Microbial Cell Factories最新文献

The increasing global demand for sustainable protein sources necessitates the exploration of alternative solutions beyond traditional livestock and crop-based proteins. Microalgae present a promising alternative due to their high protein content, rapid biomass accumulation, and minimal land and water requirements. Furthermore, their ability to thrive on non-arable land and in wastewater systems enhances their sustainability and resource efficiency. Despite these advantages, scalability and economical feasibility remain major challenges in microalgal protein production. This review explores recent advancements in microalgal protein cultivation and extraction technologies, including pulsed electric field, ultrasound-assisted extraction, enzyme-assisted extraction, and microwave-assisted extraction. These innovative techniques have significantly improved protein extraction efficiency, purity, and sustainability, while addressing cell wall disruption and protein recovery challenges. Additionally, the review examines protein digestibility and bioavailability, particularly in the context of human nutrition and aquafeed applications. A critical analysis of life cycle assessment studies highlights the environmental footprint and economical feasibility of microalgal protein production compared to conventional protein sources. Although microalgal protein production requires significant energy inputs, advancements in biorefinery approaches, carbon dioxide sequestration, and industrial integration can help mitigate these limitations. Finally, this review outlines key challenges and future research directions, emphasizing the need for cost reduction strategies, genetic engineering for enhanced yields, and industrial-scale process optimization. By integrating innovative extraction techniques with biorefinery models, microalgal proteins hold immense potential as a sustainable, high-quality protein source for food, feed, and nutraceutical applications.

Background: The industrial feasibility of photosynthetic bioproduction using cyanobacterial platforms remains challenging due to insufficient yields, particularly due to competition between product formation and cellular carbon demands across different temporal phases of growth. This study investigates how circadian clock regulation impacts carbon partitioning between storage, growth, and product synthesis in Synechococcus elongatus PCC 7942, and provides insights that suggest potential strategies for enhanced bioproduction.

Results: After entrainment to light-dark cycles, PCC 7942 cultures transitioned to constant light revealed distinct temporal patterns in sucrose production, exhibiting three-fold higher productivity during subjective night compared to subjective day despite moderate down-regulation of genes from the photosynthetic apparatus. This enhanced productivity coincided with reduced glycogen accumulation and halted cell division at subjective night time, suggesting temporal separation of competing processes. Transcriptome analysis revealed coordinated circadian clock-driven adjustment of the cell cycle and rewiring of energy and carbon metabolism, with over 300 genes showing differential expression across four time points. The subjective night was characterized by altered expression of cell division-related genes and reduced expression of genes involved in glycogen synthesis, while showing upregulation of glycogen degradation pathways, alternative electron flow components, the pentose phosphate pathway, and oxidative decarboxylation of pyruvate. These molecular changes created favorable conditions for product formation through enhanced availability of major sucrose precursors (glucose-1-phosphate and fructose-6-phosphate) and maintained redox balance through multiple mechanisms.

Conclusions: Our analysis of circadian regulatory rewiring of carbon metabolism and redox balancing suggests two potential approaches that could be developed for improving cyanobacterial bioproduction: leveraging natural circadian rhythms for optimizing cultivation conditions and timing of pathway induction, and engineering strains that mimic circadian-driven metabolic shifts through controlled carbon flux redistribution and redox rebalancing. While these strategies remain to be tested, they could theoretically improve the efficiency of photosynthetic bioproduction by enabling better temporal separation between cell growth, carbon storage accumulation, and product synthesis phases.

