Non-ribosomal peptide synthetases(NRPSs)are attractive targets for biosynthetic pathway engineering due to their modular architecture and the therapeutic relevance of their products.With catalysis mediated by specific...Non-ribosomal peptide synthetases(NRPSs)are attractive targets for biosynthetic pathway engineering due to their modular architecture and the therapeutic relevance of their products.With catalysis mediated by specific protein-protein interactions formed between the peptidyl carrier protein(PCP)and its partner enzymes,NRPS enzymology and control remains fertile ground for discovery.This review focuses on the recent efforts within structural biology by compiling high-resolution structural data that shed light into the various protein-protein interfaces formed between the PCP and its partner enzymes,including the phosphopantetheinyl transferase(PPTase),adenylation(A)domain,condensation(C)domain,thioesterase(TE)domain and other tailoring enzymes within the synthetase.Integrating our understanding of how the PCP recognizes partner proteins with the potential to use directed evolution and combinatorial biosynthetic methods will enhance future efforts in discovery and production of new bioactive compounds.展开更多
WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine(NMet-Dht)residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase(NRPS).The cytochrome P450 ...WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine(NMet-Dht)residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase(NRPS).The cytochrome P450 encoded by sas16(P450Sas)has been shown to be essential for the formation of the alkene moiety in NMet-Dht,but the timing and mechanism of the P450Sas-mediatedα,β-dehydrogenation of Dht remained unclear.Here,we show that the substrate of P450Sas is the NRPS-associated peptidyl carrier protein(PCP)-bound dipeptide intermediate(Z)-2-pent-1′-enyl-cinnamoyl-Thr-N-Me-Tyr.We demonstrate that P450Sas-mediated incorporation of the double bond follows N-methylation of the Tyr by the N-methyl transferase domain found within the NRPS,and further that P450Sas appears to be specific for substrates containing the(Z)-2-pent-1’-enyl-cinnamoyl group.A crystal structure of P450Sas reveals differences between P450Sas and other P450s involved in the modification of NRPS-associated substrates,including the substitution of the canonical active site alcohol residue with a phenylalanine(F250),which in turn is critical to P450Sas activity and WS9326A biosynthesis.Together,our results suggest that P450Sas catalyses the direct dehydrogenation of the NRPS-bound dipeptide substrate,thus expanding the repertoire of P450 enzymes that can be used to produce biologically active peptides.展开更多
Cyclodipeptides are diverse chemical scaffolds that show a broad range of bioactivities relevant for medicine,agriculture,chemical catalysis,and material sciences.Cyclodipeptides can be synthesized enzymatically throu...Cyclodipeptides are diverse chemical scaffolds that show a broad range of bioactivities relevant for medicine,agriculture,chemical catalysis,and material sciences.Cyclodipeptides can be synthesized enzymatically through two unrelated enzyme families,non-ribosomal peptide synthetases(NRPS)and cyclodipeptide synthases(CDPSs).The chemical diversity of cyclodipeptides is derived from the two amino acid side chains and the modification of those side-chains by cyclodipeptide tailoring enzymes.While a large spectrum of chemical diversity is already known today,additional chemical space-and as such potential new bioactivities-could be accessed by exploring yet undiscovered NRPS and CDPS gene clusters as well as via engineering.Further,to exploit cyclodipeptides for applications,the low yield of natural biosynthesis needs to be overcome.In this review we summarize current knowledge on NRPS and CDPS-based cyclodipeptide biosynthesis,engineering approaches to further diversity the natural chemical diversity as well as strategies for high-yield production of cyclodipeptides,including a discussion of how advancements in synthetic biology and metabolic engineering can accelerate the translational potential of cyclodipeptides.展开更多
