Microbial Cell Factories最新文献

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.

Background: Nowadays, researchers are attracted to the phyco-synthesis of selenium nanoparticles (SeNPs) for biotechnological and medical applications as they possess many advantages such as safety, nutritional value, and easy biodegradation than gold, copper, and silver nanoparticles. Spirulina platensis is the preferred microalgae for SeNPs synthesis because it contains many compounds that increase their stability making them fit for biomedical treatments.

Results: The biosynthesized Spirulina platensis selenium nanoparticles (SP-SeNPs) were spherical and crystalline, with a diameter of 65 nm and a net charge of -16.7 mV. Furthermore, they were surrounded by active groups responsible for stability. The DPPH radical scavenging test assessed the antioxidant efficacy of SP-SeNPs and exposed scavenging inhibition of 79.234% at a 100 µM dosage. ABTS and H₂O₂ radical scavenging assay is dose-dependent recording IC50 of 50.69 and 116.18 µg/ml, respectively. The antibacterial efficacy was investigated against 13 G-negative & G-positive bacteria. The study demonstrated that SP-SeNPs had antibacterial and antibiofilm efficiencies against the tested strains with MBC of 286-333 µg/ml. The highest percentages of biofilm inhibition were recorded for Bacillus subtilis and Klebsiella pneumoniae, with ratios of 78.8 and 69.9%, respectively. The prepared SP-SeNPS efficiently suppressed the tested fungi growth with MIC (350 µg/ml) and MFCs (480-950 µg/ml). Most notably, biogenic SeNPs effectively extended the clot formation period recording 170.4 S for prothrombin time (PT) and 195.6 S for the activated partial thromboplastin time (aPTT). SP-SeNPs reduced the cell viability of breast adenocarcinoma (MCF-7) and ovarian cancer (SKOV-3) cell lines with a percentage of 17.6009% and 14.9484% at a concentration of 100 ug/ml, respectively. Moreover, SP-SeNPs could effectively alleviate the inflammation in RAW 264.7 macrophages with a reduction percentage of 8.82% in Nitric oxide concentration.

Conclusion: The investigation findings reveal that SP-SeNPs are a hopeful antimicrobial, anti-tumor, anticoagulant, antioxidant, and anti-inflammatory factor that can be applied in medical cures.

Background: β-Caryophyllene, a sesquiterpenoid, holds considerable potential in pharmaceutical, nutraceutical, cosmetic, and chemical industries. In order to overcome the limitation of β-caryophyllene production by the extraction from plants or chemical synthesis, we aimed the microbial production of β-caryophyllene in non-conventional yeast Yarrowia lipolytica in this study.

Results: Two genes, tHMG1 from S. cerevisiae to boost the mevalonate pool and QHS1 from Artemisia annua, were expressed under different promoters and copy numbers in Y. lipolytica. The co-expression of 8UAS pEYK1-QHS1 and pTEF-tHMG1 in the obese strain yielded 165.4 mg/L and 201.5 mg/L of β-caryophyllene in single and double copies, respectively. Employing the same combination of promoters and genes in wild-type-based strain with two copies resulted in a 1.36-fold increase in β-caryophyllene. The introduction of an additional three copies of 8UAS pEYK1-tHMG1 further augmented the β-caryophyllene, reaching 318.5 mg/L in flask fermentation. To maximize the production titer, we optimized the carbon source ratio between glucose and erythritol as well as fermentation condition that led to 798.1 mg/L of β-caryophyllene.

Conclusions: A biosynthetic pathway of β-caryophyllene was firstly investigated in Y. lipolytica in this study. Through the modulation of key enzyme expression, we successfully demonstrated an improvement in β-caryophyllene production. This strategy suggests its potential extension to studies involving the microbial production of various industrially relevant terpenes.

Background: Scientists have faced difficulties in synthesizing natural substances with potent biological activity from cost-effective sources. Endophytic fungi metabolites with nanoparticles have been utilized to develop a friendly, suitable procedure to address this problem and ameliorate the average amount of antioxidant, antimicrobial, and anticancer materials. Therefore, this study utilized endophytic fungi as a source of the natural extract with biosynthesized manganese nanoparticles (MnNPs) in the form of nanocomposites.