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen associated with healthcare-related infections that are often challenging to treat. Conditions such as, skin and soft tissue infections, bloodstream infections, and pneumonia highlight the critical need for effective therapeutic strategies. Careful use of antibiotics under medical supervision is essential to prevent the further emergence of MRSA. Recent studies have documented the antibacterial efficacy of certain endophytic fungi extracts against MRSA, suggesting their potential as a source of novel treatments. This study investigates the metabolomic profiling of the endophytic fungus Aspergillus sp. SH1 using liquid chromatography-high-resolution electrospray ionization mass spectrometry (LC-HR-ESI-MS) and evaluates the anti-MRSA potential of the fungal extract. The metabolomic analysis identified 27 compounds (1-27) with diverse chemical natures, including polyketides, alkaloids, cyclic tripeptides, polypropionate derivatives, and sesquiterpenes. The fungal extract exhibited potent anti-MRSA activity, with an IC₅₀ value of 9.8 µg/mL, compared to ciprofloxacin (IC₅₀ = 25.7 µg/mL). To support these findings, in silico studies were performed to model the binding interactions of the identified compounds with key MRSA-related targets, including Toll-like receptor 2 (TLR2), von Willebrand factor (VWF), tumor necrosis factor (TNF), and penicillin-binding protein 2a (PBP2a). Compounds 2, 9, 15, 16, 20, 22, and 25 demonstrated enhanced binding affinities, suggesting their potential as lead molecules for developing new antibacterial agents targeting MRSA. In conclusion, this study highlights the promising anti-MRSA potential of Aspergillus sp. SH1 extract, providing a foundation for further exploration of its bioactive compounds in combating resistant bacterial infections.

Context and goal: This study aimed to isolate and optimize a high-yield lipase-producing Pseudomonas aeruginosa strain from biological samples, enhance enzyme production through random mutagenesis, and evaluate its potential anticancer activity. Fifty-one biological samples (blood, urine, sputum, wound pus) were screened, and three isolates demonstrated significant lipase activity. The isolate with the highest activity, identified as P. aeruginosa (GenBank accession number PP436388), was subjected to ethidium bromide-induced mutagenesis, resulting in a two-fold increase in lipase activity (312 U/ml). Lipase production was optimized using submerged fermentation, with critical factors identified statistically as Tween 80, peptone, and substrate concentration. The enzyme was purified via ammonium sulfate precipitation and Sephadex G-100 chromatography, and its molecular weight (53 kDa) was confirmed by SDS-PAGE.

Findings: Optimal conditions for enzyme production included a pH of 9, temperature of 20 °C, and a 24-h incubation period. The partially purified enzyme exhibited high stability at pH values up to 10 and storage temperatures of 4 °C. Anticancer activity was evaluated using the MTT assay, revealing an IC₅₀ of 78.21 U/ml against human hepatocellular carcinoma using HepG-2 cells, with no cytotoxicity observed against Vero cells. Flow cytometry confirmed that the enzyme's anticancer potential was mediated through apoptosis and necrosis. QRT-PCR data revealed that the expression of the Bcl-2 gene was significantly downregulated by 62% (P < 0.05) following the treatment of HepG-2 cells with the lipase enzyme. These findings suggest that lipase from P. aeruginosa holds promise as a novel therapeutic agent for hepatocellular carcinoma, addressing the limitations of current treatments.

Background: Vanillin is a widely utilized flavor compound of significant value in the food and pharmaceutical sectors, which can be obtained through natural extraction, chemical synthesis, or biotechnological processes. However, the yield from vanilla pods is insufficient to meet market demand, and chemically synthesized vanillin not only encounters limitations in its application within the food and pharmaceutical industries but also needs to address environmental concerns and unsustainable raw material sources. Hence, it is imperative to explore alternative approaches to develop an efficient and cost-effective green vanillin. To address the challenges encountered in vanillin biosynthesis, such as substrate uptake limitations and product-induced inhibition of cell growth,we leveraged the advantages of surface display technology and artificial multi-enzyme scaffolds to construct a hybrid surface-display biocatalytic system by assembling Eugenol oxidase (EUGO) and dioxygenase (NOV1), which can convert lignin biowaste 4-n-propylguaiacol (4-PG) into vanillin on the surface of Escherichia coli BL21(DE3).