The Antarctic represents a largely untapped source for isolation of new microorganisms with potential to produce bio- active natural products. Actinomycetes are of special interest among such microorganisms as they ar...The Antarctic represents a largely untapped source for isolation of new microorganisms with potential to produce bio- active natural products. Actinomycetes are of special interest among such microorganisms as they are known to produce a large number of natural products, many of which have clinical, pharmaceutical or agricultural applications. We isolated, characterized and classified actinomycetes from soil samples collected from different locations on Signy Island, South Orkney Islands, in the maritime Antarctic. A total of 95 putative actinomyeete strains were isolated from eight soil samples using eight types of selective isolation media. The strains were dereplicated into 16 groups based on morphology and Amplified Ribosomal DNA Restriction Analysis （ARDRA） patterns. Analysis of 16S rRNA gene sequences of representatives from each group showed that streptomy- cetes were the dominant actinomycetes isolated from these soils; however, there were also several strains belonging to diverse and rare genera in the class Actinobacteria, including Demetria, Glaciibacter, Kocuria, Marmoricola, Nakamurella and Tsukamurella. In addition, screening for antibacterial activity and non-ribosomal peptide synthetase genes showed that many of the actinomycete strains have the potential to produce antibacterial compounds.展开更多
Complex peptide natural products exhibit diverse biological functions and a wide range of physico-chemical properties.As a result,many peptides have entered the clinics for various applications.Two main routes for the...Complex peptide natural products exhibit diverse biological functions and a wide range of physico-chemical properties.As a result,many peptides have entered the clinics for various applications.Two main routes for the biosynthesis of complex peptides have evolved in nature:ribosomally synthesized and post-translationally modified peptide(RiPP)biosynthetic pathways and non-ribosomal peptide synthetases(NRPSs).Insights into both bioorthogonal peptide biosynthetic strategies led to the establishment of universal principles for each of the two routes.These universal rules can be leveraged for the targeted identification of novel peptide biosynthetic blueprints in genome sequences and used for the rational engineering of biosynthetic pathways to produce non-natural peptides.In this review,we contrast the key principles of both biosynthetic routes and compare the different biochemical strategies to install the most frequently encountered peptide modifications.In addition,the influence of the fundamentally different biosynthetic principles on past,current and future engineering ap-proaches is illustrated.Despite the different biosynthetic principles of both peptide biosynthetic routes,the arsenal of characterized peptide modifications encountered in RiPP and NRPS systems is largely overlapping.The continuous expansion of the biocatalytic toolbox of peptide modifying enzymes for both routes paves the way towards the production of complex tailor-made peptides and opens up the possibility to produce NRPS-derived peptides using the ribosomal route and vice versa.展开更多
Objective: Peptidyl alkaloids, a series of important natural products can be assembled by fungal nonribosomal peptide synthetases(NRPSs). However, many of the NRPSs associated gene clusters are silent under laboratory...Objective: Peptidyl alkaloids, a series of important natural products can be assembled by fungal nonribosomal peptide synthetases(NRPSs). However, many of the NRPSs associated gene clusters are silent under laboratory conditions, and the traditional chemical separation yields are low. In this study, we aim to discovery and efficiently prepare fungal peptidyl alkaloids assembled by fungal NRPSs.Methods: Bioinformatics analysis of gene cluster containing NRPSs from the genome of Penicillium thymicola, and heterologous expression of the putative gene cluster in Aspergillus nidulans were performed.Isolation, structural identification, and biological evaluation of the product from heterologous expression were carried out.Results: The putative tri-modular NRPS Anc A was heterologous-expressed in A. nidulans to give anacine(1) with high yield, which showed moderate and selective cytotoxic activity against A549 cell line.Conclusion: Heterologous expression in A. nidulans is an efficient strategy for mining fungal peptidyl alkaloids.展开更多
