Marine microorganisms are an important source of new drug leads. However, the discovery and sustainable production of these compounds are often hampered due to the unavailable expression of cryptic biosynthetic gene clusters or limited titer. Ribosome engineering and response surface methodology (RSM) integrated strategy was developed in this study to activate cryptic gene cluster in the deepsea-derived Streptomyces somaliensis SCSIO ZH66, and subsequently isolation, structural analysis, and the yield enhancement of the activated compound, anticancer drug lead Fredericamycin A (FDM A), were performed. In order to discover novel natural products from marine Streptomyces strains by genome mining strategy, the deepsea-derived S. somaliensis SCSIO ZH66 was subject to ribosome engineering to activate the expression of cryptic gene clusters. A resistant strain ZH66-RIF1 was thereby obtained with 300 μg/mL rifampicin, which accumulated a brown pigment with cytotoxicity on MS plate while absent in the wild type strain. After screening of fermentation conditions, the compound with pigment was purified and identified to be FDM A, indicating that the activation of a cryptic FDM A biosynthetic gene cluster was taken place in strain ZH66-RIF1, and then it was identified to be ascribed to the mutation of R444H in the β subunit of RNA polymerase. To further improve the yield efficiently, nine fermentation medium components were examined for their significance on FDM A production by Plackett–Burman design and Box-Behnken design. The optimum medium composition was achieved by RSM strategy, under which the titer of FDM A reached 679.5 ± 15.8 mg/L after 7 days of fermentation, representing a 3-fold increase compared to the original medium. In terms of short fermentation time and low-cost fermentation medium, strain ZH66-RIF1 would be an ideal alternative source for FDM A production. Our results would hasten the efforts for further development of FDM A as a drug candidate. Moreover, this ribosome engineering and RSM integrated methodology is effective, fast and efficient; it would be applicable to genome mining for novel natural products from other strains.

Actinomycete genome sequencing has disclosed a large number of cryptic secondary metabolite biosynthetic gene clusters. However, their unavailable or limited expression severely hampered the discovery of bioactive compounds. The whiB-like (wbl) regulatory genes play important roles in morphological differentiation as well as secondary metabolism; and hence the wblA so gene was probed and set as the target to activate cryptic gene clusters in deepsea-derived Streptomyces somaliensis SCSIO ZH66. wblA so from deepsea-derived S. somaliensis SCSIO ZH66 was inactivated, leading to significant changes of secondary metabolites production in the ΔwblA so mutant, from which α-pyrone compound violapyrone B (VLP B) was isolated. Subsequently, the VLP biosynthetic gene cluster was identified and characterized, which consists of a type III polyketide synthase (PKS) gene vioA and a regulatory gene vioB; delightedly, inactivation of vioB led to isolation of another four VLPs analogues, among which one was new and two exhibited improved anti-MRSA (methicillin-resistant Staphylococcus aureus, MRSA) activity than VLP B. Moreover, transcriptional analysis revealed that the expression levels of whi genes (whiD, whiG, whiH and whiI) and wbl genes (wblC, wblE, wblH, wblI and wblK) were repressed by different degrees, suggesting an intertwined regulation mechanism of wblA so in morphological differentiation and secondary metabolism of S. somaliensis SCSIO ZH66. wblA orthologues would be effective targets for activation of cryptic gene clusters in marine-derived Streptomyces strains, notwithstanding the regulation mechanisms might be varied in different strains. Moreover, the availability of the vio gene cluster has enriched the diversity of type III PKSs, providing new opportunities to expand the chemical space of polyketides through biosynthetic engineering.

Type III polyketide synthases (PKSs) are simple homodimer ketosynthases that distribute across plants, fungi, and bacteria, catalyzing formation of pyrone- and resorcinol-types aromatic polyketides with various bioactivities. The broad substrate promiscuity displayed by type III PKSs makes them wonderful candidates for expanding chemical diversity of polyketides. Violapyrone B (VLP B, 10), an α-pyrone compound produced by deepsea-derived Streptomyces somaliensis SCSIO ZH66, is encoded by a type III PKS VioA. We overexpressed VioA in three different hosts, including Streptomyces coelicolor M1146, Streptomyces sanyensis FMA as well as the native producer S. somaliensis SCSIO ZH66, leading to accumulation of different violapyrone compounds. Among them, S. coelicolor M1146 served as the host producing the most abundant violapyrones, from which five new (2–4, 7 and 12) and nine known (1, 5, 6, 8–11, 13 and 14) compounds were identified. Anti-influenza A (H1N1) virus activity of these compounds was then evaluated using ribavirin as a positive control (IC50 = 112.9 μM), revealing that compounds 11–14 showed considerable activity with IC50 values of 112.7, 26.9, 106.7 and 28.8 μM, respectively, which are significantly improved as compared to that of VLP B (10) (IC50 > 200 μM). The productions of 10 and 13 were increased by adding P450 inhibitor metyrapone. In addition, site-directed mutagenesis experiment led to demonstration of the residue S242 to be essential for the activity of VioA. Biological background of the expression hosts is an important factor impacting on the encoding products of type III PKSs. By using S. coelicolor M1146 as cell factory, we were able to generate fourteen VLPs compounds. Anti-H1N1 activity assay suggested that the lipophilic nature of the alkyl chains of VLPs plays an important role for the activity, providing valuable guidance for further structural optimization of VLPs.

