Mogrosides and steroid saponins are tetracyclic triterpenoids found in Siraitia grosvenorii.Squalene synthase(SQS) and cycloartenol synthase(CAS) are key enzymes in triterpenoid and steroid biosynthesis.In this study,...Mogrosides and steroid saponins are tetracyclic triterpenoids found in Siraitia grosvenorii.Squalene synthase(SQS) and cycloartenol synthase(CAS) are key enzymes in triterpenoid and steroid biosynthesis.In this study,full-length cDNAs of SgSQS and SgCAS were cloned by a rapid amplification of cDNA-ends with polymerase chain reaction(RACE-PCR) approach.The SgSQS cDNA has a 1254 bp open reading frame(ORF) encoding 417 amino acids,and the SgCAS cDNA contains a 2298 bp ORF encoding 765 amino acids.Bioinformatic analysis showed that the deduced SgSQS protein has two transmembrane regions in the C-terminal.Both SgSQS and SgCAS have significantly higher levels in fruits than in other tissues,suggesting that steroids and mogrosides are competitors for the same precursors in fruits.Combined in silico prediction and subcellular localization,experiments in tobacco indicated that SgSQS was probably in the cytoplasm or on the cytoskeleton,and SgCAS was likely located in the nucleus or cytosol.These results will provide a foundation for further study of SgSQS and SgCAS gene functions in S.grosvenorii,and may facilitate improvements in mogroside content in fruit by regulating gene expression.展开更多
Astragalosides are the main active constituents of traditional Chinese medicine Huang-Qi,of which cycloastragenol-type glycosides are the most typical and major bioactive compounds.This kind of compounds exhibit vario...Astragalosides are the main active constituents of traditional Chinese medicine Huang-Qi,of which cycloastragenol-type glycosides are the most typical and major bioactive compounds.This kind of compounds exhibit various biological functions including cardiovascular protective,neuroprotective,etc.Owing to the limitations of natural sources and the difficulties encountered in chemical synthesis,re-engineering of biosynthetic machinery will offer an alternative and promising approach to producing astragalosides.However,the biosynthetic pathway for astragalosides remains elusive due to their complex structures and numerous reaction types and steps.Herein,guided by transcriptome and phylogenetic analyses,a cycloartenol synthase and four glycosyltransferases catalyzing the committed steps in the biosynthesis of such bioactive astragalosides were functionally characterized from Astragalus membranaceus.AmCAS1,the first reported cycloartenol synthase from Astragalus genus,is capable of catalyzing the formation of cycloartenol;AmUGT15,AmUGT14,AmUGT13,and AmUGT7 are four glycosyltransferases biochemically characterized to catalyze 3-O-xylosylation,3-O-glucosylation,25-O-glucosylation/O-xylosylation and 2’-O-glucosylation of cycloastragenol glycosides,respectively.These findings not only clarified the crucial enzymes for the biosynthesis and the molecular basis for the structural diversity of astragalosides in Astragalus plants,also paved the way for further completely deciphering the biosynthetic pathway and constructing an artificial pathway for their efficient production.展开更多
Vitamin B_(2)is an essential water-soluble vitamin.For most prokaryotes,a bifunctional enzyme called FAD synthase catalyzes the successive conversion of riboflavin to FMN and FAD.In this study,the plasmid pNEW-AZ cont...Vitamin B_(2)is an essential water-soluble vitamin.For most prokaryotes,a bifunctional enzyme called FAD synthase catalyzes the successive conversion of riboflavin to FMN and FAD.In this study,the plasmid pNEW-AZ containing six key genes for the riboflavin synthesis was transformed into strain R2 with the deleted FMN riboswitch,yielding strain R5.The R5 strain could produce 540.23±5.40 mg/L riboflavin,which was 10.61%higher than the R4 strain containing plasmids pET-AE and pAC-Z harboring six key genes.To further enhance the production of riboflavin,homology matching and molecular docking were performed to identify key amino acid residues of FAD synthase.Nine point mutation sites were identified.By comparing riboflavin kinase activity,mutations of T203D and N210D,which respectively decreased by 29.90%and 89.32%compared to wild-type FAD synthase,were selected for CRISPR/Cas9 gene editing of the genome,generating engineered strains R203 and R210.pNEW-AZ was transformed into R203,generating R6.R6 produced 657.38±47.48 mg/L riboflavin,a 21.69%increase compared to R5.This study contributes to the high production of riboflavin in recombinant E.coli BL21.展开更多
