Immunomodulatory Effects of N-Acetyl Chitooligosaccharides on RAW264.7 Macrophages.

The ongoing development of new production methods may lead to the commercialization of N-acetyl chitooligosaccharides (NACOS), such as chitosan oligosaccharides (COS). The bioactivity of NACOS, although not well detailed, differs from that of COS, as they have more acetyl groups than COS. We used two enzymatically produced NACOS with different molecular compositions and six NACOS (NACOS1-6) with a single degree of polymerization to verify their immunomodulatory effects on the RAW264.7 macrophage cell line. We aimed to identify any differences between COS and various NACOS with a single degree of polymerization. The results showed that NACOS had similar immune enhancement effects on RAW264.7 cells as COS, including the generation of reactive oxygen species (ROS), phagocytotic activity, and the production of pro-inflammation cytokines (IL-1ß, IL-6, and TNF-α). However, unlike COS and lipopolysaccharide (LPS), NACOS1 and NACOS6 significantly inhibited nitric oxide (NO) production. Besides their immune enhancement effects, NACOS also significantly inhibited the LPS-induced RAW264.7 inflammatory response with some differences between various polymerization degrees. We confirmed that the NF-κB pathway is associated with the immunomodulatory effects of NACOS on RAW264.7 cells. This study could inform the application of NACOS with varying different degrees of polymerization in human health.

Role of major histocompatibility complex II antigen-presentation pathway genes in orange-spotted grouper infected with Cryptocaryon irritans.

Cryptocaryon irritans, a pathogen model for fish mucosal immunity, causes skin mucosal and systematic humoral immune response. Where and how MHC II antigen presentation occurs in fish infected with C. irritans remain unknown. In this study, the full-length cDNA of the grouper cysteine protease CTSS was cloned. The expression distributions of six genes (CTSB, CTSL, CTSS, GILT, MHC IIA and MHC IIB) involved in MHC II antigen presentation pathway were tested. These genes were highly expressed in systematic immune tissues and skin and gill mucosal-associated immune tissues. All six genes were upregulated in skin at most time points. Five genes expected CTSS was upregulated in spleen at most time points. CTSB, CTSL and MHC IIA were upregulated in the gill and head kidney at some time points. These results indicate that the presentation of MHC II antigen intensively occurred in local infected skin and gill. Spleen, not head kidney, had the most extensive systematic antigen presentation. In skin, six genes most likely peaked at day 2, earlier than in spleen (5-7 days), marking an earlier skin antibody peak than any recorded in serum previously. This significant and earlier mucosal antigen presentation indicates that specific immune response occurs in local mucosal tissues.

Sustainable valorizing high-protein feather waste utilization through solid-state fermentation by keratinase-enhanced Streptomyces sp. SCUT-3 using a novel promoter.

Feather waste, a rich source of proteins, has traditionally been processed through high-temperature puffing and acid-base hydrolysis, contributing to generation of greenhouse gases and H2S. To address this issue, we employed circular economy techniques to recover the nutritional value of feather waste. Streptomyces sp. SCUT-3, an efficient proteolytic and chitinolytic bacterium, was isolated for feather degradation previously. This study aimed to valorize feather waste for feed purposes by enhancing its feather transformation ability through promoter optimization. Seven promoters were identified through omics analysis and compared to a common Streptomyces promoter ermE*p. The strongest promoter, p24880, effectively enhanced the expression of three candidate keratinases (Sep39, Sep40, and Sep53). The expression efficiency of double-, triple-p24880 and sandwich p24880-sep39-p24880 promoters were further verified. The co-overexpression strain SCUT-3-p24880-sep39-p24880-sep40 exhibited a 16.21-fold increase in keratinase activity compared to the wild-type. Using this strain, a solid-state fermentation process was established that increased the feather/water ratio (w/w) to 1:1.5, shortened the fermentation time to 2.5 days, and increased soluble peptide and free amino acid yields to 0.41 g/g and 0.14 g/g, respectively. The resulting has high protein content (90.49 %), with high in vitro digestibility (94.20 %). This method has the potential to revolutionize the feather waste processing industry.

Integrative Analysis of the Microbiome and Metabolome of Broiler Intestine: Insights into the Mechanisms of Probiotic Action as an Antibiotic Substitute.

