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As a potential renewable and nitrogen-rich resource, millions of tons of feather waste are generated from poultry farming and this has been steadily increasing. Bioconversion is the most promising low-cost and environmentally benign recycling method, while the efficiency of the isolated natural feather degrading bacteria (FDB) cannot satisfy the requirements of industrial utilization. The process of bacterial degradation of feathers not only requires proteases but also involves complex reduction mechanisms that have not yet been revealed. Previous heterologous expression of keratinase genes in non-FDB hosts achieved only very limited success. In this study, the mechanism of sulfite production via cysteine dioxygenase (CDO1) oxidation was first identified in Streptomyces sp. SCUT-3, which is important for feather disulfide bond reduction. The knockdown of cdo1 significantly decreased the sulfite production (from 38.5 to 22.5 mg L−1) and feather degradation rate (from 45.7% to 39.5%) of SCUT-3 in 5% chicken feather medium (CFM) culture. The overexpression of cdo1 increased bacterial single-cell sulfite production and feather protein conversion efficiency by 3.8 and 2.5 fold, respectively, on day 2 in 5% CFM. On this basis, a new strategy is proposed for increasing feather degradation efficiency by enhancing the reducing power and keratinase activity of FDB according to their own feather degradation mechanism. Thus, the co-overexpression of CDO1 and protease Sep39 successfully increased SCUT-3's feather protein conversion efficiency (42.5% higher than the wild-type) on day 2 in 5% CFM culture. With the co-overexpression strain SCUT-Ocdo1-sep39, 57.5% of proteins in feathers were converted to soluble peptides and free amino acids (0.20 g and 0.29 g g−1 feather, respectively) by solid-state fermentation in 6 d, which was 28.6% higher than that obtained for wild-type SCUT-3. The method developed herein exhibits a much higher economic benefit and a lower ecological footprint than previously reported feather-processing methods and is powerful for industrial recycling of feather waste. This study also elucidates FDB's disulfide bond mechanism and provides a reference for further genetic modification of FDB.

The addition of heavy protein to aquafeed causes low digestibility, high cost and significant nitrogen pollution in the water. The addition of protease is an effective way of improving protein absorption, but few proteases have been developed specifically for aquafeeds. The hydrolysis efficiency of protease additives for plant protein in the neutral gut environment and their synergy with endogenous proteases has not been sufficiently investigated. In this study, the performance of the recombinant neutral protease rNpI as an aquafeed additive to reduce the raw protein materials in the diet of Carassius carassius during 56 days of growth was evaluated. rNpI effectively hydrolysed soya bean protein isolates (SPI) with a high degree of hydrolysation and a high small‐peptide yield. The rNpI‐treated SPI exhibited a notable antioxidant activity, which benefited fish health. In vitro digestibility indicated that rNpI complemented trypsin in SPI digestion better than papain or Alcalase. The growth trial showed that the addition of rNpI could warrant a reduction in the crude protein and gross energy of feed while producing a lower feed‐conversion rate (FCR) and better growth performance. Crucian that were fed an RM‐wheat‐E diet (5.5% rapeseed meal and 5% wheat reduction) showed the best performance, with the lowest FCR and highest weight gain. These results indicate that rNpI could be an effective additive for the aquafeed industry and could provide economic benefits by reducing aquafeed protein requirements and nitrogen emissions. This study provides novel information and strategies for additive protease use and development in aquafeed.

Cryptocaryon irritans is an extremely harmful ciliated obligate parasite that is responsible for large economic losses in aquaculture. C. irritans infection can cause an insect-resistant immune response in fish, and many immune cells can be observed in the local infection site. However, it is unclear whether macrophages are involved in the host defense against C. irritans infection. The Mpeg1 protein can form pores and destroy the cell membrane of invading pathogens, and is also used as a macrophage-specific marker in mammals. Therefore, a polyclonal antibody against grouper recombinant Mpeg1a was produced to mark macrophages in this study, which could recognize both isoforms of Mpeg1 (Mpeg1a/b). Immunofluorescence revealed that EcMpeg1 positive cells were mostly distributed in the head kidney and spleen in healthy grouper. Immunofluorescence and immunohistochemistry showed that the number of EcMpeg1 positive cells increased in the gills after infection with C. irritans, implying that EcMpeg1 positive cells may be involved in the process of grouper resistance against C. irritans infection.