Methods: Thirty endophytic fungi were isolated and were assessed for their antioxidant activity by 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) and antimicrobial activity. The most potent isolate was identified utilizing 18S rRNA and was applied to purify and separate their natural antimicrobial products by Flash column chromatography. In addition, the most potent product was identified based on instrumental analysis through Nuclear magnetic resonance (NMR), Fourier-transform infrared (FTIR), and Gas chromatography-mass spectrometry (GC.MS). The purified product was combined with biosynthsesized manganese nanoparticles (MnNPs) for the production of nanocomposite (MnNCs). Later on, the physicochemical features of MnNPs and its MnNCs were examined and then they were assessed for determination their biological activities.

Results: The most potent isolate was identified as Aspergillus terreus with accession number OR243300. The antioxidant and antimicrobial product produced by the strain A. terreus was identified as an amide derivative consisting of 3-(2-Hydroxy-4,4-dimethyl-6-oxo-1-cyclohexen-1-yl)-4-oxopentanoic acid (HDOCOX) with the chemical formula C₁₃H₁₈O₅. Furthermore, purified HDOCOX, MnNPs and Mn-HDOCOX-NPs nanocomposite (MnNCs) showed significant antimicrobial effectiveness. The minimum inhibitory concentrations (MICs) determined for MnNCs were 10 µg/mL against C. albicans and E.coli. Furthermore, MnNCs were reduced hepatocellular carcinoma viability.

Conclusion: The use of HDOCOX, either alone or in combination with MnNPs, is a potential candidate for inhibiting pathogenic microbes and the development of an anticancer drug pipeline.

Acetic acid, a by-product of cytidine synthesis, competes for carbon flux from central metabolism, which may be directed either to the tricarboxylic acid (TCA) cycle for cytidine synthesis or to overflow metabolites, such as acetic acid. In Escherichia coli, the acetic acid synthesis pathway, regulated by the poxB and pta genes, facilitates carbon consumption during cytidine production. To mitigate carbon source loss, the CRISPR-Cas9 gene-editing technique was employed to knock out the poxB and pta genes in E. coli, generating the engineered strains K12ΔpoxB and K12ΔpoxBΔpta. After 39 h of fermentation in 500 mL shake flasks, the cytidine yields of strains K12ΔpoxB and K12ΔpoxBΔpta were 1.91 ± 0.04 g/L and 18.28 ± 0.22 g/L, respectively. Disruption of the poxB and pta genes resulted in reduced acetic acid production and glucose consumption. Transcriptomic and metabolomic analyses revealed that impairing the acetic acid metabolic pathway in E. coli effectively redirected carbon flux toward cytidine biosynthesis, yielding a 5.26-fold reduction in acetate metabolism and an 11.56-fold increase in cytidine production. These findings provide novel insights into the influence of the acetate metabolic pathway on cytidine biosynthesis in E. coli.

Background: Antibiotics have been saving countless lives from deadly infectious diseases, which we now often take for granted. However, we are currently witnessing a significant rise in the emergence of multidrug-resistant (MDR) bacteria, making these infections increasingly difficult to treat in hospitals.

Main text: The discovery and development of new antibiotic has slowed, largely due to reduced profitability, as antibiotics often lose effectiveness quickly as pathogenic bacteria evolve into MDR strains. To address this challenge, metabolic engineering has recently become crucial in developing efficient enzymes and cell factories capable of producing both existing antibiotics and a wide range of new derivatives and analogs. In this paper, we review recent tools and strategies in metabolic engineering and synthetic biology for antibiotic discovery and the efficient production of antibiotics, their derivatives, and analogs, along with representative examples.

Conclusion: These metabolic engineering and synthetic biology strategies offer promising potential to revitalize the discovery and development of new antibiotics, providing renewed hope in humanity's fight against MDR pathogenic bacteria.

