Glucose oxidase (GOD) is an important oxidoreductase widely applied in food, pharmaceutical, biofuel, and environmental industries. Previously, we developed a GOD-hyperproducing strain, G1EeSe, by optimizing protein properties, expression cassette, and vesicle trafficking proteins EES and SEC22. However, further enhancement of GOD secretion remained challenging. Here, we developed an effective strategy to boost GOD secretion in G1EeSe by fine-tuning the expression of vesicle trafficking and protein-folding factors. Systematic replacement of AOX1 promoter and terminator regulating EES and SEC22 with various promoter–terminator combinations increased extracellular GOD production by 16% and reduced the repetitive gene sequences. Screening of endoplasmic-reticulum export factors and protein-folding genes identified HAC1, JEM1, and ERO1 as positive regulators of GOD secretion. Moderate overexpression under the P ADH2 promoter proved more effective than the strong P FDH1 promoter. Notably, ERO1 overexpression driven by P ADH2 in strain G6-AERO achieved the highest reported GOD production in shake-flask culture—72,743.9 U/g DCW and 625.5 U/mL—representing 1.5- and 1.4-fold improvements over G1EeSe, respectively. This corresponds to an extracellular GOD concentration of approximately 2.4 g/L derived from the semi-quantitative densitometry. Our work provides a practical approach for enhancing enzyme secretion in a hyperproducing yeast strain and underscores the importance of balanced gene expression when engineering protein folding and trafficking.
Ectoine, a compatible solute and valuable cosmetic additive, is synthesized from L-aspartate-beta-semialdehyde (ASA) via a three-step pathway catalyzed by EctABC. To investigate the metabolic basis of ectoine overproduction in an engineered E. coli strain E2, we employed an integrated analysis of metabolomics and quantitative PCR (qPCR). The results suggest that E2 exhibits a coordinated upregulation of central metabolism: metabolomic profiles showed elevated levels of PPP intermediates and key precursors (aspartate and oxaloacetate), consistent with increased precursor and NADPH supply. qPCR analysis confirmed the upregulated expression of genes involved in the TCA cycle, aspartate biosynthesis, and the Ectabc operon. Based on this multi-omics insight, the competing pathway of ASA toward methionine biosynthesis was selected as a potential target for metabolic intervention via knockout of the metA gene. Based on these findings, a precise metabolic intervention was implemented: knockout of the metA gene to block this diversion, which, supported by an optimized feeding strategy, resulted in a final ectoine titer of 115.52 g/L in the derived strain E2-1 after 72 hours of fermentation. This study provides insights into the metabolic basis of high-yield ectoine production and presents a paradigm of systems-level analysis for targeted engineering, providing a robust framework and a clear theoretical foundation for the rational design of advanced microbial cell factories.
Quorum sensing inhibitors (QSIs) can inhibit the quorum sensing regulated physiological functions of bacteria and might be useful in drug-resistant bacterial infection therapy. The culture medium of a mangrove derived actinobacterium Streptomyces chumphonensis HN2-56, which exhibiting higher anti-quorum sensing activity, was optimized in this study. The results showed that the GS medium was the most favourable medium for the bioactivity of HN2-56 and that the content of soluble starch in the GS medium had a significant impact on the yield of metabolites. By culturing under the optimized condition (soluble starch 40 g/L, KNO3 4 g/L, salinity of 0 and pH 6.5), both metabolites production and anti-quorum sensing activity of HN2-56 against the purple pigment (violacein) production of Chromobacterium violaceum 12472 were significantly increased (41.8% and 177.9%, respectively). Five pure compounds were obtained from the extract of HN2-56 and identified as 2,2-[bis-4-(2,3-dihydroxypropane) phenyl] propane (1), bisphenol A (2), 5-(2-methylphenyl)-4-pentenoic acid (3), 3-methyl-1H-indole-2-carboxylate (4) and dibutyl phthalate (5). Among them, compounds 2 and 3 showed anti-quorum sensing activity against C. violaceum 12472 and anti-biofilm activity against Staphylococcus aureus 43300. These findings indicated that S. chumphonensis HN2-56 should be a promising candidate for QSIs with potential anti-quorum sensing application. Although BPA was detected in the HN2-56 extract, experimental contamination could not be excluded because of the ubiquitous presence of BPA in plasticware and laboratory reagents.
The inflammatory joint diseases are characterized by high prevalence rates and significant burdens in both Bulgaria and Serbia. Regional differences in pricing policy might influence access to therapy. The goal of our study is to compare and analyze the prices, public spending, and utilization of biological disease modifying drugs for the treatment of antirheumatic diseases (bDMARDs) in Bulgaria and Serbia. It is macro costing, cross-country comparative analysis for the period 2016-2020, which is based on a retrospective review of officially published information about prices, public expenditures, and utilization. The reference prices and public expenditures are retrospectively extracted from the national regulatory bodies. Utilization is calculated as the defined daily dose (DDD)/1000 inh/day. Reference prices per DDD decline in both countries during the observed period, mostly visible for bDMARDs with biosimilar alternatives, mainly infliximab, adalimumab, and etanercept. The price differences are statistically significant for 3 International Nonproprietary Names (INNs): infliximab, golimumab, and secukinumab. Public spending and utilization are almost twice higher in Bulgaria. The medicine with the highest utilization in Serbia is infliximab, while in Bulgaria is adalimumab. Pricing policies and new molecules entrance influence utilization and expenditures for bDMARDs positively but improve patients' access. Utilization, access to new molecules and expenditures are higher in Bulgaria, but prices for most bDMARDs are lower in Serbia. Comparative price analysis supports information about the access to medicines and enlightens pricing policies. Further studies are needed in order to examine all the factors affecting medicine prices and public spending.
Snail mucus is a complex multicomponent mixture of proteoglycans, glycosaminoglycans, enzymes, hyaluronic acid, proteins, antimicrobial peptides, amino acids and metal ions. This study presents the chemical and mineral composition of mucus from the garden snail Cornu aspersum and the effectiveness of its active components on wound healing. Three samples of the mucus of the garden snail C. aspersum were analyzed: native mucus (P1), a fraction with active components with a molecular weight (MW) above 30 kDa (P2) and a MW above 10 kDa (P3). Studies with an amino acid analyser, gas chromatography and mass spectrometric analysis indicate that the snail secretion contains a significant amount of minerals and bioactive organic compounds. The cytotoxicity of the extracts on the viability of skin fibroblasts (BJ) and human keratinocytes (HaCaT) was also evaluated. The presence of important peptides and proteins, such as achacin, collagen, elastin, hemocyanin, superoxide dismutase, catalase, and glutathione peroxidase, etc., with proven healing properties, has been proven. The results showed that P1, P2 and P3 were non-toxic to normal human dermal cells at the concentrations analysed and stimulated the proliferation of BJ and HaCaT cells. Treatment of BJ cells with fraction P3 at a concentration of 120 & micro;g/mL leads to a 40% increase in proliferation and showed the strongest stimulating effect on the synthesis of membrane-associated collagen, which was 33% higher in BJ cells compared to control cells. The results confirmed that the mucus of the garden snail C. aspersum contains active components with proven effectiveness on wound healing.
