Rotavirus(RV) is a major foodborne pathogen. For RV prevention and control, it is a key to uncover the interaction mechanism between virus and its receptors. However, it is hard to specially purify the viral receptors...Rotavirus(RV) is a major foodborne pathogen. For RV prevention and control, it is a key to uncover the interaction mechanism between virus and its receptors. However, it is hard to specially purify the viral receptors, including histo-blood group antigens(HBGAs). Previously, the protruding domain protein(P protein) of human norovirus(genotype Ⅱ.4) was displayed on the surface of Escherichia coli, and it specifically recognized and captured the viral ligands. In order to further verify the feasibility of the system, P protein was replaced by VP8* of RV(G9 P[8]) in this study. In the system, VP8*could be correctly released by thrombin treatment with antigenicity retaining, which was confirmed by Western blot and Enzyme-Linked Immunosorbent Assays. Type A HBGAs from porcine gastric mucin(PGM) were recognized and captured by this system. From saliva mixture, the captured viral receptor bound with displayed VP8* was confirmed positive with monoclonal antibody against type A HBGAs. It indicated that the target ligands could be easily separated from the complex matrix. These results demonstrate that the bacterial surface display system will be an effective platform to explore viral receptors/ligands from cell lines or food matrix.展开更多
Triacylglycerol(TAG)is crucial for antibiotic biosynthesis derived from Streptomyces,as it serves as an important carbon source.In this study,the supplementation of exogenous TAG led to a 3.92-fold augmentation in spi...Triacylglycerol(TAG)is crucial for antibiotic biosynthesis derived from Streptomyces,as it serves as an important carbon source.In this study,the supplementation of exogenous TAG led to a 3.92-fold augmentation in spinosad production.The impact of exogenous TAG on the metabolic network of Saccharopolyspora spinosa were deeply analyzed through comparative proteomics.To optimize TAG metabolism and enhance spinosad biosynthesis,the lipase-encoding genes lip886 and lip385 were overexpressed or co-expressed.The results shown that the yield of spinosad was increased by 0.8-fold and 0.4-fold when lip886 and lip385 genes were overexpressed,respectively.Synergistic co-expression of these genes resulted in a 2.29-fold increase in the yield of spinosad.Remarkably,the combined overexpression of lip886 and lip385 in the presence of exogenous TAG elevated spinosad yields by 5.5-fold,led to a drastic increase in spinosad production from 0.036 g/L to 0.234 g/L.This study underscores the modification of intracellular concentrations of free fatty acids(FFAs),short-chain acyl-CoAs,ATP,and NADPH as mechanisms by which exogenous TAG modulates spinosad biosynthesis.Overall,the findings validate the enhancement of TAG catabolism as a beneficial strategy for optimizing spinosad production and provide foundational insights for engineering secondary metabolite biosynthesis pathways in another Streptomyces.展开更多
基金the National Key Research and Development Program of China(2017YFF0210200)the National Natural Science Foundation of China(31772078).
文摘Rotavirus(RV) is a major foodborne pathogen. For RV prevention and control, it is a key to uncover the interaction mechanism between virus and its receptors. However, it is hard to specially purify the viral receptors, including histo-blood group antigens(HBGAs). Previously, the protruding domain protein(P protein) of human norovirus(genotype Ⅱ.4) was displayed on the surface of Escherichia coli, and it specifically recognized and captured the viral ligands. In order to further verify the feasibility of the system, P protein was replaced by VP8* of RV(G9 P[8]) in this study. In the system, VP8*could be correctly released by thrombin treatment with antigenicity retaining, which was confirmed by Western blot and Enzyme-Linked Immunosorbent Assays. Type A HBGAs from porcine gastric mucin(PGM) were recognized and captured by this system. From saliva mixture, the captured viral receptor bound with displayed VP8* was confirmed positive with monoclonal antibody against type A HBGAs. It indicated that the target ligands could be easily separated from the complex matrix. These results demonstrate that the bacterial surface display system will be an effective platform to explore viral receptors/ligands from cell lines or food matrix.
基金supported by funding from the National Natural Science Foundation of China(31770106,32200062)the Natural Science Foundation of Hunan Province(2024JJ5258).
文摘Triacylglycerol(TAG)is crucial for antibiotic biosynthesis derived from Streptomyces,as it serves as an important carbon source.In this study,the supplementation of exogenous TAG led to a 3.92-fold augmentation in spinosad production.The impact of exogenous TAG on the metabolic network of Saccharopolyspora spinosa were deeply analyzed through comparative proteomics.To optimize TAG metabolism and enhance spinosad biosynthesis,the lipase-encoding genes lip886 and lip385 were overexpressed or co-expressed.The results shown that the yield of spinosad was increased by 0.8-fold and 0.4-fold when lip886 and lip385 genes were overexpressed,respectively.Synergistic co-expression of these genes resulted in a 2.29-fold increase in the yield of spinosad.Remarkably,the combined overexpression of lip886 and lip385 in the presence of exogenous TAG elevated spinosad yields by 5.5-fold,led to a drastic increase in spinosad production from 0.036 g/L to 0.234 g/L.This study underscores the modification of intracellular concentrations of free fatty acids(FFAs),short-chain acyl-CoAs,ATP,and NADPH as mechanisms by which exogenous TAG modulates spinosad biosynthesis.Overall,the findings validate the enhancement of TAG catabolism as a beneficial strategy for optimizing spinosad production and provide foundational insights for engineering secondary metabolite biosynthesis pathways in another Streptomyces.