Background: Continuous fermentation offers advantages in improving production efficiency and reducing costs, making it highly competitive for industrial ethanol production. A key requirement for Saccharomyces cerevisiae strains used in this process is their tolerance to high ethanol concentrations, which enables them to adapt to continuous fermentation conditions. To explore how yeast cells respond to varying levels of ethanol stress during fermentation, a two-month continuous fermentation was conducted. Cells were collected at different ethanol concentrations (from 60 g/L to 100 g/L) for comparative transcriptomic analysis.

Results: During continuous fermentation, as ethanol concentration increased, the expression of genes associated with cytoplasmic ribosomes, translation, and fatty acid biosynthesis progressively declined, while the expression of genes related to heat shock proteins (HSPs) and ubiquitin-mediated protein degradation gradually increased. Besides, cells exhibited distinct responses to varying ethanol concentrations. At lower ethanol concentrations (nearly 70 g/L), genes involved in mitochondrial ribosomes, oxidative phosphorylation, the tricarboxylic acid (TCA) cycle, antioxidant enzymes, ergosterol synthesis, and glycerol biosynthesis were specifically upregulated compared to those at 60 g/L. This suggests that cells enhanced respiratory energy production, ROS scavenging capacity, and the synthesis of ergosterol and glycerol to counteract stress. At relatively higher ethanol concentrations (nearly 80 g/L), genes involved in respiration and ergosterol synthesis were inhibited, while those associated with glycolysis and glycerol biosynthesis were notably upregulated. This suggests a metabolic shift from respiration towards enhanced glycerol synthesis. Interestingly, the longevity-regulating pathway seemed to play a pivotal role in mediating the cellular adaptations to different ethanol concentrations. Upon reaching an ethanol concentration of 100 g/L, the aforementioned metabolic activities were largely inhibited. Cells primarily focused on enhancing the clearance of denatured proteins to preserve cellular viability.

Conclusions: This study elucidated the mechanisms by which an ethanol-tolerant S. cerevisiae strain adapts to increasing ethanol concentrations during continuous fermentation. The findings suggest that the longevity-regulating pathway may play a critical role in adapting to varying ethanol stress by regulating mitochondrial respiration, glycerol synthesis, ergosterol synthesis, antioxidant enzyme, and HSPs. This work provides a novel and valuable understanding of the mechanisms that govern ethanol tolerance during continuous fermentation.

Previous studies showed that the female genital tract microbiome plays a crucial role in regulating the host's immune defense mechanisms. Our previous research has shown that Lactobacillus gasseri LGV03 (L. gasseri LGV03) isolated from cervico-vagina of HPV-cleared women contributes to clearance of HPV infection and beneficially regulate immune response. However, the mechanisms behind the regulation of L. gasseri LGV03 in immune response remain unclear. To better understand the interaction between female genital tract microbiome and immune function, the immunomodulatory activities of L. gasseri LGV03 were investigated in zebrafish models of neutropenia, macrophage and T cells deficiency. L. gasseri LGV03 showed higher potent activities in ameliorating vinorelbine-induced neutropenia, macrophage and T cells deficiency, and significantly enhanced mRNA expressions of cytokines TNF-α, TNF-β and IFN-α. Moreover, the transcriptome sequencing results indicated L. gasseri LGV03 might alleviate vinorelbine-induced immunosuppression in zebrafish. Non-targeted detection and analysis revealed that indole derivatives including phenylacetaldehyde, 3-phenyllactic acid, N-acetylserotonin and indole-3-lactic acid were significantly increased in the lysate and supernatant of L. gasseri LGV03. Meanwhile, L. gasseri LGV03 supernatant and indole-3-lactic acid ameliorated the vinorelbine-induced reduction in abundance of macrophages, neutrophils and T cells. However, the alleviating effects of L. gasseri LGV03 supernatant or indole-3-lactic acid were eliminated by aryl hydrocarbon receptor (AHR) antagonist CH-223,191. Furthermore, L. gasseri LGV03 supernatant and indole-3-lactic acid significantly increased the secretion of IFN-α, IFN-β and chemokines (MIP-1α, MIP-1β) in Ect1/E6E7 cells, meanwhile, these benefits were eliminated by CH-223,191 treatment. In summary, L. gasseri LGV03-derived indole-3-lactic acid can activate AHR-mediated immune response.