Bacteroides thetaiotaomicron colonizes the human gastrointestinal tract and establishes a symbiotic relationship with the host,contributing to reducing intestinal inflammation and enhancing resistance against foreign ...Bacteroides thetaiotaomicron colonizes the human gastrointestinal tract and establishes a symbiotic relationship with the host,contributing to reducing intestinal inflammation and enhancing resistance against foreign pathogens.Recent reports have revealed that diverse lipid species such as glycerophospholipids,sphingolipids,and N-acyl amines exist in B.thetaiotaomicron and play essential roles in the immune process.In this research,total lipids obtained from B.thetaiotaomicron were purified via thin-layer chromatography,and the species and molecular structures of visible lipids in different hydrophobic regions were qualitatively characterized by high-performance liquid chromatography-mass spectrometry.The results indicated that seven lipid species were primarily displayed on the plate,including phosphatidylethanolamine,ethanolamine phosphoryl dihydroceramide,inositol phosphoryl dihydroceramide,glycyl-serine phosphoryl dihydroceramide,phosphatidylglycerol,cardiolipin,and glycyl-serine phosphoryl diacylglycerol.The phosphatidylethanolamine,ethanolamine phosphoryl dihydroceramide,and inositol phosphoryl dihydroceramide species corresponding to ion peaks at m/z 676.48,691.53,and 796.53 exhibited significantly high abundance compared to other species,suggesting their prevalent presence in total lipids.The molecular structures of phosphatidylethanolamine and ethanolamine phosphoryl dihydroceramide were derived from the modification of diacylglycerol and dihydroceramide with phosphoethanolamine,while the structure of inositol phosphoryl dihydroceramide was derived from the modification of dihydroceramide with phosphoinositol.The phosphatidylglycerol and cardiolipin species corresponding to m/z 721.51 and 1323.94 have been detected in the membrane lipids of B.thetaiotaomicron,although they were not mentioned in previous studies.These findings are important for understanding the molecular mechanisms of B.thetaiotaomicron colonization in mammalian gut.展开更多
2′-Fucosyllactose is the most abundant human milk oligosaccharides and one of the three essential nutrients for infant growth.Corynebacterium glutamicum is one of the most common industrial fermentation bacteria but ...2′-Fucosyllactose is the most abundant human milk oligosaccharides and one of the three essential nutrients for infant growth.Corynebacterium glutamicum is one of the most common industrial fermentation bacteria but cannot synthesize 2′-fucosyllactose.In this study,C.glutamicum ATCC13032 was engineered for 2′-fucosyllactose production from fucose and lactose.The gene futC from Helicobacter pylori encodingα-1,2-fucosyltransferase was codon optimized and mutated at four amino acids(F40S/Q150H/C151R/Q239S).The modified gene futC and the gene fkp from Bacteroides thetaiotaomicron encoding fucokinase/GDP-fucose pyrophosphorylase were overexpressed in plasmid pEC and transformed into C.glutamicum,resulting in CW002.CW002 did not synthesize 2′-fucosyllactose possibly because the substrates fucose and/or lactose did not pass through the cell membrane.Therefore,the gene lacY encoding lactose permease and the gene fucP encoding fucose permease from Escherichia coli were overexpressed in plasmid pXTuf and transformed into CW002,resulting in CW006.CW006 did synthesize 2′-fucosyllactose as expected.It is interesting that the production of 2′-fucosyllactose was decreased or stopped when the expression combination of these four genes was changed,suggesting that the expression levels of the four genes in CW006 might have to well balanced.C.glutamicum CW006 produced 2.07 g/L 2′-fucosyllactose in a 2.4 L bioreactor.展开更多
The human intestinal symbiotic microorganism Bacteroides thetaiotaomicron has a unique lipooligosaccharide structure,which promotes its beneficial symbiosis with the host.But its synthesis mechanism is not fully under...The human intestinal symbiotic microorganism Bacteroides thetaiotaomicron has a unique lipooligosaccharide structure,which promotes its beneficial symbiosis with the host.But its synthesis mechanism is not fully understood.In this study,protein sequence alignment revealed that the protein encoded by B.thetaiotaomicron VPI 5482 BT_2747 gene shares 24%sequence identity with Escherichia coli WaaA.The expression vector was used to overexpress BT_2747 in E.coli lipopolysaccharide mutant strains constructed by knocking out the waaC-waaF,lpxL or lpxM genes,resulting in the recombinant strains WH001(ΔwaaA)/pBT2747,WL003(ΔwaaAwaaC-F)/pBT2747,WL004(ΔwaaAwaaC-FlpxL)/pBT2747,WL005(ΔwaaAwaaC-FlpxM)/pBT2747 and WL006(ΔwaaAwaaC-FlpxLlpxM)/pBT2747.Lipid A/Kdo-lipid A were extracted from these recombinant strains and analyzed by liquid chromatography-mass spectrometry.The results showed that BT_2747 could convert a portion of the lipid IVA into Kdo-lipid IVA,but no Kdo2-lipid IVA structure was detected in E.coli.Small amounts of hexa-acylated Kdo-lipid A were also detected in the recombinant strains WH001/pBT2747 and WL003/pBT2747,and a small amount of penta-acylated Kdo-lipid A was found in WL004/pBT2747.The recombinant strain was further modified by introducing lpxF that enabled to synthesize Kdo-lipid IVA-1-phosphate.The results revealed that the BT_2747 gene in B.thetaiotaomicron VPI 5482 encodes the Kdo transferase of lipid A which uses lipid IVA as a substrate and only transfers single Kdo residue to lipid IVA.This study extends our understanding of the Kdo-lipid A structure and synthesis mechanism of B.thetaiotaomicron,which might provide a new perspective on how intestinal commensal bacteria regulate host immune homeostasis through unique Kdo-lipid A structure.展开更多
基金supported by the Research Program of State Key Laboratory of Food Science and Resources,Jiangnan University(SKLF-ZZB-202417).