Results: To assemble bioactive macromolecules of EUGO and NOV1 on the surface of E. coli BL21(DE3), we utilized Lpp-OmpA-SpyCatcher (LOAS) as an anchoring motif and displayed EUGO-linker-NOV1-SpyTag (ELNS) by covalent interaction between SpyTag andSpyCatcher to allow their spatial proximity. After optimization of the reaction system, our self-assembly display system exhibited highly efficiency in converting 4-PG into vanillin and reached a final concentration of vanillin at 12.58 g/L, 2.5 times higher than that achieved by thewhole-cell biocatalytic system. The LOAS-ELNS display system was applied to the sustainable biosynthesis of vanillin from lignin-derived 4-n-propylguaiacol at least 10 times.

Conclusions: This work provided a generalized approach to co-expressing proteins and offered an efficient, eco-friendly, and renewable method for the biosynthesis of vanillin from 4-PG.

Background: Biotechnological applications are steadily growing and have become an important tool to reinvent the synthesis of chemicals and pharmaceuticals for lower dependence on fossil resources. In order to sustain this progression, new feedstocks for biotechnological hosts have to be explored. One-carbon (C₁-)compounds, including formate, derived from CO₂ or organic waste are accessible in large quantities with renewable energy, making them promising candidates. Previous studies showed that introducing the formate assimilation machinery from Methylorubrum extorquens into Escherichia coli allows assimilation of formate through the C₁-tetrahydrofolate (C₁-H₄F) metabolism. Applying this route for formate assimilation, we here investigated utilisation of formate for the synthesis of value-added building blocks in E. coli using S-adenosylmethionine (SAM)-dependent methyltransferases (MT).

Results: We first used a two-vector system to link formate assimilation and SAM-dependent methylation with three different MTs in E. coli BL21. By feeding isotopically labelled formate, methylated products with 51-81% ¹³C-labelling could be obtained without substantial changes in conversion rates. Focussing on improvement of product formation with one MT, we analysed the engineered C₁-auxotrophic E. coli strain C₁S. Screening of different formate concentrations allowed doubling of the conversion rate in comparison to the not formate-supplemented BL21 strain with a share of more than 70% formate-derived methyl groups.

Conclusions: Within this study transformation of formate into methyl groups is demonstrated in E. coli. Our findings support that feeding formate can improve the availability of usable C₁-compounds and, as a result, increase whole-cell methylation with engineered E. coli. Using this as a starting point, the introduction of additional auxiliary enzymes and ideas to make the system more energy-efficient are discussed for future applications.

Background: A variety of probiotics have been utilized as chassis strains and engineered to develop the synthetic probiotics for disease treatment. Among these probiotics, Lactobacilli, which are generally viewed as safe and capable of colonizing the gastrointestinal tract effectively, are widely used. We review recent advancements in the engineering of Lactobacilli for disease treatment. Specifically, the Lactobacilli that are used for the construction of synthetic probiotics, the application of these engineered strains for diseases treatment, and the therapeutic outcomes of these engineered microbes are summarized in this review. Moreover, the applications of these engineered strains for disease treatment are categorized based on their engineering strategies. Of note, we compare the advantages and disadvantages of various engineering strategies and offer insights for the future development of genetically modified Lactobacillus strains with stable and safe properties.

Short conclusion: Our study comprehensively reviews researches on engineering diverse Lactobacillus strains for disease treatment, categorized by their engineering strategies, and emphasizes the importance of developing synthetic probiotics with stable and safe characteristics to enhance their therapeutic applications.

