Tsukubarubicin is a nogalamycin-type anthracycline antibiotic with potent cytotoxic activity against human breast cancer cells, making it a promising antitumor candidate. However, its native producer produces only trace amounts of tsukubarubicin and is slow-growing and genetically intractable, limiting further pharmacological evaluation and potential application. To overcome this bottleneck, we developed a multi-layered engineering strategy to improve tsukubarubicin production. The complete tsukubarubicin biosynthetic gene cluster (tsu-BGC) was initially isolated from a PAC library, followed by vector backbone replacement and insertion of an integration module using Red/ET recombineering. The engineered tsu-BGC was introduced into the heterologous host Streptomyces albidoflavus Del14 by conjugation, generating the initial strain D1401. To optimize pathway-specific regulation, the role of the regulatory gene tsuR1 was investigated by gene deletion and overexpression using promoters of different strengths. In situ replacement of the native tsuR1 promoter with the P21 promoter subsequently increased tsukubarubicin production from 3.9 to 37.8 mg/L. Electron beam irradiation mutagenesis was subsequently employed to further improve production, and transcriptomic analysis of the resulting high-producing mutant revealed metabolic changes associated with enhanced tsukubarubicin biosynthesis. Guided by these findings, candidate metabolic pathways were rationally engineered, further improving tsukubarubicin production. The final engineered strain D1404-1 produced 153.0 mg/L tsukubarubicin, representing a 39-fold increase over D1401. This study establishes an efficient heterologous production platform for tsukubarubicin and substantially alleviates the supply limitation associated with its native producer. The combination of regulatory engineering, mutagenesis, and rational metabolic optimization provides an effective strategy for improving the production of tsukubarubicin, which provides a valuable foundation for pharmacological evaluation. Furthermore, this multi-level engineering paradigm offers a versatile reference for the heterologous overproduction of other actinomycete-derived polyketide natural products.
Brevicidine is a promising non-ribosomal antimicrobial peptide with potent activity against Gram-negative pathogens, but its practical development is limited by low natural productivity and insufficient understanding of the physiological basis underlying high-yield phenotypes. In this study, an integrated strategy combining fermentation optimization, atmospheric and room-temperature plasma mutagenesis, and mechanistic analysis was established to improve brevicidine production in Brevibacillus laterosporus 811. Single-factor optimization increased the brevicidine titer to 257.32 mg/L, and subsequent Plackett–Burman and Box–Behnken designs further improved production to 326.05 mg/L. A temperature-shift strategy combined with Tween-80 supplementation further increased the titer to 605.1 mg/L in shake-flask fermentation. In parallel, mutagenesis generated a stable high-yield mutant designated as T3, which showed a 26.6
Maintaining cell envelope integrity and intracellular enzyme stability while alleviating transmembrane mass-transfer limitations is important for sustained γ-aminobutyric acid (GABA) production by whole-cell biocatalysis. We investigated the effects of ethanol on the apparent whole-cell glutamate decarboxylase (GAD) activity of Lactiplantibacillus plantarum SS5 and optimized the corresponding GABA production process. Co-incubation with 6–8
The tumor microenvironment (TME) comprises malignant, stromal, immune, and microbial components. Spatial imaging and molecular profiling have identified intracellular bacteria or bacterial signals in selected tumors, although localization, viability, and functional significance vary across tumor types and experimental settings. Certain tumor-associated bacteria can influence cancer progression and treatment response, motivating strategies for selective microbial intervention. Bacteriophages offer bacterial specificity and opportunities for engineering, but their access to intracellular targets remains a major challenge. This narrative review examines three proposed strategies: lytic elimination of intracellular bacteria, modification of bacterial functions, and bacterial production of therapeutic payloads following phage-mediated delivery. Evidence from mammalian-cell uptake studies, intracellular infection models, and emerging cancer cell-bacterium co-cultures supports individual components of these strategies. However, the complete sequence linking systemic engineered phage delivery, intracellular bacterial clearance, and antitumor immune reprogramming has not been established in orthotopic tumor models or human tumor organoid systems. We therefore present the intracellular scalpel as a testable hypothesis and discuss its potential combinations with established cancer treatments and exploratory adjuvants. Particular attention is given to delivery barriers, evidence limitations, biosafety, and the preclinical measurements required to determine whether intracellular bacterial targeting can improve cancer treatment.
Ethanol production from biomass-derived and gaseous substrates (CO, H2 and CO2) is essential for achieving a carbon-neutral society. The thermophilic acetogen Neomoorella thermoacetica offers notable industrial advantages for converting such diverse feedstocks; however, its physiology and metabolic engineering strategies remain insufficiently understood. Using stoichiometric analysis, we evaluated the metabolic pathways for ethanol production from sugars and gaseous substrates, with particular attention to redox balance and ATP conservation. This analysis suggested that introducing an NADPH-dependent route for reducing acetyl-CoA to ethanol could maintain redox-balanced metabolism under conditions that support sufficient energy generation. Consistent with this prediction, disruption of the major acetate-forming pathway, together with the introduction of heterologous NADPH-dependent reductases, enabled high-selectivity ethanol production (> 90