文摘Bacteroides thetaiotaomicron colonizes the human gastrointestinal tract and establishes a symbiotic relationship with the host,contributing to reducing intestinal inflammation and enhancing resistance against foreign pathogens.Recent reports have revealed that diverse lipid species such as glycerophospholipids,sphingolipids,and N-acyl amines exist in B.thetaiotaomicron and play essential roles in the immune process.In this research,total lipids obtained from B.thetaiotaomicron were purified via thin-layer chromatography,and the species and molecular structures of visible lipids in different hydrophobic regions were qualitatively characterized by high-performance liquid chromatography-mass spectrometry.The results indicated that seven lipid species were primarily displayed on the plate,including phosphatidylethanolamine,ethanolamine phosphoryl dihydroceramide,inositol phosphoryl dihydroceramide,glycyl-serine phosphoryl dihydroceramide,phosphatidylglycerol,cardiolipin,and glycyl-serine phosphoryl diacylglycerol.The phosphatidylethanolamine,ethanolamine phosphoryl dihydroceramide,and inositol phosphoryl dihydroceramide species corresponding to ion peaks at m/z 676.48,691.53,and 796.53 exhibited significantly high abundance compared to other species,suggesting their prevalent presence in total lipids.The molecular structures of phosphatidylethanolamine and ethanolamine phosphoryl dihydroceramide were derived from the modification of diacylglycerol and dihydroceramide with phosphoethanolamine,while the structure of inositol phosphoryl dihydroceramide was derived from the modification of dihydroceramide with phosphoinositol.The phosphatidylglycerol and cardiolipin species corresponding to m/z 721.51 and 1323.94 have been detected in the membrane lipids of B.thetaiotaomicron,although they were not mentioned in previous studies.These findings are important for understanding the molecular mechanisms of B.thetaiotaomicron colonization in mammalian gut.
文摘2′-Fucosyllactose is the most abundant human milk oligosaccharides and one of the three essential nutrients for infant growth.Corynebacterium glutamicum is one of the most common industrial fermentation bacteria but cannot synthesize 2′-fucosyllactose.In this study,C.glutamicum ATCC13032 was engineered for 2′-fucosyllactose production from fucose and lactose.The gene futC from Helicobacter pylori encodingα-1,2-fucosyltransferase was codon optimized and mutated at four amino acids(F40S/Q150H/C151R/Q239S).The modified gene futC and the gene fkp from Bacteroides thetaiotaomicron encoding fucokinase/GDP-fucose pyrophosphorylase were overexpressed in plasmid pEC and transformed into C.glutamicum,resulting in CW002.CW002 did not synthesize 2′-fucosyllactose possibly because the substrates fucose and/or lactose did not pass through the cell membrane.Therefore,the gene lacY encoding lactose permease and the gene fucP encoding fucose permease from Escherichia coli were overexpressed in plasmid pXTuf and transformed into CW002,resulting in CW006.CW006 did synthesize 2′-fucosyllactose as expected.It is interesting that the production of 2′-fucosyllactose was decreased or stopped when the expression combination of these four genes was changed,suggesting that the expression levels of the four genes in CW006 might have to well balanced.C.glutamicum CW006 produced 2.07 g/L 2′-fucosyllactose in a 2.4 L bioreactor.
基金funded by the Basic Research Program of Jiangsu and supported by the Jiangsu Basic Research Center for Synthetic Biology(Grant No.BK20233003)by National Natural Science Foundation of China(NSFC32270102).
文摘The human intestinal symbiotic microorganism Bacteroides thetaiotaomicron has a unique lipooligosaccharide structure,which promotes its beneficial symbiosis with the host.But its synthesis mechanism is not fully understood.In this study,protein sequence alignment revealed that the protein encoded by B.thetaiotaomicron VPI 5482 BT_2747 gene shares 24%sequence identity with Escherichia coli WaaA.The expression vector was used to overexpress BT_2747 in E.coli lipopolysaccharide mutant strains constructed by knocking out the waaC-waaF,lpxL or lpxM genes,resulting in the recombinant strains WH001(ΔwaaA)/pBT2747,WL003(ΔwaaAwaaC-F)/pBT2747,WL004(ΔwaaAwaaC-FlpxL)/pBT2747,WL005(ΔwaaAwaaC-FlpxM)/pBT2747 and WL006(ΔwaaAwaaC-FlpxLlpxM)/pBT2747.Lipid A/Kdo-lipid A were extracted from these recombinant strains and analyzed by liquid chromatography-mass spectrometry.The results showed that BT_2747 could convert a portion of the lipid IVA into Kdo-lipid IVA,but no Kdo2-lipid IVA structure was detected in E.coli.Small amounts of hexa-acylated Kdo-lipid A were also detected in the recombinant strains WH001/pBT2747 and WL003/pBT2747,and a small amount of penta-acylated Kdo-lipid A was found in WL004/pBT2747.The recombinant strain was further modified by introducing lpxF that enabled to synthesize Kdo-lipid IVA-1-phosphate.The results revealed that the BT_2747 gene in B.thetaiotaomicron VPI 5482 encodes the Kdo transferase of lipid A which uses lipid IVA as a substrate and only transfers single Kdo residue to lipid IVA.This study extends our understanding of the Kdo-lipid A structure and synthesis mechanism of B.thetaiotaomicron,which might provide a new perspective on how intestinal commensal bacteria regulate host immune homeostasis through unique Kdo-lipid A structure.
