Hao Cui

In Streptomyces ahygroscopicus, the homologous PAS-LuxR regulators NysRIV and TtmRIV govern nystatin and tetramycin biosynthesis, respectively. To confirm their functional relationship within the same strain, gene deletion, complementation, domain swapping, electrophoretic mobility shift assays, transcriptomics, qRT-PCR, and CoA-derived metabolite measurements were combined to characterize their regulatory roles. Deletion of nysRIV abolished nystatin production, and native nysRIV complementation restored nystatin partially (about 17% of the parental level) in the ΔttmS₁ΔnysRIV background and not at all in ΔnysRIV, indicating that NysRIV is required for full nystatin production. TtmRIV and the two chimeric regulators failed to substitute for NysRIV, indicating limited regulatory interchangeability. Their DNA-binding preference regulators largely followed the origin of their HTH domains, supporting an important contribution of the C-terminal HTH region to differential promoter recognition in vitro. Their effects on polyene production also depended on genetic background. Transcriptomics of the nysRIV deletion mutant revealed changes in lipid, polyketide, pyruvate, and CoA-related metabolism, accompanied by altered acc-gene expression and intracellular CoA-derived metabolite pools. Together, these results indicate distinct but partially overlapping roles of NysRIV and TtmRIV in polyene biosynthesis and highlight the influence of regulator identity and host genetic background on PAS-LuxR regulatory outcomes.

Disaccharide polyene macrolides exhibit superior water solubility and significantly reduced hemolytic toxicity compared to their monosaccharide counterparts, making them promising candidates for safer antifungal therapeutics. In this study, we engineered a Streptomyces gilvosporeus (pSET152-nppY) capable of producing disaccharide-pimaricin (DSP) through heterologous expression of the nppY gene, which encodes a glycosyltransferase responsible for the second sugar extension in the biosynthetic pathway. The novel compound was structurally characterized and designated disaccharide-pimaricin (DSP), featuring an aglycone identical to pimaricin and a unique disaccharide moiety (mycosaminyl-α1–4-N-acetylglucosamine). A purification protocol for DSP was established. Compared to pimaricin, DSP demonstrated a 50% reduction in antifungal activity, a 12.6-fold decrease in hemolytic toxicity, and a remarkable 107.6-fold increase in water solubility. Growth analysis revealed a delayed growth cycle in the mutant strain, suggesting that nppY expression may impose additional metabolic burden. Optimization of the fermentation medium using a statistical design identified an optimal formulation, with a maximum DSP titer of 138.168 mg/L. This study underscores the potential of disaccharide polyene macrolides as safer antifungal agents and establishes a robust framework for engineering strains to produce these compounds. The findings provide critical insights into balancing biosynthetic efficiency and strain fitness, advancing the development of next-generation polyene antibiotics.

The principal objectives of this study encompass three distinct aspects: firstly, to streamline the extraction process of Phragmites communis (Cav) Trin essential oil; secondly, to systematically analyze the essential oil’s chemical composition; and finally, to evaluate biological activity. Through the system optimization experiment, the optimal extraction process was determined: the crushing size was set to 10 mesh, the total solid-liquid ratio was 19:1 (mL/g), and the extraction time limit was 5 hours. Under this condition, the maximum yield of essential oil was 0.233% (mass fraction). In addition, the GC-MS (gas chromatography-mass spectrometry) composition of pure essential reed oil (PCT-Eos) was analysed, and 27 different compounds were detected. The major components included: 2-(2-ethylhexoxycarbonyl) benzoic acid (15.78%), 2,2'-methylenebis (6-tert-butyl- 4-methylphenol) (9.96%), pentacosane (4.97%), triacontane (4.96%), dibutyl phthalate (4.92%), dotriacontane (4.64%), and nonacosane (4.17%). The findings of the activity test demonstrated that this essential oil possessed a moderate antioxidant capacity, with IC50/EC50 values of 2.42 ± 0.311, 3.5 ± 0.306, 6.5 ± 0.395, and 9.8 ± 0.557 mg/mL. In both microdilution assessments, concurrently exhibiting significant inhibitory effects on various pathogenic bacteria, the respective minimum inhibitory concentrations (MICs) were determined to be 72.00, 72.00, 43.20, 9.33, and 120.00 mg/mL. These findings indicate that PCT-Eos possesses significant potential for applications in the food, pharmaceutical, and chemical sectors due to its notable antimicrobial and antioxidant properties.