As a potential renewable and nitrogen-rich feedstock, tons of shrimp shell waste is generated from the increasing consumption of seafood. However, its traditional processing is costly and creates pollution. The bioconversion of shell waste into valuable nitrogen-containing chemicals via enzymatic hydrolysis is a promising technology. However, intact shells are poorly hydrolyzed by chitinases unless pre-demineralization and deproteinization with chemicals or proteases are performed. In this study, three carbohydrate-binding modules (CBMs) were fused to the C-terminal of a chitinase Chit46 to enhance its hydrolysis of shrimp shell waste. The addition of CBMs significantly improved the chitinase activity and substrate-binding activity of Chit46. Chimeric chitinases could directly hydrolyze shrimp shells without pretreatment and their hydrolysis efficiency was much higher than that of previously reported chitinases. With the modified chitinase (‘Chit46-CBM3’), we developed a one-step shrimp shell processing method. The method could convert 46.5% of chitin in shrimp shells to chitin oligomers by hydrolysis in 12 h. Partial protein release accompanied chitin hydrolysis. Consisting of 8.8 g l−1 chitin oligomers and 11.3 g l−1 protein, the hydrolysate could support robust microbial growth and the residue was more digestible by mammals than conventional shrimp shell powder. This process is superior to previously reported methods in cost, conversion efficiency and labour. The one-step process also exhibited a much lower ecological footprint than conventional processing and can be applied to other types of chitinous waste in addition to shrimp shells.

Phycosphere hosts the boundary of unique holobionts harboring dynamic algae-bacteria interactions. During our investigating the microbial consortia composition of phycosphere microbiota (PM) derived from diverse harmful algal blooms (HAB) dinoflagellates, a novel rod-shaped, motile and faint yellow-pigmented bacterium, designated as strain LZ-6 T, was isolated from HAB Alexandrium catenella LZT09 which produces high levels paralytic shellfish poisoning toxins. Phylogenetic analysis based on 16S rRNA gene and two housekeeping genes, rpoA and pheS sequences showed that the novel isolate shared the highest gene similarity with Marinobacter shengliensis CGMCC 1.12758 T (99.6%) with the similarity values of 99.6%, 99.9% and 98.5%, respectively. Further phylogenomic calculations of average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values between strains LZ-6 T and the type strain of M. shengliensis were 95.9%, 96.4% and 68.5%, respectively. However, combined phenotypic and chemotaxonomic characterizations revealed that the new isolate was obviously different from the type strain of M. shengliensis. The obtained taxonomic evidences supported that strain LZ-6 T represents a novel subspecies of M. shengliensis, for which the name is proposed, Marinobacter shengliensis subsp. alexandrii subsp. nov. with the type strain LZ-6 T (= CCTCC AB 2018388TT = KCTC 72197 T). This proposal automatically creates Marinobacter shengliensis subsp. shengliensis for which the type strain is SL013A34A2T (= LMG 27740 T = CGMCC 1.12758 T).

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.

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., Li et a. report a new Streptromyces isolate, SCUT-3 which can efficiently degrade feather into products with high amino acid content, useful as feed for plants, animals and microbes. Using multiple omics and other techniques, they report how SCUT-3 turns on its feather utilization machinery and suggest a number of expressed genes most likely implicated in feather degradation.

Antimicrobial peptides (AMPs) are small proteins showing broad-spectrum antimicrobial activity that have been known to be powerful agents against a variety of pathogens (bacteria, fungi and viruses). In this study, the effects of AMPs from Bacillus subtilis on Epinephelus coioides were examined. E. coioides were fed with diets containing AMPs (0, 100, 200, 400 or 800 mg/kg) for four weeks. Results showed that the levels of total protein (TP), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and blood glucose (GLU) and lipopolysaccharide (LPS) in the serum of E. coioides changed than those of the control group; compared to the control group, the levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and lysozyme (LZM) levels in E. coioides fed with different dosages AMP diets were also different; in addition, the mRNA expression of tumor necrosis factor alpha (TNF-α), interleukin-1-beta (IL-1β), and heat shock protein 90 (Hsp90) in the tissues of E. coioides were measured, the three genes in the tissues examined were significantly upregulated. The results demonstrated that diets containing AMPs can enhance the antioxidant capacity and innate immune ability of E. coioides, indicating that AMPs might be a potential alternative to antibiotics in E. coioides.