Non-ribosomal peptides are a group of structurally diverse natural products with various important therapeutic and agrochemical applications.Bacterial pyrrolizidine alkaloids(PAs),containing a scaffold of two fused fi...Non-ribosomal peptides are a group of structurally diverse natural products with various important therapeutic and agrochemical applications.Bacterial pyrrolizidine alkaloids(PAs),containing a scaffold of two fused five-membered ring system with a nitrogen atom at the bridgehead,have been found to originate from a multidomain non-ribosomal peptide synthetase to generate indolizidine intermediates,followed by multistep oxidation,catalysed by single Bayer-Villiger(BV)enzymes,to yield PA scaffolds.Although bacterial PAs are rare in natural product inventory,bioinformatics analysis suggested that the biosynthetic gene clusters(BGCs)that are likely to be responsible for the production of PA-like metabolites are widely distributed in bacterial genomes.However,most of the strains containing PA-like BGCs are not deposited in the public domain,therefore preventing further assessment of the chemical spaces of this group of bioactive metabolites.Here,we report a genomic scanning strategy to assess the potential of PA metabolites production in our culture collection without prior knowledge of genome information.Among the strains tested,we found fifteen contain the key BV enzymes that are likely to be involved in the last step of PA ring formation.Subsequently one-strain-many-compound(OSMAC)method,supported by a combination of HR-MS,NMR,SMART 2.0 technology,and GNPS analysis,allowed identification and characterization of a new[5+7]heterobicyclic carbamate,legoncarbamate,together with five known PAs,bohemamine derivatives,from Streptomyces sp.CT37,a Ghanaian soil isolate.The absolute stereochemistry of legoncarbamate was determined by comparison of measured and calculated ECD spectra.Legoncarbamate displays antibacterial activity against E.coli ATCC 25922 with an MIC value of 3.1μg/mL.Finally,a biosynthetic model of legoncarbamate and other bohemamines was proposed based on the knowledge we have gained so far.展开更多
基金J.C.C.was supported by the National Institute of General Medical Science(NIGMS)of the National Institutes of Health(NIH)under award number 1F31GM13761601A1J.O.S.was supported by the ACS Bridge Program and The Genentech FoundationThis work was supported by the NIGMS of the NIH under award number R01GM095970.
文摘Non-ribosomal peptide synthetases(NRPSs)are attractive targets for biosynthetic pathway engineering due to their modular architecture and the therapeutic relevance of their products.With catalysis mediated by specific protein-protein interactions formed between the peptidyl carrier protein(PCP)and its partner enzymes,NRPS enzymology and control remains fertile ground for discovery.This review focuses on the recent efforts within structural biology by compiling high-resolution structural data that shed light into the various protein-protein interfaces formed between the PCP and its partner enzymes,including the phosphopantetheinyl transferase(PPTase),adenylation(A)domain,condensation(C)domain,thioesterase(TE)domain and other tailoring enzymes within the synthetase.Integrating our understanding of how the PCP recognizes partner proteins with the potential to use directed evolution and combinatorial biosynthetic methods will enhance future efforts in discovery and production of new bioactive compounds.
基金supported by the BBSRC(MIBTP studentship to Daniel J.Leng)the Monash Warwick Alliance(Seed Fund Award to Manuela Tosin and Max J.Cryle)+6 种基金the University of Warwick(Career Support Award to Manuela Tosin)Monash University,EMBL Australia,the Australian Research Council(Discovery Project DP210101752 to Max J.Cryle)the National Health and Medical Research Council(APP1140619 to Max J.Cryle)the Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science(CE200100012)funded by the Australian Governmentfunded by the National Natural Science Foundation of China(82104044 to Songya Zhang)the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-PTJS-003-07)。
文摘WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine(NMet-Dht)residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase(NRPS).The cytochrome P450 encoded by sas16(P450Sas)has been shown to be essential for the formation of the alkene moiety in NMet-Dht,but the timing and mechanism of the P450Sas-mediatedα,β-dehydrogenation of Dht remained unclear.Here,we show that the substrate of P450Sas is the NRPS-associated peptidyl carrier protein(PCP)-bound dipeptide intermediate(Z)-2-pent-1′-enyl-cinnamoyl-Thr-N-Me-Tyr.We demonstrate that P450Sas-mediated incorporation of the double bond follows N-methylation of the Tyr by the N-methyl transferase domain found within the NRPS,and further that P450Sas appears to be specific for substrates containing the(Z)-2-pent-1’-enyl-cinnamoyl group.A crystal structure of P450Sas reveals differences between P450Sas and other P450s involved in the modification of NRPS-associated substrates,including the substitution of the canonical active site alcohol residue with a phenylalanine(F250),which in turn is critical to P450Sas activity and WS9326A biosynthesis.Together,our results suggest that P450Sas catalyses the direct dehydrogenation of the NRPS-bound dipeptide substrate,thus expanding the repertoire of P450 enzymes that can be used to produce biologically active peptides.
