Microbial Cell Factories最新文献

In recent years, the integration of atmospheric and room-temperature plasma (ARTP) mutagenesis with droplet-based microfluidic (DBMF) technology has enabled the development of a novel high-efficiency mutagenesis and screening system. This system not only enhances microbial mutagenesis efficiency but also achieves precise screening and high-throughput detection, demonstrating broad applications in biosynthesis, fermentation engineering, biological feed production, edible fungus breeding, and environmental remediation. This review comprehensively elaborates on the principles and advantages of the system and discusses its diverse applications across multiple fields.

Background: Used motor oil (UMO) is a dangerous environmental pollutant that needs to be treated effectively. This work introduces a novel approach for producing bioelectricity and UMO biodegradation simultaneously in a single-chamber microbial fuel cell (SCMFC) using native mixed bacterial cultures.

Results: Under certain conditions (2% oil, 1% peptone, 4% inoculum, 21 days), the optimized bacterial culture degraded UMO by about 80%. Through bioelectrochemical studies, a maximum voltage of 257 mV and a power density of 36.6 mW/m² were demonstrated, showing a strong correlation between UMO removal and electricity generation. Moreover, metagenomic data showed that Firmicutes, particularly Bacillus, dominated the biofilm at roughly 65%. Fourier Transform Infrared (FTIR) and Gas Chromatography-Mass Spectroscopy (GC-MS) verified the breakdown of complex hydrocarbon molecules, highlighting their crucial role in UMO biodegradation and bioenergy production. The effective elimination of UMOs and simultaneous power generation, supported by metagenomic and biochemical tests, showed the microbial activity and hydrocarbon breakdown.

Conclusions: The results suggest SCMFC technology as a sustainable solution for managing petroleum waste while producing renewable energy.

Background: Filamentous fungi produce a broad spectrum of colored secondary metabolites that are largely used in various industries, including food, cosmetics, fabrics, and medications. This study explores, for the first time, the potential of Aspergillus frequens to produce pigmented secondary metabolites and their application in various biotechnological treatments.

Results: Aspergillus frequens (Asmaa 2024) produced the highest concentration of pigmented secondary metabolites among the 20 tested fungal rhizospheric fungi, reaching 21.36 ± 1.8 AU/mL in potato dextrose broth (PDB) medium. Scanning electron microscopy (SEM) revealed that the extracted pigment has an irregular shape and particle size, ranging from 40 to 184 nm. The elemental composition revealed the presence of high ratios of carbon and oxygen using energy-dispersive X-ray (EDX). Many functional groups and chromophore compounds have been detected in the extracted pigment using Fourier-transform infrared spectroscopy (FT-IR) and gas chromatography-mass spectrometry (GC-MS). Thirteen pathogenic species of bacteria were significantly inhibited in their development by the colored metabolites, whose minimum bactericidal concentrations (MBCs) varied from 4.5 to 16.7 mg/mL. The most notable percentages in suppression biofilm development, suggesting a major influence, were 66.8% for Klebsiella pneumoniae and 64.8% for Bacillus subtilis using the microtiter plate technique. Following assessment of zeta potential, particle size, and polydispersity index (PDI) of the target bacteria, the effective antibacterial efficacy of the pigmented secondary metabolites was confirmed. The viability of the osteosarcoma (HOS) and lung cancer (A549) cell lines was significantly diminished by the A. frequens' secondary metabolites, with IC50 values of 43.3 and 77.1 µg/mL, respectively. In contrast, the skin cancer cell line (A431) showed no signs of impact, using the MTT assay.

Conclusion: Based on the obtained findings, A. frequens pigmented secondary metabolites have promising potential in the biological control of pathogenic and biofilm-forming bacteria, as well as in the treatment of bone and lung cancer. While numerous studies have investigated pigment production in Aspergillus species, this research represents the first investigation into pigment synthesis by A. frequens.

Escherichia coli strains are widely utilized as cell factories for recombinant protein production. However, acetate overflow remains a significant challenge that negatively impacts both biomass yield and protein expression. Here, we evaluated a previously engineered E. coli K 12 BW25113 strain with pka and arcA deletions (RV04) for the expression of a single-chain variable fragments (scFv) derived from 4D5MOC-B, a monoclonal antibody that binds to epithelial cell adhesion molecule (EpCAM) as a biologically important marker for tumor immunotherapy. According to our results, RV04 strain demonstrated a significant growth advantage over both BW25113 and BL21 strains. In minimal M9 medium, RV04 exhibited a maximum cell density that was 44% higher than wild‑type and 11% higher than BL21. In enriched M9 medium, RV04 achieved a remarkable maximum specific growth rate (µ_max) of 0.775 ± 0.003 h⁻¹ and a maximum cell density of 2.1095 ± 0.0205, even under metabolic load. Regarding acetate accumulation, RV04 fully eliminated acetate accumulation within 24 h, whereas BW25113 accumulated acetate up to 0.521 g L^- 1 under the same minimal medium conditions. Similarly, in enriched M9 medium, RV04 maintained significantly lower acetate levels (1.65 g L^- 1 at 24 h) compared to BW25113 (3.99 g L^- 1), despite increased biomass and protein production. These results confirm that RV04 can control acetate overflow more efficiently than the wild type under both minimal and enriched conditions. The combination of using the genetically modified strain and medium enrichment strategy resulted in significantly increased recombinant protein production. In LB medium, RV04 produced 5% more protein than BL21 and 44.8% more than the wild‑type, while in enriched synthetic M9 medium, it outperformed BL21 and BW25113 by 7.1% and 59.5%, respectively. Furthermore, RV04 demonstrated markedly enhanced protein expression compared to other commercial strains; it produced approximately 33.8% more protein than SHuffle, 145.7% more than Rosetta, and over sevenfold more than Origami B. Our findings demonstrate that RV04 effectively mitigates acetate overflow, enhances growth, and substantially increases the recombinant protein titer under both minimal and enriched culture conditions. These features make RV04 a strong candidate for large-scale industrial bioprocessing operations.

