Microbial Cell Factories最新文献

Background: Pseudomonas putida KT2440, a non-pathogenic soil bacterium, is a key platform strain in synthetic biology and industrial applications due to its robustness and metabolic versatility. Various systems have been developed for genome editing in P. putida, including transposon modules, integrative plasmids, recombineering systems, and CRISPR/Cas systems. However, rapid iterative genome editing is limited by complex and lengthy processes.

Results: We discovered that the pBBR1MCS2 plasmid carrying the CRISPR/Cas9 module could be easily cured in P. putida KT2440 at 30 ^oC. We then developed an all-in-one CRISPR/Cas9 system for yqhD and ech-vdh-fcs deletions, respectively, and further optimized the editing efficiency by varying homology arm lengths and target sites. Sequential gene deletions of vdh and vanAB were carried out rapidly using single-round processing and easy plasmid curing. This system's user-friendliness was validated by 3 researchers from two labs for 9 deletions, 3 substitutions, and 2 insertions. Finally, iterative genome editing was used to engineer P. putida for valencene biosynthesis, achieving a 10-fold increase in yield.

Conclusions: We developed and applied a rapid all-in-one plasmid CRISPR/Cas9 system for genome editing in P. putida. This system requires less than 1.5 days for one edit due to simplified plasmid construction, electroporation and curing processes, thus accelerating the cycle of genome editing. To our knowledge, this is the fastest iterative genome editing system for P. putida. Using this system, we rapidly engineered P. putida for valencene biosynthesis for the first time, showcasing the system's potential for expanding biotechnological applications.

Background: Ribosome engineering is a semi-empirical technique used to select antibiotic-resistant mutants that exhibit altered secondary metabolism. This method has been demonstrated to effectively select mutants with enhanced synthesis of natural products in many bacterial species, including actinomycetes. Myxobacteria are recognized as fascinating producers of natural active products. However, it remains uncertain whether this technique is similarly effective in myxobacteria, especially for the heterologous production of epothilones in Myxococcus xanthus.

Results: Antibiotics that target the ribosome and RNA polymerase (RNAP) were evaluated for ribosome engineering of the epothilone-producing strain M. xanthus ZE9. The production of epothilone was dramatically altered in different resistant mutants. We screened the mutants resistant to neomycin and rifampicin and found that the yield of epothilones in the resistant mutant ZE9N-R22 was improved by sixfold compared to that of ZE9. Our findings indicate that the improved growth of the mutants, the upregulation of epothilone biosynthetic genes, and specific mutations identified through genome re-sequencing may collectively contribute to the yield improvement. Ultimately, the total titer of epothilones achieved in a 10 L bioreactor reached 93.4 mg/L.

Conclusions: Ribosome engineering is an efficient approach to obtain M. xanthus strains with enhanced production of epothilones through various interference mechanisms. Here, we discuss the potential mechanisms of the semi-empirical method.

17β-estradiol (E2) is an endocrine disruptor, and even trace concentrations (ng/L) of environmental estrogen can interfere with the endocrine system of organisms. Lignin holds promise in enhancing the microbial degradation E2. However, the mechanisms by which lignin facilitates this process remain unclear, which is crucial for understanding complex environmental biodegradation in nature. In this study, we conducted a comprehensive analysis using cellular and lipidomics approaches to investigate the relationship between E2-degrading strain, Rhodococcus sp. RCBS9, and lignin. Our findings demonstrate that lignin significantly enhances E2 degradation efficiency, reaching 94.28% within 5 days with the addition of 0.25 mM lignin. This enhancement is associated with increased microbial growth and activity, reduced of membrane damages, and alleviation of oxidative stress. Fourier Transform Infrared Spectroscopy (FTIR) results indicate that lignin addition alters lipid peaks. Consequently, by analyzing lipid metabolism changes, we further elucidate how lignin addition promotes E2 degradation.

