The ginsenoside Rgfound in Panax species has extensive pharmacological properties,in particular anti-cancer effects.However,its natural yield in Panax plants is limited.Here,we report a multimodular strategy to improv...The ginsenoside Rgfound in Panax species has extensive pharmacological properties,in particular anti-cancer effects.However,its natural yield in Panax plants is limited.Here,we report a multimodular strategy to improve yields of Rgin a Panax ginseng chassis,combining engineering of triterpene metabolism and overexpression of a lignin biosynthesis gene,phenylalanine ammonia lyase(PAL).We first performed semi-rational design and site mutagenesis to improve the enzymatic efficiency of Pq3-O-UGT2,a glycosyltransferase that directly catalyzes the biosynthesis of Rgfrom Rh.Next,we used clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)gene editing to knock down the branch pathway of protopanaxatriol-type ginsenoside biosynthesis to enhance the metabolic flux of the protopanaxadiol-type ginsenoside Rg.Overexpression of PAL accelerated the formation of the xylem structure,significantly improving ginsenoside Rgaccumulation(to 6.19-fold higher than in thecontrol).Wecombinedoverexpression of the ginsenoside aglycon synthetic genes squalene epoxidase,Pq3-O-UGT2,and PAL with CRISPR/Cas9-based knockdown of CYP716A53v2 to improve ginsenoside Rgaccumulation.Finally,we produced ginsenoside Rgat a yield of 83.6 mg/L in a shake flask(7.0 mg/g dry weight,21.12-fold higher than with wild-type cultures).The highproduction system established in this study could be a potential platform to produce the ginsenoside Rgcommercially for pharmaceutical use.展开更多
Rheumatoid arthritis(RA)is exacerbated by TNF-alpha signaling.However,it remains unclear whether TNF-α-activated TNFR1 and TNFR2 are regulated by extracellular factors.Here,we showed that soluble glycosylated interle...Rheumatoid arthritis(RA)is exacerbated by TNF-alpha signaling.However,it remains unclear whether TNF-α-activated TNFR1 and TNFR2 are regulated by extracellular factors.Here,we showed that soluble glycosylated interleukin-17 receptor D(sIL-17RD),which was produced by proteolytic cleavage,enhanced TNF-α-induced RA.We revealed that IL-17RD shedding was induced by the proteolytic enzyme TACE and enhanced by TNF-αexpression in macrophages.Intriguingly,sIL-17RD was elevated in the sera of arthritic mice and rats.Recombinant sIL-17RD significantly enhanced the TNF-α-induced proinflammatory response by promoting TNF-α-TNFR-sIL-17RD complex formation and receptor clustering,leading to the accelerated development of collagen-induced arthritis.Our observations revealed that ectodomain shedding of IL-17RD occurred in RA to boost the TNF-α-induced inflammatory response.Targeting sIL-17RD may provide a new strategy for the therapy of RA.展开更多
基金financial support from the National Key Research and Development Program(2020YFA0907903)a key project at the central government level:“The ability to establish a sustainable use for valuable Chinese medicine resources”(2060302)+2 种基金the National Science Foundation of China(91954112 and 31900501)the Young Elite Scientists Sponsorship Program of Tianjin(TJSQNTJ-2020-19)the Scientific Research Transformation Foundation of the Wenzhou Safety(Emergency)Institute of Tianjin University。
文摘The ginsenoside Rgfound in Panax species has extensive pharmacological properties,in particular anti-cancer effects.However,its natural yield in Panax plants is limited.Here,we report a multimodular strategy to improve yields of Rgin a Panax ginseng chassis,combining engineering of triterpene metabolism and overexpression of a lignin biosynthesis gene,phenylalanine ammonia lyase(PAL).We first performed semi-rational design and site mutagenesis to improve the enzymatic efficiency of Pq3-O-UGT2,a glycosyltransferase that directly catalyzes the biosynthesis of Rgfrom Rh.Next,we used clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)gene editing to knock down the branch pathway of protopanaxatriol-type ginsenoside biosynthesis to enhance the metabolic flux of the protopanaxadiol-type ginsenoside Rg.Overexpression of PAL accelerated the formation of the xylem structure,significantly improving ginsenoside Rgaccumulation(to 6.19-fold higher than in thecontrol).Wecombinedoverexpression of the ginsenoside aglycon synthetic genes squalene epoxidase,Pq3-O-UGT2,and PAL with CRISPR/Cas9-based knockdown of CYP716A53v2 to improve ginsenoside Rgaccumulation.Finally,we produced ginsenoside Rgat a yield of 83.6 mg/L in a shake flask(7.0 mg/g dry weight,21.12-fold higher than with wild-type cultures).The highproduction system established in this study could be a potential platform to produce the ginsenoside Rgcommercially for pharmaceutical use.
基金This work was supported by grants from the Chinese National Major Scientific Research Program(2016YFA0500301)from the National Natural Science Foundation of China(NSFC)(81872244,81830092,and 81572729).
文摘Rheumatoid arthritis(RA)is exacerbated by TNF-alpha signaling.However,it remains unclear whether TNF-α-activated TNFR1 and TNFR2 are regulated by extracellular factors.Here,we showed that soluble glycosylated interleukin-17 receptor D(sIL-17RD),which was produced by proteolytic cleavage,enhanced TNF-α-induced RA.We revealed that IL-17RD shedding was induced by the proteolytic enzyme TACE and enhanced by TNF-αexpression in macrophages.Intriguingly,sIL-17RD was elevated in the sera of arthritic mice and rats.Recombinant sIL-17RD significantly enhanced the TNF-α-induced proinflammatory response by promoting TNF-α-TNFR-sIL-17RD complex formation and receptor clustering,leading to the accelerated development of collagen-induced arthritis.Our observations revealed that ectodomain shedding of IL-17RD occurred in RA to boost the TNF-α-induced inflammatory response.Targeting sIL-17RD may provide a new strategy for the therapy of RA.