Antibiotic substitutes have become a research focus due to restrictions on antibiotic usage. Among the antibiotic substitutes on the market, probiotics have been extensively researched and used. However, the mechanism by which probiotics replace antibiotics remains unclear. In this study, we aimed to investigate this mechanism by comparing the effects of probiotics and antibiotics on broiler growth performance and intestinal microbiota composition. Results shown that both probiotics and antibiotics increased daily weight gain and reduced feed conversion rate in broilers. Analysis of ileum and cecum microorganisms via 16S rRNA gene sequencing revealed that both interventions decreased intestinal microbial diversity. Moreover, the abundance of Bacteroides increased in the mature ileum, while that of Erysipelatoclostridium decreased in the cecum in response to both probiotics and antibiotics. The main metabolites of probiotics and antibiotics in the intestine were found to be organic acids, amino acids, and sugars, which might play comparable roles in growth performance. Furthermore, disaccharides and trisaccharides may be essential components in the ileum that enable probiotics to replace antibiotics. These findings provide important insights into the mechanisms underlying the use of probiotics as antibiotic substitutes in broiler breeding.

Degradation of indole via a two-component indole oxygenase system from Enterococcus hirae GDIAS-5.

Animal farming copiously generates indoles, which contribute to odor and pose a challenge for deodorization. While biodegradation is widely accepted, there is a lack of suitable indole-degrading bacteria for animal husbandry. In this study, we aimed to construct genetically engineered strains with indole-degrading abilities. Enterococcus hirae GDIAS-5 is a highly efficient indole-degrading bacterium, which functions via a monooxygenase YcnE presumably contributes to indole oxidation. However, the efficiency of engineered Escherichia coli expressing YcnE for indole degradation is lower than that of GDIAS-5. To improve its efficacy, the underlying indole-degradation mechanisms in GDIAS-5 were analyzed. An ido operon that responds to a two-component indole oxygenase system was identified. In vitro experiments showed that the reductase component of YcnE, YdgI, can improve the catalytic efficiency. The reconstruction of the two-component system in E. coli exhibited higher indole removal efficiency than GDIAS-5. Furthermore, isatin, the key intermediate metabolite in indole degradation, might be degraded via a novel isatin-acetaminophen-aminophenol pathway involving an amidase whose coding gene is located near the ido operon. The two-component anaerobic oxidation system, upstream degradation pathway, and engineering strains investigated in this study provide important insights into indole degradation metabolism and offer efficient resources for achieving bacterial odor elimination.

Synergic chitin degradation by Streptomyces sp. SCUT-3 chitinases and their applications in chitinous waste recycling and pathogenic fungi biocontrol.

The genus Streptomyces comprises the most important chitin decomposers in soil and revealing their chitinolytic machinery is beneficial for the conversion of chitinous wastes. Streptomyces sp. SCUT-3, a chitin-hydrolyzing and a robust feather-degrading bacterium, was isolated previously. The potential chitin-degrading enzymes produced by SCUT-3 were analyzed in the present study. Among these enzymes, three chitinases were successfully expressed in Pichia pastoris at comparatively high yields of 4.8 U/mL (SsExoChi18A), 11.2 U/mL (SsExoChi18B), and 17.8 U/mL (SsEndoChi19). Conserved motifs and constructive 3D structures of these three exo- and endochitinases were also analyzed. These chitinases hydrolyzed colloidal chitin to chitin oligomers. SsExoChi18A showed apparent synergic effects with SsEndoChi19 in colloidal chitin and shrimp shell hydrolysis, with an improvement of 29.3 % and 124.9 %, respectively. Compared with SsExoChi18B and SsEndoChi19, SsExoChi18A exhibited the strongest antifungal effects against four plant pathogens by inhibiting mycelial growth and spore germination. This study provided good candidates for chitinous waste-processing enzymes and antifungal biocontrol agents. These synergic chitin-degrading enzymes of SCUT-3 are good targets for its further genetical modification to construct super chitinous waste-degrading bacteria with strong abilities to hydrolyze both protein and chitin, thereby providing a direction for the future path of the chitinous waste recycling industry.

Indole metabolism mechanisms in a new, efficient indole-degrading facultative anaerobe isolate Enterococcus hirae GDIAS-5.