Background: Vitamin K2 is an essential nutrient for blood coagulation and cardiovascular health and mainly produced by bacteria strain like B. subtilis. researchers have explored producing strain improvement, cultivation mode, environmental optimization, increased secretion, and using cheaper carbon and nitrogen sources in order to increase vitamin K2 productivity. This study examines the impact of varioius concentration of soapstock, which is a by-product of vegetable oil refining, as an alternative carbon source with lower pirce, in the fermentation medium instead of glycerol on the microbial synthesis of vitamin K2 using B. subtilis natto ATCC 23857.

Results: The results demonstrate that when the glycerol in fermentation medium was substituted with soapstock, by 75% concentartion, the fermentation process produced a yield of 158.16 mg/L of vitamin K2 after 72 h; This was 3.8 times more than the control medium containing glycerol. When the entire culture medium was replaced with wastewater, the vitamin K2 concentration reached 21.18 mg/L, 52% of the control medium's concentration. If the carbon sources in the fermentation medium consisted of 20% soapstock and 47.4 g/L glycerol (maintaining the same final glycerol concentration as the control medium), the vitamin K2 concentration reached 35.7 mg/L or 85.8% of the control medium. The analysis of soapstock fermentation medium characteristics reveals that after fermentation with B. subtilis, the COD of soapstock fermentation medium was dramatically reduced from 259,500 mg/L to 57,830 mg/L.

Conclusions: Using soapstock as an alternative carbon source for fermentation did not negatively impact the bioprocess and increased vitamin K2 production. Therefore, this research introduces an alternative carbon resource for vitamin K2 production and paves the way for the biorefinement of soapstock.

Background: Fire blight, caused by Erwinia amylovora, poses a significant threat to global agriculture, with antibiotic-resistant strains necessitating alternative solutions such as phage therapy. Scaling phage therapy to an industrial level requires efficient mass-production methods, particularly in optimizing the seed culture process. In this study, we investigated large-scale E. amylovora phage production by optimizing media supplementation and fermenter conditions, focusing on minimizing seed phages and pathogenic strains to reduce risks and improve the seed culture process.

Results: We optimized the phage inoculum concentrations and media supplements to achieve higher phage yields comparable to or exceeding conventional methods. Laboratory-scale validation and refinement for fermenter-scale production allowed us to reduce bacterial and phage inoculum levels to 10⁵ CFU/mL and 10³ PFU/mL, respectively. Using fructose and sucrose supplements, the yields were comparable to conventional methods that use 10⁸ CFU/mL host bacteria and 10⁷ PFU/mL phages. Further pH adjustments in the fermenter increased yields by 16-303% across all phages tested.

Conclusions: We demonstrated the successful optimization and scale-up of E. amylovora phage production, emphasizing the potential for industrial bioprocessing with the reduced use of host cells and phage seeds. Overall, by refining key production parameters, we established a robust and scalable method for enhancing phage production efficiency.

Background: Biocatalysis offers a potentially greener alternative to chemical processes. For biocatalytic systems requiring cofactor recycling, hydrogen emerges as an attractive reducing agent. Hydrogen is attractive because all the electrons can be fully transferred to the product, and it can be efficiently produced from water using renewable electricity. In this article, resting cells of Cupriavidus necator H16 harboring a NAD-dependent hydrogenase were employed for cofactor recycling to reduce D-xylose to xylitol, a commonly used sweetener. To enable this bioconversion, D-xylose reductase from Scheffersomyces stipitis was heterologously expressed in C. necator.

Results: D-xylose reductase was successfully expressed in C. necator, enabling almost complete bioconversion of 30 g/L of D-xylose into xylitol. It was found that over 90% of the energy and protons derived from hydrogen were spent for the bioconversion, demonstrating the efficiency of the system. The highest xylitol productivity reached was 0.7 g/L/h. Additionally, the same chassis efficiently produced L-arabitol and D-ribitol from L-arabinose and D-ribose, respectively.

Conclusions: This study highlights the efficient utilization of renewable hydrogen as a reducing agent to power cofactor recycling. Hydrogen-oxidizing bacteria, such as C. necator, can be promising hosts for performing hydrogen-driven biocatalysis.

