De novo lipogenesis(DNL)plays an important role in the pathogenesis of hepatic steatosis and also appears to be implicated in hepatic inflammation and fibrosis.Accordingly,the inhibition of acetyl-CoA carboxylase,whic...De novo lipogenesis(DNL)plays an important role in the pathogenesis of hepatic steatosis and also appears to be implicated in hepatic inflammation and fibrosis.Accordingly,the inhibition of acetyl-CoA carboxylase,which catalyzes the ratelimiting step of DNL,might represent a useful approach in the management of patients with nonalcoholic fatty liver disease(NAFLD).Animal studies and preliminary data in patients with NAFLD consistently showed an improvement in steatosis with the use of these agents.However,effects on fibrosis were variable and an increase in plasma triglyceride levels was observed.Therefore,more longterm studies are needed to clarify the role of these agents in NAFLD and to determine their risk/benefit profile.展开更多
Objective:To characterize the Entamoeba histolytica(E.histolytica)antigen(s)recognized by moribound amoebic liver abscess hamsters.Methods:Crude soluble antigen of E.histolytica was probed with sera of moribund hamste...Objective:To characterize the Entamoeba histolytica(E.histolytica)antigen(s)recognized by moribound amoebic liver abscess hamsters.Methods:Crude soluble antigen of E.histolytica was probed with sera of moribund hamsters in 1D-and 2D-Westem blot analyses.The antigenic protein was then sent for tandem mass spectrometry analysis.The corresponding gene was cloned and expressed in Escherichia coli BL21-AI to produce the recombinant E.histolytica ADP-forming acetyl-CoA synthetase(EhACS)protein.A customised ELISA was developed to evaluate the sensitivity and specificity of the recombinant protein.Results:A^75 kDa protein band with a pl value of 5.91-6.5 was found to be antigenic;and not detected by sera of hamsters in the control group.Tandem mass spectrometry analysis revealed the protein to be the 77 kDa E.histolytica ADP-forming acetyl-CoA synthetase(EhACS).The customised ELISA results revealed 100%sensitivity and 100%specificity when tested against infected(n=31)and control group hamsters(n=5)serum samples,respectively.Conclusions:This rinding suggested the significant role of EhACS as a biomarker for moribund hamsters with acute amoebic liver abscess(ALA)infection.It is deemed pertinent that future studies explore the potential roles of EhACS in better understanding the pathogenesis of ALA;and in the development of vaccine and diagnostic tests to control ALA in human populations.展开更多
The entire gene of carboxyltransferase（CT） domain of acetyl-CoA carboxylase（ACCase） from Chinese Spring wheat（CSW） plastid was cloned firstly, and the 2.3 kb gene was inserted into PET28a^＋ vector and expressed...The entire gene of carboxyltransferase（CT） domain of acetyl-CoA carboxylase（ACCase） from Chinese Spring wheat（CSW） plastid was cloned firstly, and the 2.3 kb gene was inserted into PET28a^＋ vector and expressed in E. coil in a soluble state. The （His）6 fusion protein was identified by SDS-PAGE and Western blot. The recombinant protein was purified by affinity chromatography, and the calculated molecular mass（Mr） was 88000. The results of the sequence analysis indicate that the cloned gene（GeneBank accession No. EU124675） was a supplement and revision of the reported ACCase CT partial cDNA from Chinese Spring wheat plastid. The recombinant protein will be significant for us to investigate the recognizing mechanism between ACCase and herbicides, and further to screen new herbicides.展开更多
Synthetic biology is constantly making progress for producing compounds on demand.Recently,Yocum and collaborators have developed an outstanding approach based on the anchoring of biosynthetic enzymes to the peroxisom...Synthetic biology is constantly making progress for producing compounds on demand.Recently,Yocum and collaborators have developed an outstanding approach based on the anchoring of biosynthetic enzymes to the peroxisomal membrane.This allowed access to an untapped resource of acetyl-CoA and stimulated the synthesis of a valuable polyketide.展开更多