Streptomyces ahygroscopicus ΔnysB is a genetically engineered strain that produces tetramycin by blocking the competition nystatin pathway. Response Surface Methodology (RSM) was used to optimize its fermentation medium composition. Taking the yield of tetramycin as the response value, the effects of corn starch, glucose, fish peptone, (NH₄)₂SO₄, KH₂PO_4, and CH₃COONa on the yield of tetramycin were investigated by the Plackett-Burman method and three main factors of corn starch, glucose and (NH₄)₂SO₄, were determined. The path of the steepest ascent test was used to approach the maximum response region and the center point was determined. Based on this, the three-factor three-level test was designed by the Box-Behnken center combination. Quadratic regression analysis was performed to calculate the medium with the highest yield of tetramycin and the optimal fermentation medium for tetramycin was formulated as corn starch 25.08 g/L, soybean cake meal 30 g/L, glucose 37.70 g/L, NaCl 0.2 g/L, MgSO4 0.2 g/L, K₂HPO₄ 0.1 g/L, FeSO₄ 0.2 g/L, CaCO₃ 5 g/L, (NH₄)₂SO₄ 0.24 g/L. The yield of tetramycin in the optimized medium reached 167.19 mg/L, which was close to the predicted value of the model. The yield of tetramycin was significantly increased, which was 5.5 times of that before optimization. The results provide a basis for improving the production level of tetramycin.

To explore the flavonoids from Morus alba L. leaves (MLF), the process of extracting was optimized by a response surface methodology and the antimicrobial and antioxidant activities were evaluated in vitro. The yield of flavonoids reached 50.52 mg g⁻¹ under the optimized extraction conditions (i.e., extraction temperature, 70.85 °C; solvent concentration, 39.30%; extraction time, 120.18 min; and liquid/solid ratio, 34.60:1). The total flavonoids were extracted in organic solvents with various polarities, including petroleum ether (MLF_p), ethyl acetate (MLF_e), and n-butanol (MLF_b). In vitro, the four MLF samples exhibited good antioxidant activities for scavenging of 2, 2′-azinobis-(3-ethylbenz-thiazoline-6-sulphonate) radical, 1, 1-diphenyl-2-picrylhydrazyl radical, and total reducing power. Regarding antimicrobial efficacy, the MLF samples suppressed the development of Staphylococcus aureus, Bacillus subtilis, and Bacillus pumilus. The MLF samples inhibited α-amylase activity to a certain extent. The analytical hierarchy process (AHP) was used to evaluate comprehensively the bioactivities of the MLF samples. The AHP results revealed that the bioactivity comprehensive score (78.83 μg mL⁻¹) of MLF_e was optimal among the four MLF samples. Morus alba L. leaves also exhibited non-hemolytic properties. All bioactivities suggested the potential of MLF_e as a candidate resource in the food and drug industries.

Isepamicin is a weakly toxic but highly active aminoglycoside antibiotic derivative of gentamicin B. Gentamicin B is a naturally occurring minor component isolated from Micromonospora echinospora. 2ʹ-NH2-containing gentamicin C complex is a dominant compound produced by wild-type M. echinospora; by contrast, 2ʹ-OH-containing gentamicin B is produced as a minor component. However, the biosynthetic pathway of gentamicin B remains unclear. Considering that gentamicin B shares a unique C2ʹ hydroxyl group with kanamycin A, we aimed to design a new biosynthetic pathway of gentamicin B by combining twelve steps of gentamicin biosynthesis and two steps of kanamycin biosynthesis. We blocked the biosynthetic pathway of byproducts and generated a strain overproducing JI-20A, which was used as a precursor of gentamicin B biosynthesis, by disrupting genK and genP. The amount of JI-20A produced in M. echinospora ∆K∆P reached 911 μg/ml, which was 14-fold higher than that of M. echinospora ∆P. The enzymes KanJ and KanK necessary to convert 2ʹ-NH2 into 2ʹ-OH from the kanamycin biosynthetic pathway were heterologously expressed in M. echinospora ΔKΔP to transform JI-20A into gentamicin B. The strain with kanJK under PermE* could produce 80 μg/ml of gentamicin B, which was tenfold higher than that of the wild-type strain. To enhance gentamicin B production, we employed different promoters and gene integration combinations. When a PhrdB promoter was used and kanJ and kanK were integrated in the genome through gene replacement, gentamicin B was generated as the major product with a maximum yield of 880 μg/ml. We constructed a new biosynthetic pathway of high-level gentamicin B production; in this pathway, most byproducts were removed. This method also provided novel insights into the biosynthesis of secondary metabolites via the combinatorial biosynthesis.