Bacillus cereus 0-9 is a biocontrol microorganism that antagonizes Gram-positive bacteria and pathogenic fungi, such as Staphylococcus aureus and Gaeumannomyces graminis, through the secretion of antimicrobial peptides. However, its low antibacterial activity limits its biocontrol application. In this study, a significant enhancement in antibacterial activity against S. aureus was achieved by overexpressing glucose dehydrogenase from Bacillus subtilis (BsGDH) in B. cereus 0-9, expanding the activity from 6.98 to 11.59 U/mL, representing a 66% improvement. To further improve its biocontrol capability, we aimed to improve the catalytic efficiency of BsGDH by screening 11 low-conserved residues in the protein's second-shell via conservation analysis and molecular docking. Following three rounds of saturation mutagenesis, the specific enzyme activity and K_cat/K_m value of the variant N97F/N192S/E198G reached to 289.74 U/mg and 4.95 µM⁻¹·min⁻¹, representing 5.66 and 11.38 times greater than that of the wild-type BsGDH, respectively. Molecular docking suggested that residues Gly94, Gly14, and Ile191 form a triangular region enhancing substrate affinity and enzymatic activity. Furthermore, the Root Mean Square Fluctuation analysis from molecular dynamics showed significant conformational changes in five regions of the mutants (α2 helix, α3 helix, α5 helix + β4 sheet, α8 helix + β5 sheet, and α13-14 helix), increasing the flexibility of the active pocket. Ultimately, the antibacterial activity of B. cereus 0-9 expressing N97F/N192S/E198G reached 22.79 U/mL, 2.26 times higher than that of B. cereus 0-9. This study offers a promising candidate for enhancing NAD(P)⁺ metabolic cycling and antimicrobial peptide synthesis in cells for industrial applications.

Background: N-glycosylation is a prevalent post-translational modification in eukaryotes, essential for regulating protein secretion. In Saccharomyces cerevisiae, glycosylation mutants have been shown to enhance the secretion of heterologous glycosylated proteins. However, whether these mutants can also increase the secretion of non-glycosylated proteins and whether the growth defects associated with glycosylation mutations can be mitigated remains unclear. This study aimed to characterize and optimize enhanced secretory expression in the promising yeast host Kluyveromyces marxianus by deleting MNN11, which encodes a subunit of the mannose polymerase II complex responsible for elongating α-1,6-linked mannose chains.

Results: Compared to wild-type cells, the mnn11Δ cells significantly increased the secretion activities of four glycosylated enzymes and three non-glycosylated enzymes in flasks, with increases ranging from 29 to 668%. Transcriptomic analysis of mnn11Δ mutant revealed upregulation of genes related to essential protein secretion processes, including vesicle coating and tethering, protein folding, translocation, and glycosylation. Additionally, genes involved in vacuolar amino acid transport and amino acid biosynthesis were upregulated, suggesting an amino acid shortage, which might contribute to the observed severe growth defect of the mnn11Δ mutant in a synthetic medium with inorganic nitrogen. Supplementation of the synthetic medium with amino acids or low concentrations of yeast extract alleviated this growth defect, reducing the specific growth rate difference between wild-type strain and mnn11Δ cells from 65% to as little as 2%. During high-density fermentation, the addition of 0.5% yeast extract substantially reduced the lag phase of mnn11Δ mutants and increased the secretory activities of α-galactosidase, endoxylanase, and β-glucanase, by 11%, 18%, and 36%, respectively, compared to mnn11Δ mutant grown without yeast extract.

Conclusion: In K. marxianus, deletion of MNN11 enhances the secretion of both glycosylated and non-glycosylated proteins by improving key protein secretion processes. The growth defect in the mnn11Δ mutant is closely tied to insufficient amino acid supply. Supplementing the synthetic medium with low concentrations of organic nitrogen sources effectively alleviates this growth defect and enhances secretory expression. This strategy could be applied to optimize the expression of other glycosylation mutants.

Nattokinase, the thrombolytically active substance in the health food natto, nevertheless, its lower thermostability restricts its use in food and pharmaceutical applications. In this study, two heat-resistant variants of nattokinase, designated 50-109 (M1) and 15-271 (M2), were successfully obtained by introducing a disulfide bonding strategy. Their half-lives at 55℃ were found to be 2.50-fold and 5.17-fold higher, respectively, than that of the wild type. Furthermore, the specific enzyme activities of the variants, M1 and M2, were also increased by 2.37 and 1.66-fold, respectively. Meanwhile, the combination of two mutants increased the thermostability of nattokinase by 8.0-fold. Bioinformatics analyses indicated that the enhanced thermostability of the M1 and M2 variants was due to the increased rigidity and structural contraction of the overall structure. Finally, the fermentation process of mutant M1 was optimized to increase the expression of nattokinase. Study provides substantial molecular and theoretical support for the industrial production and application of nattokinase.