This paper focuses on the group of metalloproteins/metalloenzymes in the acetyl-coenzyme A synthesis pathway of anaerobic microbes called Wood-Ljungdahl pathway,including formate dehydrogenase (FDH),corrinoid iron sul...This paper focuses on the group of metalloproteins/metalloenzymes in the acetyl-coenzyme A synthesis pathway of anaerobic microbes called Wood-Ljungdahl pathway,including formate dehydrogenase (FDH),corrinoid iron sulfur protein (CoFeSP),acetyl-CoA synthase (ACS) and CO dehydrogenase (CODH). FDH,a key metalloenzyme involved in the conversion of carbon dioxide to methyltetrahydrofolate,catalyzes the reversible oxidation of formate to carbon dioxide. CoFeSP,as a methyl group transformer,accepts the methyl group from CH3-H4 folate and then transfers it to ACS. CODH reversibly catalyzes the reduction of CO2 to CO and ACS functions for acetyl-coenzyme A synthesis through condensation of the methyl group,CO and coenzyme A,to finish the whole pathway. This paper introduces the structure,function and reaction mechanisms of these enzymes.展开更多
10-DeacetylbaccatinⅢ(10-DAB)C10 acetylation is an indispensable procedure for Taxol semi-synthesis,which often requires harsh conditions.10-DeacetylbaccatinⅢ-10-β-O-acetyltransferase(DBAT)catalyzes the acetylation ...10-DeacetylbaccatinⅢ(10-DAB)C10 acetylation is an indispensable procedure for Taxol semi-synthesis,which often requires harsh conditions.10-DeacetylbaccatinⅢ-10-β-O-acetyltransferase(DBAT)catalyzes the acetylation but acetyl-CoA supply remains a key limiting factor.Here we refactored the innate biosynthetic pathway of acetyl-CoA in Escherichia coli and obtained a chassis with acetyl-CoA productivity over three times higher than that of the host cell.Then,we constructed a microbial cell factory by introducing DBAT gene into this chassis for efficiently converting 10-DAB into baccatinⅢ.We found that baccatinⅢcould be efficiently deacetylated into 10-DAB by DBAT with CoASH and K+under alkaline condition.Thus,we fed acetic acid to the engineered strain both for serving as a substrate of acetyl-CoA biosynthesis and for alleviating the deacetylation of baccatinⅢ.The fermentation conditions were optimized and the baccatinⅢtiters reached 2,3 and 4.6 g/L,respectively,in a 3-L bioreactor culture when 2,3 and 6 g/L of 10-DAB were supplied.Our study provides an environmentfriendly approach for the large scale 10-DAB acetylation without addition of acetyl-CoA in the industrial Taxol semi-synthesis.The finding of DBAT deacetylase activity may broaden its application in the structural modification of pharmaceutically important lead compounds.展开更多
文摘De novo lipogenesis(DNL)plays an important role in the pathogenesis of hepatic steatosis and also appears to be implicated in hepatic inflammation and fibrosis.Accordingly,the inhibition of acetyl-CoA carboxylase,which catalyzes the ratelimiting step of DNL,might represent a useful approach in the management of patients with nonalcoholic fatty liver disease(NAFLD).Animal studies and preliminary data in patients with NAFLD consistently showed an improvement in steatosis with the use of these agents.However,effects on fibrosis were variable and an increase in plasma triglyceride levels was observed.Therefore,more longterm studies are needed to clarify the role of these agents in NAFLD and to determine their risk/benefit profile.
基金Supported by Malaysia Ministry of Education Long-Term Research Grant Scheme(LRGS)No.203/PSK/6722002
文摘Objective:To characterize the Entamoeba histolytica(E.histolytica)antigen(s)recognized by moribound amoebic liver abscess hamsters.Methods:Crude soluble antigen of E.histolytica was probed with sera of moribund hamsters in 1D-and 2D-Westem blot analyses.The antigenic protein was then sent for tandem mass spectrometry analysis.The corresponding gene was cloned and expressed in Escherichia coli BL21-AI to produce the recombinant E.histolytica ADP-forming acetyl-CoA synthetase(EhACS)protein.A customised ELISA was developed to evaluate the sensitivity and specificity of the recombinant protein.Results:A^75 kDa protein band with a pl value of 5.91-6.5 was found to be antigenic;and not detected by sera of hamsters in the control group.Tandem mass spectrometry analysis revealed the protein to be the 77 kDa E.histolytica ADP-forming acetyl-CoA synthetase(EhACS).The customised ELISA results revealed 100%sensitivity and 100%specificity when tested against infected(n=31)and control group hamsters(n=5)serum samples,respectively.Conclusions:This rinding suggested the significant role of EhACS as a biomarker for moribund hamsters with acute amoebic liver abscess(ALA)infection.It is deemed pertinent that future studies explore the potential roles of EhACS in better understanding the pathogenesis of ALA;and in the development of vaccine and diagnostic tests to control ALA in human populations.
