously produces PHB in large amounts. We choose a genetically amenable filamentous cyanobacterium of the genus Nostoc sp., which is a diazotrophic cyanobacterium, capable of atmospheric nitrogen (N₂) fixation but naturally does not produce PHB. We transformed this Nostoc strain with various constructs containing the constitutive promotor P_psbA and the PHB synthesis operon phaC1AB from Cupriavidus necator H16. In fact, while the transformants initially produced PHB, the PHB-producing strains rapidly lost cell viability. Therefore, we next attempted further optimization of the biosynthetic gene cluster. The PHB operon was expanded with phasin gene phaP1 from Cupriavidus necator H16 in combination with the native intergenic region of apcBA from Nostoc sp. 7120. Finally, we succeeded in stabilized PHB production, whilst simultaneously avoiding decreasing cell viability. In conclusion, the recombinant Nostoc strain constructed in the present work constitutes the first example of a continuous and stable PHB production platform in cyanobacteria, which has been decoupled from nitrogen starvation and, hence, harbours great potential for sustainable, industrial PHB production.

Cell-free expression is a technique used to synthesize proteins without utilising living cells. This technique relies mainly on the cellular machinery -ribosomes, enzymes, and other components - extracted from cells to produce proteins in vitro. Thus far, cell-free expression systems have been used for an array of biologically important purposes, such as studying protein functions and interactions, designing synthetic pathways, and producing novel proteins and enzymes. In this review article, we aim to provide bacteriophage (phage) researchers with an understanding of the cell-free expression process and the potential it holds to accelerate phage production and engineering for phage therapy and other applications. Throughout the review, we summarize the system's main steps and components, both generally and particularly for the self-assembly and engineering of phages and discuss their potential optimization for better protein and phage production. Cell-free expression systems have the potential to serve as a platform for the biosynthetic production of personalized phage therapeutics. This is an area of in vitro biosynthesis that is becoming increasingly attractive, given the current high interest in phages and their promising potential role in the fight against antimicrobial resistant infections.

The commercial growth factors (GFs) and serum proteins (SPs) contribute to the high cost associated with the serum-free media for cultivated meat production. Producing recombinant GFs and SPs in scale from microbial cell factories can reduce the cost of culture media. Escherichia coli is a frequently employed host in the expression recombinant GFs and SPs. This review explores critical strategies for cost reduction in GFs and SPs production, focusing on yield enhancement, product improvement, purification innovation, and process innovation. Firstly, the review discusses the use of fusion tags to increase the solubility and yield of GFs & SPs, highlighting various studies that have successfully employed these tags for yield enhancement. We then explore how tagging strategies can streamline and economize the purification process, further reducing production costs. Additionally, we address the challenge of low half-life in GFs and SPs and propose potential strategies that can enhance their stability. Furthermore, improvements in the E. coli chassis and cell engineering strategies are also described, with an emphasis on the key areas that can improve yield and identify areas for cost minimization. Finally, we discuss key bioprocessing areas which can facilitate easier scale-up, enhance yield, titer, and productivity, and ultimately lower long-term production costs. It is crucial to recognize that not all suggested approaches can be applied simultaneously, as their relevance varies with different GFs and SPs. However, integrating of multiple strategies is anticipated to yield a cumulative effect, significantly reducing production costs. This collective effort is expected to substantially decrease the price of cultivated meat, contributing to the broader goal of developing sustainable and affordable meat.

Lipases are biocatalysts of significant industrial and medical relevance, owing to their ability to hydrolyze lipid substrates and catalyze esterification reactions under mild conditions. This review provides a comprehensive overview of microbial lipases' production, purification, and biochemical properties. It explores optimized fermentation strategies to enhance enzyme yield, including using agro-industrial residues as substrates. The challenges associated with purification techniques such as ultrafiltration, chromatography, and precipitation are discussed, alongside methods to improve enzyme stability and specificity. Additionally, the review addresses the growing importance of genetic engineering approaches for improving lipase characteristics, such as activity, stability, and specificity.Additionally, this review highlights the diverse applications of microbial lipases in industries, including food, pharmaceuticals, biofuels, and cosmetics. The enzyme's role in bioremediation, biodegradation, and the synthesis of bioactive compounds is analyzed, emphasizing its potential in sustainable and eco-friendly technologies. The biocatalytic properties of lipases make them ideal candidates for the green chemistry initiatives in these industries. In the biomedical domain, lipase has shown promise in drug delivery systems, anti-obesity treatments, and diagnostics.This review provides insights into the strategic development of microbes as microbial cell factories for the sustainable production of lipases, paving the way for future research and industrial innovations in enzyme technology.