文摘Cyclodipeptides are diverse chemical scaffolds that show a broad range of bioactivities relevant for medicine,agriculture,chemical catalysis,and material sciences.Cyclodipeptides can be synthesized enzymatically through two unrelated enzyme families,non-ribosomal peptide synthetases(NRPS)and cyclodipeptide synthases(CDPSs).The chemical diversity of cyclodipeptides is derived from the two amino acid side chains and the modification of those side-chains by cyclodipeptide tailoring enzymes.While a large spectrum of chemical diversity is already known today,additional chemical space-and as such potential new bioactivities-could be accessed by exploring yet undiscovered NRPS and CDPS gene clusters as well as via engineering.Further,to exploit cyclodipeptides for applications,the low yield of natural biosynthesis needs to be overcome.In this review we summarize current knowledge on NRPS and CDPS-based cyclodipeptide biosynthesis,engineering approaches to further diversity the natural chemical diversity as well as strategies for high-yield production of cyclodipeptides,including a discussion of how advancements in synthetic biology and metabolic engineering can accelerate the translational potential of cyclodipeptides.
基金supported by grants from the University of Malaya(Grant no.PS334/2007B)the Malaysian Ministry of Higher Education(Grant no.FP007-2009)
文摘The Antarctic represents a largely untapped source for isolation of new microorganisms with potential to produce bio- active natural products. Actinomycetes are of special interest among such microorganisms as they are known to produce a large number of natural products, many of which have clinical, pharmaceutical or agricultural applications. We isolated, characterized and classified actinomycetes from soil samples collected from different locations on Signy Island, South Orkney Islands, in the maritime Antarctic. A total of 95 putative actinomyeete strains were isolated from eight soil samples using eight types of selective isolation media. The strains were dereplicated into 16 groups based on morphology and Amplified Ribosomal DNA Restriction Analysis （ARDRA） patterns. Analysis of 16S rRNA gene sequences of representatives from each group showed that streptomy- cetes were the dominant actinomycetes isolated from these soils; however, there were also several strains belonging to diverse and rare genera in the class Actinobacteria, including Demetria, Glaciibacter, Kocuria, Marmoricola, Nakamurella and Tsukamurella. In addition, screening for antibacterial activity and non-ribosomal peptide synthetase genes showed that many of the actinomycete strains have the potential to produce antibacterial compounds.
基金the LOEWE Center for Translational Biodiversity Genomics(LOEWE-TBG).
文摘Complex peptide natural products exhibit diverse biological functions and a wide range of physico-chemical properties.As a result,many peptides have entered the clinics for various applications.Two main routes for the biosynthesis of complex peptides have evolved in nature:ribosomally synthesized and post-translationally modified peptide(RiPP)biosynthetic pathways and non-ribosomal peptide synthetases(NRPSs).Insights into both bioorthogonal peptide biosynthetic strategies led to the establishment of universal principles for each of the two routes.These universal rules can be leveraged for the targeted identification of novel peptide biosynthetic blueprints in genome sequences and used for the rational engineering of biosynthetic pathways to produce non-natural peptides.In this review,we contrast the key principles of both biosynthetic routes and compare the different biochemical strategies to install the most frequently encountered peptide modifications.In addition,the influence of the fundamentally different biosynthetic principles on past,current and future engineering ap-proaches is illustrated.Despite the different biosynthetic principles of both peptide biosynthetic routes,the arsenal of characterized peptide modifications encountered in RiPP and NRPS systems is largely overlapping.The continuous expansion of the biocatalytic toolbox of peptide modifying enzymes for both routes paves the way towards the production of complex tailor-made peptides and opens up the possibility to produce NRPS-derived peptides using the ribosomal route and vice versa.