Ubiquinones (UQ) are important electron transporters and play lot of important roles in most organisms. In different species, UQ was classified to be UQ-6, 7, 8, 9, 10 according to their polyprenyl side chain length. The side chain's length is determined by the enzyme named Poly-Prenyl diphosphate synthases (PPPS). Bacteria are usually reconstructed to producing UQ-10 used in human's food additive, medicine or cosmetics, such as using decaprenyl Diphosphate Synthase (DPS) gene from R. radiobacter to substitute E.coli's octaprenyl diphosphate synthase gene , just like E. coli BL21 (�� ispB::ddsA) used in this study. It is interesting that not only in these reconstructed bacteria, but in human-being, DPS can synthesize UQ-9 besides UQ-10. The mechanism of this phenomenon is still unknown. In this study, the effects of culture conditions, including the temperature, dissolved oxygen, pH and culture medium, on the DPS characteristics in E. coli BL21 (�� ispB::ddsA) were examined. Results show that temperature greatly affects the ratio of UQ-9/UQ-10, but not the total ubiquinone's production. Increasing dissolved oxygen and protein concentration in culture medium can promote total ubiquinone's production, but not the ratio of UQ-9/UQ-10. These results may give reference for UQ-10's industrial produce and the mechanism of these conditions' effect on DPS will be discussed.

A truncated neutral protease I (NpI) from Aspergillus oryzae 3.042 was expressed in Pichia pastoris with a high enzyme yield of 43101 U/mL. Its optimum pH was about 8.0, and it was stable in the pH range of 5.0-9.0. Its optimum temperature was about 55 °C and retained90% activity at 50 °C for 120 min. Recombinant NpI (rNpI) was inhibited by Cu(2+) and EDTA. Eight cleavage sites of rNpI in oxidized insulin B-chain were determined by mass spectrometry, and five of them had high hydrophobic amino acid affinity, which makes it efficient in producing antihypertensive peptide IPP from β-casein and a potential debittering agent. The high degree of hydrolysis (DH) of rNpI to soybean protein (8.8%) and peanut protein (11.1%) compared to papain and alcalase makes it a good candidate in the processing of oil industry byproducts. The mutagenesis of H(429), H(433), and E(453) in the deduced zinc-binding motif confirmed rNpI as a gluzincin. All of these results show the great potential of rNpI to be used in the protein hydrolysis industry.

A leucine aminopeptidase Lap1 was cloned from Aspergillus sojae GIM3.30. The truncated Lap1 without a signal peptide was over-expressed in P. pastoris, and the enzymatic characteristics of recombinant Lap1 (rLap1) were tested. The rLap1 was about 36.7 kDa with an optimal pH 8.0 and optimal temperature 50 °C for substrate Leu-p-nitroanilide and it sustained 50 % activity after 1 h incubation at 50 °C. The activity of rLap1 was significantly inhibited by EDTA, whereas Co(2+), Mn(2+), and Ca(2+) ions, but not Zn(2+) ions, restored its activity. rLap1 showed the highest activity against Arg-pNA and then Leu-, Lys-, Met-, and Phe-pNA. The 3D structure of rLap1 showed it had a conserved functional charge/dipole complex and a hydrogen bond network of Zn2-D179-S228-Q177-D229-S158 around its active center. An acidic Asp residue was found at the bottom of the substrate binding pocket, which explains its preference for basic N-terminal amino acid substrates such as Arg and Lys. rLap1 improved the degree of hydrolysis of casein and soy protein hydrolysates and also decreased their bitterness, indicating its potential utility in food production.