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The canine anti‐tumor necrosis factor‐alpha (TNF‐α) monoclonal antibody is a potential therapeutic option for treating canine arthritis. The current treatments for arthritis in dogs have limitations due to side effects, emphasizing the need for safer and more effective therapies. The crystal structure of canine TNF‐α (cTNF‐α) was successfully determined at a resolution of 1.85 Å, and the protein was shown to assemble as a trimer, with high similarity to the functional quaternary structure of human TNF‐α (hTNF‐α). Adalimumab (Humira), a known TNF‐α inhibitor, effectively targets and neutralizes TNF‐α to reduce inflammation and has been used to manage autoimmune conditions such as rheumatoid arthritis. By comparing the structure of cTNF‐α with the complex structure of hTNF‐α and adalimumab‐Fab, the epitope of adalimumab on cTNF‐α was identified. The significant structural similarities of epitopes in cTNF‐α and hTNF‐α indicate the potential of using adalimumab to target cTNF‐α. Therefore, a canine/human chimeric antibody, Humivet‐R1, was created by grafting the variable domain of adalimumab onto a canine antibody framework derived from ranevetmab. Humivet‐R1 exhibits potent neutralizing ability (IC50 = 0.05 nM) and high binding affinity (EC50 = 0.416 nM) to cTNF‐α, comparable to that of adalimumab for both hTNF‐α and cTNF‐α. These results strongly suggest that Humivet‐R1 has the potential to provide effective treatment for canine arthritis with reduced side effects. Here, we propose a structure‐guided antibody design for the use of a chimeric antibody to treat canine inflammatory disease. Our successful development strategy can speed up therapeutic antibody discovery for animals and has the potential to revolutionize veterinary medicine. [ABSTRACT FROM AUTHOR]

Fishmeal (FM) is considered a preferred source of protein for aquatic feed due to its good palatability and digestibility, a balanced amino acid profile, and a low level of anti-nutritional factors (ANFs). Shortage of FM supply and increases in price, however, are major challenges faced by feed producers and aquaculture farmers. Fermented soybean meal (FSBM) has been proposed to partially replace FM because of its high nutritional value and low cost. The optimal FSBM concentration for substituting FM and effects of FSBM substitution on growth performance, energy metabolism, and immune response nevertheless remain unclear. We evaluated the effects of shrimp diets containing FSBM at 0% (FM), 25% (FSBM25), 50% (FSBM50), 75% (FSBM75), and 100% (FSBM100) on the shrimp Penaeus vannamei Boone, 1931. FSBM50 led to a significantly higher weight gain (postlarvae 1927.36 ± 46.64%, young juveniles 237.00 ± 8.25%, and juveniles 153.96 ± 2.18%) than the other experimental and control diets in different stages of the shrimp. The FM group consumed less oxygen than the experimental diet groups. FSBM75 and FSBM100 led to a significantly higher ammonia-N excretion than FM, FSBM25, and FBSM50. All experimental diet groups exhibited a significantly higher O:N ratio (8.3 ± 0.8–9.4 ± 1.7) than the FM group (7.1 ± 0.2). Shrimp fed with FSBM25- and FSBM50-containing diets exhibited an increased total hemocyte count, and FSBM25, FBSM50, and FSBM100 led to a significantly higher superoxide anion production than FSBM75 and FM. FSBM substitution in shrimp diets resulted in a significantly higher phagocytic activity than the FM diet, suggesting that FSBM substitution improves the cell-mediated immune response in shrimps. In challenge trials with white spot syndrome virus (WSSV), all experimental diets led to a higher survival rate than the FM diet, and in Vibrio parahaemolyticus challenge trials, FSBM50 led to a higher survival rate than the other experimental and control diets, indicating that FSBM substitution in shrimp diets could improve the defense capacity of shrimps against WSSV and V. parahaemolyticus. [ABSTRACT FROM AUTHOR]