基金supported financially by the Drug Innovation Major Project (No. 2018ZX09711001-006)the CAMS Initiative for Innovative Medicine (No. 2017-I2M-4-004)。
文摘Objective: Peptidyl alkaloids, a series of important natural products can be assembled by fungal nonribosomal peptide synthetases(NRPSs). However, many of the NRPSs associated gene clusters are silent under laboratory conditions, and the traditional chemical separation yields are low. In this study, we aim to discovery and efficiently prepare fungal peptidyl alkaloids assembled by fungal NRPSs.Methods: Bioinformatics analysis of gene cluster containing NRPSs from the genome of Penicillium thymicola, and heterologous expression of the putative gene cluster in Aspergillus nidulans were performed.Isolation, structural identification, and biological evaluation of the product from heterologous expression were carried out.Results: The putative tri-modular NRPS Anc A was heterologous-expressed in A. nidulans to give anacine(1) with high yield, which showed moderate and selective cytotoxic activity against A549 cell line.Conclusion: Heterologous expression in A. nidulans is an efficient strategy for mining fungal peptidyl alkaloids.
基金QF and HD are grateful to the University of Aberdeen Elphinstone Scholarship and Scottish Funding Council/ScotCHEM for financial support through the PEER/PERCE Funding.HD and SW thank the financial supports of Biotechnology and Biological Sciences Research Council UK(BBSRC,BB/P00380X/1)HD,SAM and CP thank Business Interaction Vouchers(BIV009)from BBSRC funded Natural Products discovery and bioengineering Network(NPRONET)+2 种基金H.D.and K.K.thank the financial supports of Leverhulme Trust-Royal Society Africa award(AA090088)the jointly funded UK Medical Research Council-UK Department for International Development(MRC/DFID)Concordat agreement African Research Leaders Award(MR/S00520X/1)YZ and HD thank National Natural Science Foundation of China(31929001).
文摘Non-ribosomal peptides are a group of structurally diverse natural products with various important therapeutic and agrochemical applications.Bacterial pyrrolizidine alkaloids(PAs),containing a scaffold of two fused five-membered ring system with a nitrogen atom at the bridgehead,have been found to originate from a multidomain non-ribosomal peptide synthetase to generate indolizidine intermediates,followed by multistep oxidation,catalysed by single Bayer-Villiger(BV)enzymes,to yield PA scaffolds.Although bacterial PAs are rare in natural product inventory,bioinformatics analysis suggested that the biosynthetic gene clusters(BGCs)that are likely to be responsible for the production of PA-like metabolites are widely distributed in bacterial genomes.However,most of the strains containing PA-like BGCs are not deposited in the public domain,therefore preventing further assessment of the chemical spaces of this group of bioactive metabolites.Here,we report a genomic scanning strategy to assess the potential of PA metabolites production in our culture collection without prior knowledge of genome information.Among the strains tested,we found fifteen contain the key BV enzymes that are likely to be involved in the last step of PA ring formation.Subsequently one-strain-many-compound(OSMAC)method,supported by a combination of HR-MS,NMR,SMART 2.0 technology,and GNPS analysis,allowed identification and characterization of a new[5+7]heterobicyclic carbamate,legoncarbamate,together with five known PAs,bohemamine derivatives,from Streptomyces sp.CT37,a Ghanaian soil isolate.The absolute stereochemistry of legoncarbamate was determined by comparison of measured and calculated ECD spectra.Legoncarbamate displays antibacterial activity against E.coli ATCC 25922 with an MIC value of 3.1μg/mL.Finally,a biosynthetic model of legoncarbamate and other bohemamines was proposed based on the knowledge we have gained so far.
