Inorganic polyphosphate(polyP),a linear polymer of orthophosphate residues,plays critical roles in diverse biological processes spanning blood coagulation,immunomodulation,and post-translational protein modifica-tions...Inorganic polyphosphate(polyP),a linear polymer of orthophosphate residues,plays critical roles in diverse biological processes spanning blood coagulation,immunomodulation,and post-translational protein modifica-tions in eukaryotes.Notably,long-chain polyP(>100 phosphate units)exhibits distinct biological functionalities compared to shorter-chain counterparts.While Saccharomyces cerevisiae serves as a promising microbial platform for polyP biosynthesis,the genetic regulatory mechanisms underlying polyP metabolism remain poorly eluci-dated.Here,we systematically investigated the genetic determinants governing intracellular polyP levels and chain length dynamics in yeast.Through screening a library of 55 single-gene knockout strains,we identified six mutants(Δddp1,Δvip1,Δppn1,Δppn2,Δecm33,andΔccr4)exhibiting elevated polyP accumulation,whereas deletions of vtc1,kcs1,vma22,vma5,pho85,vtc4,vma2,vma3,ecm14,and vph2 resulted in near-complete polyP depletion.Subsequent combinatorial deletions in theΔppn1 background revealed that theΔppn1Δvip1 double mutant achieved synergistic enhancement in both polyP concentration(53.01 mg-P/g-DCW)and chain length,attributable to increased ATP availability and reduced polyphosphatase activity.Leveraging CRISPR/Cas9-mediated overexpression inΔppn1Δvip1,we engineered strain PP2(vtc4 overexpression),which demonstrated a 2-fold increase in polyP yield(62.6 mg-P/g-DCW)relative to wild-type BY4741,with predominant synthesis of long-chain species.Mechanistically,qRT-PCR analysis confirmed that PP2 exhibited 46-fold up-regulation of vtc4 coupled with down-regulation of polyphosphatases encoding genes,ppn2,ddp1,and ppx1.This study performed a systematic study of regulating inorganic polyphosphates production in yeast and provides a synthetic biology strategy to engineer high-yield polyP-producing strains,advancing both fundamental understanding and biotechnological applications.展开更多
Zhigancao decoction is a traditional prescription for treating irregular pulse and palpitations in China.As the monarch drug of Zhigancao decoction,the bioactive molecules of licorice against heart diseases remain elu...Zhigancao decoction is a traditional prescription for treating irregular pulse and palpitations in China.As the monarch drug of Zhigancao decoction,the bioactive molecules of licorice against heart diseases remain elusive.We established the HRESIMS-guided method leading to the isolation of three novel bicyclic peptides,glycnsisitins A-C(1-3),with distinctive C-C and C-O-C side-chain-toside-chain linkages from the roots of Glycyrrhiza uralensis(Licorice).Glycnsisitin A demonstrated stronger cardioprotective activity than glycnsisitins B and C in an in vitro model of doxorubicin(DOX)-induced cardiomyocyte injury.Glycnsisitin A treatment not only reduced the mortality of heart failure(HF)mice in a dose-dependent manner but also significantly attenuated DOX-induced cardiac dysfunction and myocardial fibrosis.Gene set enrichment analysis(GSEA)of the differentially expressed genes indicated that the cardioprotective effect of glycnsisitin A was mainly attributed to its ability to maintain iron homeostasis in the myocardium.Mechanistically,glycnsisitin A interacted with transferrin and facilitated its binding to the transferrin receptor(TFRC),which caused increased uptake of iron in cardiomyocytes.These findings highlight the key role of bicyclic peptides as bioactive molecules of Zhigancao decoction for the treatment of HF,and glycnsisitin A constitutes a promising therapeutic agent for the treatment of HF.展开更多
基金supported by the National Key Research Program 2019YFA0905800Other financial support was provided by the National Nature Science Foundation of China(22025701 to J.Z.,22177048 to W.W.and 22293052 to J.Z.)+1 种基金Natural Science Foundation of Jiangsu Province(BK20232020 to J.Z.)the Fundamental Research Funds for the Central Universities,and Nanjing Science and Technology Programme(202305003).