基金Supported by the National Natural Science Foundation of China(Nos. 20432010, 20672045 and 30570405)
文摘The entire gene of carboxyltransferase（CT） domain of acetyl-CoA carboxylase（ACCase） from Chinese Spring wheat（CSW） plastid was cloned firstly, and the 2.3 kb gene was inserted into PET28a^＋ vector and expressed in E. coil in a soluble state. The （His）6 fusion protein was identified by SDS-PAGE and Western blot. The recombinant protein was purified by affinity chromatography, and the calculated molecular mass（Mr） was 88000. The results of the sequence analysis indicate that the cloned gene（GeneBank accession No. EU124675） was a supplement and revision of the reported ACCase CT partial cDNA from Chinese Spring wheat plastid. The recombinant protein will be significant for us to investigate the recognizing mechanism between ACCase and herbicides, and further to screen new herbicides.
基金funding from the EU Horizon 2020 research and innovation program(MIAMi project-Grant agreement N°814645)ARD-CVL Biopharmaceutical program of the Region Centre Val de Loire(ETOPOCentre project),Le Studium Institute(Consortium Fellowship)and ANR(project MIACYC-ANR-20-CE43-0010).
文摘Synthetic biology is constantly making progress for producing compounds on demand.Recently,Yocum and collaborators have developed an outstanding approach based on the anchoring of biosynthetic enzymes to the peroxisomal membrane.This allowed access to an untapped resource of acetyl-CoA and stimulated the synthesis of a valuable polyketide.
基金Supported by the National Natural Science Foundation of China (Grant No. 20771029)Shanghai Pujiang Talent Project (Grant No. 08PJ14017)Shanghai Leading Academic Discipline Project (Grant No. B108)
文摘This paper focuses on the group of metalloproteins/metalloenzymes in the acetyl-coenzyme A synthesis pathway of anaerobic microbes called Wood-Ljungdahl pathway,including formate dehydrogenase (FDH),corrinoid iron sulfur protein (CoFeSP),acetyl-CoA synthase (ACS) and CO dehydrogenase (CODH). FDH,a key metalloenzyme involved in the conversion of carbon dioxide to methyltetrahydrofolate,catalyzes the reversible oxidation of formate to carbon dioxide. CoFeSP,as a methyl group transformer,accepts the methyl group from CH3-H4 folate and then transfers it to ACS. CODH reversibly catalyzes the reduction of CO2 to CO and ACS functions for acetyl-coenzyme A synthesis through condensation of the methyl group,CO and coenzyme A,to finish the whole pathway. This paper introduces the structure,function and reaction mechanisms of these enzymes.
基金supported by the National Key Research and Development Program of China(grant Nos.2018YFA0901900 and 2020YFA0908003)the Drug Innovation Major Project(grant No.2018ZX09711001-006-001,China)+2 种基金the National Natural Science Foundation of China(grant No.81573325)the CAMS Innovation Fund for Medical Sciences(CIFMS,(grant No.2017-I2M-2-004,2019-I2M-1-005,China)PUMC Disciplinary Development of Synthetic Biology(201920100801,China)。
文摘10-DeacetylbaccatinⅢ(10-DAB)C10 acetylation is an indispensable procedure for Taxol semi-synthesis,which often requires harsh conditions.10-DeacetylbaccatinⅢ-10-β-O-acetyltransferase(DBAT)catalyzes the acetylation but acetyl-CoA supply remains a key limiting factor.Here we refactored the innate biosynthetic pathway of acetyl-CoA in Escherichia coli and obtained a chassis with acetyl-CoA productivity over three times higher than that of the host cell.Then,we constructed a microbial cell factory by introducing DBAT gene into this chassis for efficiently converting 10-DAB into baccatinⅢ.We found that baccatinⅢcould be efficiently deacetylated into 10-DAB by DBAT with CoASH and K+under alkaline condition.Thus,we fed acetic acid to the engineered strain both for serving as a substrate of acetyl-CoA biosynthesis and for alleviating the deacetylation of baccatinⅢ.The fermentation conditions were optimized and the baccatinⅢtiters reached 2,3 and 4.6 g/L,respectively,in a 3-L bioreactor culture when 2,3 and 6 g/L of 10-DAB were supplied.Our study provides an environmentfriendly approach for the large scale 10-DAB acetylation without addition of acetyl-CoA in the industrial Taxol semi-synthesis.The finding of DBAT deacetylase activity may broaden its application in the structural modification of pharmaceutically important lead compounds.