The wblA_so gene functions as a global regulatory gene in a negative manner in deepsea-derived Streptomyces somaliensis SCSIO ZH66. A new dioic acid (1) as well as two known butenolides (2 and 3) were isolated from the ΔwblA_so mutant strain of S. somaliensis SCSIO ZH66. The structure of 1 was elucidated by a combination of spectroscopic analyses, including MS and NMR techniques. In the cell growth inhibitory evaluation, compound 3 exhibited moderate activity against the human hepatic carcinoma cell line (Huh7.5) with an IC₅₀ value of 19.4 μg/mL, while compounds 1 and 2 showed null activity up to 100 μg/mL.

Owing to their remarkable biological activities and health benefits, probiotics have gained widespread application in enhancing pet health and welfare. Host-derived probiotics are considered optimal due to their unique digestive tract environments. This study isolated Lactobacillus acidophilus L1 from the feces of a 2-month-old Shiba Inu puppy and conducted a comprehensive evaluation of its potential as a probiotic candidate for pet health. Strain L1 demonstrated high tolerance to acidic conditions (survival rates of 90.41%, 92.90% and 98.81% at pH 2, 2.5, and 3.0, respectively) and bile salts (survival rates of 98.05%, 95.68%, and 82.21% at 0.1%, 0.2%, and 0.3% concentrations, respectively). Adhesion to Caco-2 intestinal epithelial cells reached 38.33%, with hydrophobicity of 97.81% and auto-aggregation of 32.28%. L1 also displayed pronounced antioxidant activity, with DPPH and ABTS radical scavenging rates of 71.15% and 83.20%. Both the bacterial suspension and the cell-free supernatant had potent inhibition of pathogenic bacteria, while the strain showed a non-hemolytic phenotype and remained sensitive to clinically relevant antibiotics (e.g., penicillin). On the other hand, animal experiments conducted in ICR mice (randomly divided into four groups) demonstrated that oral administration of L1 had no toxic effects on the mice and increased serum SOD and CAT levels, while reducing MDA levels. Furthermore, whole-genome sequencing revealed that L1 is 2,106,895 bp in size and contains 2098 coding sequences, two CRISPR arrays, ten genomic islands, and two prophage regions. Collectively, the in vitro and in vivo data presented here indicate that L. acidophilus L1, originally isolated from canine feces, supports further evaluation as a candidate strain for incorporation into functional pet foods.

Breast milk is a major source of probiotics, particularly lactic acid bacteria (LAB), which are known to regulate the intestinal microbial community and exert antibacterial effects. However, little is known about the preventive effects of feline milk-derived LAB against Salmonella infection in vivo. In this study, a strain of Pediococcus acidilactici (M22) was isolated from feline milk and evaluated for its protective potential in C57BL/6 mice challenged with Salmonella Typhimurium SL1344 (VNP20009). Following oral administration of M22, mice were infected with S. Typhimurium, and protective efficacy was assessed through body weight changes, bacterial loads in tissues, histopathological examination of the colon, oxidative stress markers, cytokine profiles, and 16S rRNA gene sequencing of cecal microbiota. The results showed that pretreatment with M22 significantly reduced bacterial loads in the liver, spleen, and cecum compared with controls. M22 administration enhanced antioxidant capacity, alleviated infection-induced inflammation, and preserved intestinal barrier integrity by restoring villus morphology and upregulating tight junction proteins (ZO-1 and occludin). Microbiota analysis further revealed that M22 enriched short-chain fatty acid-producing beneficial taxa (e.g., lactic acid bacteria) while suppressing pro-inflammatory genera. Collectively, these findings provide scientific evidence that feline milk-derived P. acidilactici M22 is a safe and effective probiotic candidate. By enhancing gut health and host resistance to infection, M22 offers a promising strategy to improve companion animal health, reduce reliance on antibiotics, and mitigate zoonotic transmission of pathogens.