Indole is an inter-species and inter-kingdom signaling molecule widespread in the natural world. A large amount of indole in livestock wastes makes it difficult to be degraded, which causes serious malodor. Identifying efficient and eco-friendly ways to eliminate it is an urgent task for the sustainable development of husbandry. While bioconversion is a widely accepted means, the mechanism of indole microbial degradation is little understood, especially under anaerobic conditions. Herein, a new Enterococcus hirae isolate GDIAS-5, effectively degraded 100 mg/L indole within 28 h aerobically or 5 days anaerobically. Three intermediates (oxindole, isatin, and catechol) were identified in indole degradation, and catechol was further degraded by a meta-cleavage catabolic pathway. Two important processes for GDIAS-5 indole utilization were discovered. One is Fe(III) uptake and reduction, which may be a critical process that is coupled with indole oxidation, and the other is the entire pathway directly involved in indole oxidation and metabolism. Furthermore, monooxygenase ycnE responsible for indole oxidation via the indole-oxindole-isatin pathway was identified for the first time. Bioinformatic analyses showed that ycnE from E. hirae formed a phylogenetically separate branch from monooxygenases of other species. These findings provide new targets and strategies for synthetic biological reconstruction of indole-degrading bacteria.

The feather degradation mechanisms of a new Streptomyces sp. isolate SCUT-3.

Feather waste is the highest protein-containing resource in nature and is poorly reused. Bioconversion is widely accepted as a low-cost and environmentally benign process, but limited by the availability of safe and highly efficient feather degrading bacteria (FDB) for its industrial-scale fermentation. Excessive focuses on keratinase and limited knowledge of other factors have hindered complete understanding of the mechanisms employed by FDB to utilize feathers and feather cycling in the biosphere. Streptomyces sp. SCUT-3 can efficiently degrade feather to products with high amino acid content, useful as a nutrition source for animals, plants and microorganisms. Using multiple omics and other techniques, we reveal how SCUT-3 turns on its feather utilization machinery, including its colonization, reducing agent and protease secretion, peptide/amino acid importation and metabolism, oxygen consumption and iron uptake, spore formation and resuscitation, and so on. This study would shed light on the feather utilization mechanisms of FDBs.

Heterologous expression and characterization of an antifungal chitinase (Chit46) from Trichoderma harzianum GIM 3.442 and its application in colloidal chitin conversion.

In this study, a chitinase gene, Chit46 from a mycoparasitic fungus Trichoderma harzianum was successfully expressed in Pichia pastoris with a high heterologous chitinase production of 31.4 U/mL, much higher than the previous reports. The active center and substrate binding pocket of the recombinant Chit46 (rChit46) were analyzed and the effects of pH, temperature, metal ions and glycosylation on its activity were tested. rChit46 effectively hydrolyzed colloidal chitin with a high conversion rate of 80.5% in 3 h and the chitin hydrolysates were mainly composed of (GlcNAc)2 (94.8%), which make it a good candidate for the green recycling of chitinous waste. rChit46 could also significantly inhibit growth of the phytopathogenic fungus Botrytis cinerea, which endowed it with the potential as a biocontrol agent.

Biocontrol activity of recombinant aspartic protease from Trichoderma harzianum against pathogenic fungi.

The use of cell wall degrading enzymes of Trichoderma is a promising alternative for improving food storage. The aspartic protease P6281 secreted by the fungus Trichoderma harzianum plays an important role in mycoparasitism on phytopathogenic fungi. In this study, recombinant P6281 (rP6281) expressed in Pichia pastoris showed high activity of 321.8 U/mL. Maximum activity was observed at pH 2.5 and 40 °C, and the enzyme was stable in the pH range of 2.5-6.0. rP6281 significantly inhibited spore germination and growth of plant and animal pathogenic fungi such as Botrytis cinerea, Mucor circinelloides, Aspergillus fumigatus, Aspergillus flavus, Rhizoctonia solani, and Candida albicans. Transmission electron microscopy revealed that rP6281 efficiently damages the cell wall of Botrytis cinerea. In addition, the protease significantly inhibited the development of grey mold that causes rotting of apple, orange, and cucumber, indicating that rP6281 may be developed as an effective anti-mold agent for fruit storage.