Abstract Background To achieve an economical cellulosic ethanol production, a host that can do both cellulosic saccharification and ethanol fermentation is desirable. However, to engineer a non-cellulolytic yeast to be such a host requires synthetic biology techniques to transform multiple enzyme genes into its genome. Results A technique, named Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO), that employs overlapping oligonucleotides for recombinatorial assembly of gene cassettes with individual promoters, was developed. PGASO was applied to engineer Kluyveromycesmarxianus KY3, which is a thermo- and toxin-tolerant yeast. We obtained a recombinant strain, called KR5, that is capable of simultaneously expressing exoglucanase and endoglucanase (both of Trichodermareesei), a beta-glucosidase (from a cow rumen fungus), a neomycin phosphotransferase, and a green fluorescent protein. High transformation efficiency and accuracy were achieved as ~63% of the transformants was confirmed to be correct. KR5 can utilize beta-glycan, cellobiose or CMC as the sole carbon source for growth and can directly convert cellobiose and beta-glycan to ethanol. Conclusions This study provides the first example of multi-gene assembly in a single step in a yeast species other than Saccharomyces cerevisiae. We successfully engineered a yeast host with a five-gene cassette assembly and the new host is capable of co-expressing three types of cellulase genes. Our study shows that PGASO is an efficient tool for simultaneous expression of multiple enzymes in the kefir yeast KY3 and that KY3 can serve as a host for developing synthetic biology tools.

Abstract Background Cellulose, which is the most abundant renewable biomass on earth, is a potential bio-resource of alternative energy. The hydrolysis of plant polysaccharides is catalyzed by microbial cellulases, including endo-β-1,4-glucanases, cellobiohydrolases, cellodextrinases, and β-glucosidases. Converting cellobiose by β-glucosidases is the key factor for reducing cellobiose inhibition and enhancing the efficiency of cellulolytic enzymes for cellulosic ethanol production. Results In this study, a cDNA encoding β-glucosidase was isolated from the buffalo rumen fungus Neocallimastix patriciarum W5 and is named NpaBGS. It has a length of 2,331 bp with an open reading frame coding for a protein of 776 amino acid residues, corresponding to a theoretical molecular mass of 85.1 kDa and isoelectric point of 4.4. Two GH3 catalytic domains were found at the N and C terminals of NpaBGS by sequence analysis. The cDNA was expressed in Pichia pastoris and after protein purification, the enzyme displayed a specific activity of 34.5 U/mg against cellobiose as the substrate. Enzymatic assays showed that NpaBGS was active on short cello-oligosaccharides from various substrates. A weak activity in carboxymethyl cellulose (CMC) digestion indicated that the enzyme might also have the function of an endoglucanase. The optimal activity was detected at 40°C and pH 5 ~ 6, showing that the enzyme prefers a weak acid condition. Moreover, its activity could be enhanced at 50°C by adding Mg2+ or Mn2+ ions. Interestingly, in simultaneous saccharification and fermentation (SSF) experiments using Saccharomyces cerevisiae BY4741 or Kluyveromyces marxianus KY3 as the fermentation yeast, NpaBGS showed advantages in cell growth, glucose production, and ethanol production over the commercial enzyme Novo 188. Moreover, we showed that the KY3 strain engineered with the NpaNGS gene can utilize 2 % dry napiergrass as the sole carbon source to produce 3.32 mg/ml ethanol when Celluclast 1.5 L was added to the SSF system. Conclusion Our characterizations of the novel β-glucosidase NpaBGS revealed that it has a preference of weak acidity for optimal yeast fermentation and an optimal temperature of ~40°C. Since NpaBGS performs better than Novo 188 under the living conditions of fermentation yeasts, it has the potential to be a suitable enzyme for SSF.

Abstract: Cellulose is a renewable feedstock for green industry. It is therefore important to develop a technique to construct a host with a high cellulolytic efficiency to digest cellulose. In this study, we developed a convenient host‐engineering technique to adjust the expression levels of heterologous genes in the host by promoter rearrangement and gene copy number adjustment. Using genes from different glycoside hydrolase (GH) families including GH2, GH3, GH5, GH6, GH7, and GH12 from Aspergillus niger, Trichoderma reesei, and Neocallimastix patriciarum, we constructed a cellulolytic Kluyveromyces marxianus with eight cellulase gene‐cassettes that produced a cellulase cocktail with a high cellulolytic efficiency, leading to a significant reduction in enzyme cost in a rice straw saccharification process. Our technique can be used to design a host that can efficiently convert biomass feedstock to biofuel. [ABSTRACT FROM AUTHOR]