[目的]克隆浙贝母环阿屯醇合成酶(Cycloartenol synthase,CAS)5`末端cDNA序列。[方法]利用简并引物及RT-PCR方法获得CAS的核心基因,应用cDNA末端快速扩增(Rapid-amplification of cDNA ends,RACE)技术快速克隆5`末端片段,采用NCBI网站上...[目的]克隆浙贝母环阿屯醇合成酶(Cycloartenol synthase,CAS)5`末端cDNA序列。[方法]利用简并引物及RT-PCR方法获得CAS的核心基因,应用cDNA末端快速扩增(Rapid-amplification of cDNA ends,RACE)技术快速克隆5`末端片段,采用NCBI网站上的Protein Blast软件进行氨基酸同源性比对。[结果]从浙贝母叶片中成功地克隆出一条长691bp的CAS基因5`末端cDNA片段,含起始密码子及163bp的5`延伸区。对5`末端的150个氨基酸同源性分析表明,该片段与C.asiatica,G.glabra,A.thaliana,W.somnifera的CAS同源性分别达72%,66%,71%,71%。[结论]上述结果表明所克隆的片段为浙贝母CAS的5`末端序列,为下一步CAS全长基因的克隆及CAS在浙贝母次生代谢中的功能研究打下基础。展开更多
基金supported by the National Nature Science Foundation of China(No.81373914)National Key Technology Support Program(No.2011BAI01B03)Guangxi Natural Science Fundation(Nos.2013GXNSFDA019021 and 2012GXNSFAA053043)
文摘Mogrosides and steroid saponins are tetracyclic triterpenoids found in Siraitia grosvenorii.Squalene synthase(SQS) and cycloartenol synthase(CAS) are key enzymes in triterpenoid and steroid biosynthesis.In this study,full-length cDNAs of SgSQS and SgCAS were cloned by a rapid amplification of cDNA-ends with polymerase chain reaction(RACE-PCR) approach.The SgSQS cDNA has a 1254 bp open reading frame(ORF) encoding 417 amino acids,and the SgCAS cDNA contains a 2298 bp ORF encoding 765 amino acids.Bioinformatic analysis showed that the deduced SgSQS protein has two transmembrane regions in the C-terminal.Both SgSQS and SgCAS have significantly higher levels in fruits than in other tissues,suggesting that steroids and mogrosides are competitors for the same precursors in fruits.Combined in silico prediction and subcellular localization,experiments in tobacco indicated that SgSQS was probably in the cytoplasm or on the cytoskeleton,and SgCAS was likely located in the nucleus or cytosol.These results will provide a foundation for further study of SgSQS and SgCAS gene functions in S.grosvenorii,and may facilitate improvements in mogroside content in fruit by regulating gene expression.
基金supported by the National Key Research and Development Program of China(2020YFA0908000)CAMS Innovation fund for Medical Sciences(CIFMS,No.2021-I2M-1029,China)Beijing Key Laboratory of non-Clinical Drug Metabolism and PK/PD Study(Z141102004414062)。
文摘Astragalosides are the main active constituents of traditional Chinese medicine Huang-Qi,of which cycloastragenol-type glycosides are the most typical and major bioactive compounds.This kind of compounds exhibit various biological functions including cardiovascular protective,neuroprotective,etc.Owing to the limitations of natural sources and the difficulties encountered in chemical synthesis,re-engineering of biosynthetic machinery will offer an alternative and promising approach to producing astragalosides.However,the biosynthetic pathway for astragalosides remains elusive due to their complex structures and numerous reaction types and steps.Herein,guided by transcriptome and phylogenetic analyses,a cycloartenol synthase and four glycosyltransferases catalyzing the committed steps in the biosynthesis of such bioactive astragalosides were functionally characterized from Astragalus membranaceus.AmCAS1,the first reported cycloartenol synthase from Astragalus genus,is capable of catalyzing the formation of cycloartenol;AmUGT15,AmUGT14,AmUGT13,and AmUGT7 are four glycosyltransferases biochemically characterized to catalyze 3-O-xylosylation,3-O-glucosylation,25-O-glucosylation/O-xylosylation and 2’-O-glucosylation of cycloastragenol glycosides,respectively.These findings not only clarified the crucial enzymes for the biosynthesis and the molecular basis for the structural diversity of astragalosides in Astragalus plants,also paved the way for further completely deciphering the biosynthetic pathway and constructing an artificial pathway for their efficient production.
基金the Natural Science Foundation of Zhejiang Province,China(No.LY21C200006)。
文摘Vitamin B_(2)is an essential water-soluble vitamin.For most prokaryotes,a bifunctional enzyme called FAD synthase catalyzes the successive conversion of riboflavin to FMN and FAD.In this study,the plasmid pNEW-AZ containing six key genes for the riboflavin synthesis was transformed into strain R2 with the deleted FMN riboswitch,yielding strain R5.The R5 strain could produce 540.23±5.40 mg/L riboflavin,which was 10.61%higher than the R4 strain containing plasmids pET-AE and pAC-Z harboring six key genes.To further enhance the production of riboflavin,homology matching and molecular docking were performed to identify key amino acid residues of FAD synthase.Nine point mutation sites were identified.By comparing riboflavin kinase activity,mutations of T203D and N210D,which respectively decreased by 29.90%and 89.32%compared to wild-type FAD synthase,were selected for CRISPR/Cas9 gene editing of the genome,generating engineered strains R203 and R210.pNEW-AZ was transformed into R203,generating R6.R6 produced 657.38±47.48 mg/L riboflavin,a 21.69%increase compared to R5.This study contributes to the high production of riboflavin in recombinant E.coli BL21.