Background: Conventional genetic engineering approaches for bacterial metabolic pathway manipulation, although highly applicable, still face limitations including metabolic burden, irreversibility, and dependency on host cellular machinery. Cell-penetrating peptide-peptide nucleic acid conjugates (CPP-PNAs), known for their applicability as antibacterial tools and in the elucidation of protein function, offer a promising alternative to overcome such limitations. Since the application of CPP-PNA in metabolic engineering and pathway elucidation remains largely unexplored, we developed and validated a CPP-PNA platform using Synechocystis sp. PCC 6803 as a model system to demonstrate targeted metabolic pathway evaluation.

Results: High compatibility and dose-dependent permeation efficiency in strain PCC 6803 was first observed when the amphipathic CPP (KFF)₃K was employed, achieving clear cell growth inhibition at 10 µM and above. Specific targeting of D-lactate dehydrogenase (Ddh) using CPP-Syn6803ddh conjugates achieved near-complete protein translation knockdown within 24 h, as confirmed by Western blot analysis. Metabolomics analysis using LC-MS on predetermined metabolites revealed that CPP-PNA treatment produced metabolic effects comparable to stable genetic knockout strains, with both approaches showing a significant 2.5-fold increase in pyruvate accumulation compared to wild-type controls. Further elucidating the reason for pyruvate accumulation, we observed compensatory activation of the glyoxalase pathway at 48 h post-treatment, resulting in 3-fold increased D-lactate production presumably through methylglyoxal detoxification. Validating this observation, RT-qPCR analysis confirmed 2-3-fold upregulation of the glyII gene, encoding for the glyoxalase II (GlyII) enzyme, in both CPP-PNA treated and knockout strains, while double CPP-PNA inhibition experiments targeting both Ddh and glyoxalase pathways suppressed D-lactate accumulation.

Conclusions: This study establishes CPP-PNAs as efficient tools for rapid, and simple metabolic pathway investigation. The approach produces results comparable to conventional genetic knockouts while offering dose-dependent control and avoiding permanent genomic alterations. Our findings reveal unexpected metabolic complexity in Synechocystis sp. PCC 6803 D-lactate synthesis under light conditions and demonstrate the utility of CPP-PNA for uncovering compensatory pathway activation. This platform represents a valuable addition to bacterial genetic engineering, addressing some of the critical limitations faced by conventional approaches, while showing potential for further understanding the biochemistry of metabolite-producing bacteria.

Continuous two-stage E. coli fermentations offer potential for high-efficiency bioprocesses but are often limited by plasmid instability, genetic mutations, and unintended expression during seed phases. In this study, we aimed to overcome these limitations by employing a plasmid-dependent auxotrophic selection system, specifically using a thymidine auxotrophy (thyA deletion), in conjunction with a series of plasmid modifications to enhance stability and expression control. We engineered the E. coli strain enGenes-e^Xpress V2 ΔthyA and constructed modified plasmids containing thyA along with regulatory elements to maintain plasmid stability and minimize basal expression. The modified strains were evaluated in continuous two-stage fermentations under carbon-limited conditions. Our results indicate significant reduction in plasmid loss, improved population homogeneity, and suppressed basal expression in non-induced phases. The addition of elements such as the cer (ColE1 resolution) site and a modified T7 promoter further enhanced plasmid stability and reduced basal expression levels. High-throughput screenings in microbioreactor setups confirmed that optimized constructs maintained a homogeneous producing population and suppressed non-producing cells over extended periods, which was validated by fed-batch cultivations and single-cell analyses. Finally, the two most promising constructs demonstrated high robustness in continuous two-stage chemostat fermentations lasting over 1000 h, maintaining stable GFP titers, plasmid concentrations, and cell dry mass throughout the process. Our findings demonstrate that the auxotrophic marker thyA-based selection system, combined with strategic plasmid modifications, can substantially improve the genetic stability and productivity of E. coli in continuous bioprocesses. This approach provides a robust platform for sustainable, antibiotic- free production in industrial biotechnology, highlighting its potential for scale-up in long-term continuous fermentations.