Tumor-targeting engineered probiotic Escherichia coli Nissle 1917 inhibits colorectal tumorigenesis and modulates gut microbiota homeostasis in mice.

AIMS: Preliminary studies have identified the use of probiotics as a potential treatment strategy against colorectal cancer (CRC). However, natural probiotics lack direct tumor-targeting and tumor-killing activity in the intestine. This study aimed to construct a tumor-targeting engineered probiotic to combat CRC. MAIN METHODS: Standard adhesion assay was performed to analyze the adherence ability of tumor-binding protein HlpA to CT26 cells. CCK-8 assay, Hoechst 33258 staining and flow cytometry analysis were used for examining cytotoxicity of tumoricidal protein azurin toward CT26 cells. An engineered probiotic Ep-AH harboring azurin and hlpA genes was developed using Escherichia coli Nissle 1917 (EcN) chassis. Antitumor effects of Ep-AH were evaluated in the azoxymethane (AOM) and dextran sodium sulfate salt (DSS)-induced CRC mice. Moreover, analysis of gut microbiota was conducted via fecal 16S rRNA gene sequencing and shotgun metagenomic sequencing. KEY FINDINGS: Azurin caused a dose-dependent increase of apoptosis in CT26 cells. Ep-AH treatment reversed weight loss (p < 0.001), fecal occult blood (p < 0.01), and shortening of colon length (p < 0.001) than model group, as well as reducing tumorigenesis by 36 % (p < 0.001). Both Ep-H and Ep-A (EcN expressing HlpA or azurin) were less effective than Ep-AH. Furthermore, Ep-AH enriched the members of beneficial bacteria (e.g., Blautia and Bifidobacterium) and reversed abnormal changes of genes associated with several metabolic pathways (e.g., lipopolysaccharide biosynthesis). SIGNIFICANCE: These results demonstrated that Ep-AH had excellent therapeutic benefits on cancer remission and gut microbiota modulation. Our study provides an effective strategy for anti-CRC treatment.

Transcriptome analysis reveals self-redox mineralization mechanism of azo dyes and novel decolorizing hydrolases in Aspergillus tabacinus LZ-M.

Bio-degradation is the most affordable method of azo dye decontamination, while its drawbacks such as aromatic amines accumulation and low degradation efficiency must be overcome. In this study, a novel mechanism of azo dye degradation by a fungus was discovered. At a concentration of 400 mg/L, the decolorization efficiency of Acid Red 73 (AR73) by Aspergillus tabacinus LZ-M was 90.28%. Metabolite analysis and transcriptome sequencing analysis revealed a self-redox process of AR73 degradation, where the electrons generated in carbon oxidation were transferred to the reduction of -C-N = and -NN. The metabolites, 2-hydroxynaphthalene and N-phenylnitrous amide were mineralized into CO2 through catechol pathway and a glycolytic process. Furthermore, the mineralization ratio of dye was computed to be 31.8% by the carbon balance and electron balance. By using comparative transcriptome, a novel decoloring enzyme Ord95 was discovered in unknown genes through gene cloning. It hydrolyzed AR73 into 2-hydroxynaphthalene and N-phenylnitrous amide, containing a glutathione S-transferase domain with three arginines as key active sites. Here the new mechanism of azo dye degradation was discovered with identification of a novel enzyme in Aspergillus tabacinus LZ-M.

Insights into methanogenesis of mesophilic-psychrophilic varied anaerobic digestion of municipal sludge with antibiotic stress.

Methane production through anaerobic digestion (AD) of municipal sludge is economic and eco-friendly, which is commonly affected by temperature and pollutants residues. However, little is known about methanogenesis in psychrophilic AD (PAD) with temperature variations that simulating seasonal variations and with antibiotic stress. Here, two groups of AD systems with oxytetracycline (OTC) were operated with temperature maintained at 35 °C and 15 °C or variation to explore the influence to methanogenesis. The acetic acid was noticeably accumulated in OTC groups initially (P < 0.001). Methane production was noticeably inhibited initially in PAD with OTC, but had been stimulated later at 35 °C. The dominant acetoclastic methanogens Methanosaeta gradually decreased to 15.48% and was replaced by methylotrophic Methanomethylovorans (73.43%) in PAD with OTC. Correspondingly, the abundances of functional genes related to methylotrophic methanogenesis were also higher in these groups. Besides, genes involving in methane oxidation had over 50 times higher abundances in PAD with OTC groups in the second phase. Further investigation is essential to understand the main dynamics of methanogenesis in PAD and to clear the related molecular mechanism for future methane production regulation in sludge systems.

Reduction of metronidazole in municipal wastewater and protection of activated sludge system using a novel immobilized Aspergillus tabacinus LZ-M.

Metronidazole (MNZ) accumulation inhibits municipal wastewater treatment bio-systems, and an effective solution to augment anaerobic activated sludge (AAS) is required. This research discovered that Aspergillus tabacinus LZ-M could degrade 77.39% of MNZ at 5 mg/L. MNZ was metabolized into urea, and the enzymes involved in its degradation were aminotransferase, methyltransferase, monooxygenase, and CN cleavage hydrolase. The strain was immobilized in polyurethane foam and used in AAS for the treatment of MNZ-containing municipal wastewater. The results showed that, using immobilized LZ-M, MNZ was completely removed, and the degradation efficiency of wastewater's chemical oxygen demand (COD) was increased from 11.7% to 83.31%. The extracellular polymer and ROS levels indicated that MNZ's toxicity on AAS was reduced. Furthermore, bioaugmentation stabilized its microbial community, and decreased MNZ resistance genes. These observations confirm that the immobilized fungi are effective in protecting AAS against antibiotic contamination in the treatment process of municipal wastewater.