This comprehensive review explores the emergence of titanium dioxide nanoparticles (TiO₂-NPs) as versatile nanomaterials, particularly exploring their biogenic synthesis methods through different biological entities such as plants, bacteria, fungi, viruses, and algae. These biological entities provide eco-friendly, cost-effective, biocompatible, and rapid methods for TiO₂-NP synthesis to overcome the disadvantages of traditional approaches. TiO₂-NPs have distinctive properties, including high surface area, stability, UV protection, and photocatalytic activity, which enable diverse applications. Through detailed analysis, this review demonstrates significant applications of green fabricated TiO₂-NPs in biomedicine, explicitly highlighting their antimicrobial, anticancer, and antioxidant activities, along with applications in targeted drug delivery, photodynamic therapy, and theragnostic cancer treatment. Additionally, the review underscores their pivotal significance in biosensors, bioimaging, and agricultural applications such as nanopesticides and nanofertilizers. Also, this review proves valuable incorporation of TiO₂-NPs in the treatment of contaminated soil and water with various environmental contaminants such as dyes, heavy metals, radionuclides, agricultural effluents, and pathogens. These comprehensive findings establish the foundation for future innovations in nanotechnology, underscoring the importance of further investigating bio-based synthetic approaches and bioactivity mechanisms to enhance their efficacy and safety across healthcare, agricultural, and environmental applications.

Bacterial biofilms pose significant challenges, from healthcare-associated infections to biofouling in industrial systems, resulting in significant health impacts and financial losses globally. Classic antimicrobial methods often fail to eradicate sessile microbial communities within biofilms, requiring innovative approaches. This review explores the structure, formation, and role of biofilms, highlighting the critical importance of exopolysaccharides in biofilm stability and resistance mechanisms. We emphasize the potential of microbial enzymatic approaches, particularly focusing on glycosidases, proteases, and deoxyribonucleases, which can disrupt biofilm matrices effectively. We also delve into the importance of enzymes such as cellobiose dehydrogenase, which disrupts biofilms by degrading polysaccharides. This enzyme is mainly sourced from Aspergillus niger and Sclerotium rolfsii, with optimized production strategies enhancing its efficacy. Additionally, we explore levan hydrolase, alginate lyase, α-amylase, protease, and lysostaphin as potent antibiofilm agents, discussing their microbial origins and production optimization strategies. These enzymes offer promising avenues for combating biofilm-related challenges in healthcare, environmental, and industrial settings. Ultimately, enzymatic strategies present environmentally friendly solutions with high potential for biofilm management and infection control.

The conversion of CO₂ into methanol depicts one of the most promising emerging renewable routes for the chemical and biotech industry. Under this regard, native methylotrophs have a large potential for converting methanol into value-added products but require targeted engineering approaches to enhance their performances and to widen their product spectrum. Here we use a systems-based approach to analyze and engineer M. extorquens TK 0001 for production of glycolic acid. Application of constraint-based metabolic modeling reveals the great potential of M. extorquens for that purpose, which is not yet described in literature. In particular, a superior theoretical product yield of 1.0 C-mol_{Glycolic acid} C-mol_Methanol^-1 is predicted by our model, surpassing theoretical yields of sugar fermentation. Following this approach, we show here that strain engineering is viable and present 1st generation strains producing glycolic acid via a heterologous NADPH-dependent glyoxylate reductase. It was found that lactic acid is a surprising by-product of glycolic acid formation in M. extorquens, most likely due to a surplus of available NADH upon glycolic acid synthesis. Finally, the best performing strain was tested in a fed-batch fermentation producing a mixture of up to total 1.2 g L^-1 glycolic acid and lactic acid. Several key performance indicators of our glycolic acid producer strain are superior to state-of-the-art synthetic methylotrophs. The presented results open the door for further strain engineering of the native methylotroph M. extorquens and pave the way to produce two promising biopolymer building blocks from green methanol, i.e., glycolic acid and lactic acid.