文摘Inorganic polyphosphate(polyP),a linear polymer of orthophosphate residues,plays critical roles in diverse biological processes spanning blood coagulation,immunomodulation,and post-translational protein modifica-tions in eukaryotes.Notably,long-chain polyP(>100 phosphate units)exhibits distinct biological functionalities compared to shorter-chain counterparts.While Saccharomyces cerevisiae serves as a promising microbial platform for polyP biosynthesis,the genetic regulatory mechanisms underlying polyP metabolism remain poorly eluci-dated.Here,we systematically investigated the genetic determinants governing intracellular polyP levels and chain length dynamics in yeast.Through screening a library of 55 single-gene knockout strains,we identified six mutants(Δddp1,Δvip1,Δppn1,Δppn2,Δecm33,andΔccr4)exhibiting elevated polyP accumulation,whereas deletions of vtc1,kcs1,vma22,vma5,pho85,vtc4,vma2,vma3,ecm14,and vph2 resulted in near-complete polyP depletion.Subsequent combinatorial deletions in theΔppn1 background revealed that theΔppn1Δvip1 double mutant achieved synergistic enhancement in both polyP concentration(53.01 mg-P/g-DCW)and chain length,attributable to increased ATP availability and reduced polyphosphatase activity.Leveraging CRISPR/Cas9-mediated overexpression inΔppn1Δvip1,we engineered strain PP2(vtc4 overexpression),which demonstrated a 2-fold increase in polyP yield(62.6 mg-P/g-DCW)relative to wild-type BY4741,with predominant synthesis of long-chain species.Mechanistically,qRT-PCR analysis confirmed that PP2 exhibited 46-fold up-regulation of vtc4 coupled with down-regulation of polyphosphatases encoding genes,ppn2,ddp1,and ppx1.This study performed a systematic study of regulating inorganic polyphosphates production in yeast and provides a synthetic biology strategy to engineer high-yield polyP-producing strains,advancing both fundamental understanding and biotechnological applications.
基金supported by Chinese Academy of Medical Sciences(CAMS)Innovation Fund for Medical Sciences(2022-I2M-2-002,2021-I2M-1-016,and 2022-I2M-1-014,China)the Beijing Outstanding Young Scientist Program(BJJWZYJH01201910023028,China)Chinese Academy of Medical Sciences(CAMS)Central Public-interest Scientific Institution Basal Research Fund(2018PT35004,Molecular Mechanism and Target Discovery of Metabolic Disorder and Tumorigenesis,CAMS Key Laboratory).
文摘Zhigancao decoction is a traditional prescription for treating irregular pulse and palpitations in China.As the monarch drug of Zhigancao decoction,the bioactive molecules of licorice against heart diseases remain elusive.We established the HRESIMS-guided method leading to the isolation of three novel bicyclic peptides,glycnsisitins A-C(1-3),with distinctive C-C and C-O-C side-chain-toside-chain linkages from the roots of Glycyrrhiza uralensis(Licorice).Glycnsisitin A demonstrated stronger cardioprotective activity than glycnsisitins B and C in an in vitro model of doxorubicin(DOX)-induced cardiomyocyte injury.Glycnsisitin A treatment not only reduced the mortality of heart failure(HF)mice in a dose-dependent manner but also significantly attenuated DOX-induced cardiac dysfunction and myocardial fibrosis.Gene set enrichment analysis(GSEA)of the differentially expressed genes indicated that the cardioprotective effect of glycnsisitin A was mainly attributed to its ability to maintain iron homeostasis in the myocardium.Mechanistically,glycnsisitin A interacted with transferrin and facilitated its binding to the transferrin receptor(TFRC),which caused increased uptake of iron in cardiomyocytes.These findings highlight the key role of bicyclic peptides as bioactive molecules of Zhigancao decoction for the treatment of HF,and glycnsisitin A constitutes a promising therapeutic agent for the treatment of HF.
