The Experts below are selected from a list of 2079 Experts worldwide ranked by ideXlab platform
José A. Salas - One of the best experts on this subject based on the ideXlab platform.
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New Sipanmycin Analogues Generated by Combinatorial Biosynthesis and Mutasynthesis Approaches Relying on the Substrate Flexibility of Key Enzymes in the Biosynthetic Pathway
Applied and environmental microbiology, 2020Co-Authors: Mónica G. Malmierca, José A. Salas, Carmen Méndez, Ignacio Pérez-victoria, Jesús Martín, Fernando Reyes, Carlos OlanoAbstract:The appearance of new infectious diseases, the increase in multidrug-resistant bacteria, and the need for more effective chemotherapeutic agents have oriented the interests of researchers toward the search for metabolites with novel or improved bioactivities. Sipanmycins are disaccharyl glycosylated macrolactams that exert antibiotic and cytotoxic activities. By applying Combinatorial Biosynthesis and mutasynthesis approaches, we have generated eight new members of the sipanmycin family. The introduction of plasmids harboring genes responsible for the Biosynthesis of several deoxysugars into sipanmycin-producing Streptomyces sp. strain CS149 led to the production of six derivatives with altered glycosylation patterns. After structural elucidation of these new metabolites, we conclude that some of these sugars are the result of the combination of the enzymatic machinery encoded by the introduced plasmids and the native enzymes of the d-sipanose biosynthetic pathway of the wild-type CS149 strain. In addition, two analogues of the parental compounds with a modified polyketide backbone were generated by a mutasynthesis approach, feeding cultures of a mutant strain defective in sipanmycin Biosynthesis with 3-aminopentanoic acid. The generation of new sipanmycin analogues shown in this work relied on the substrate flexibility of key enzymes involved in sipanmycin Biosynthesis, particularly the glycosyltransferase pair SipS9/SipS14 and enzymes SipL3, SipL1, SipL7, and SipL2, which are involved in the incorporation of the polyketide synthase starting unit.IMPORTANCE Combinatorial Biosynthesis has proved its usefulness in generating derivatives of already known compounds with novel or improved pharmacological properties. Sipanmycins are a family of glycosylated macrolactams produced by Streptomyces sp. strain CS149, whose antiproliferative activity is dependent on the sugar moieties attached to the aglycone. In this work, we report the generation of several sipanmycin analogues with different deoxysugars, showing the high degree of flexibility exerted by the glycosyltransferase machinery with respect to the recognition of diverse nucleotide-activated sugars. In addition, modifications in the macrolactam ring were introduced by mutasynthesis approaches, indicating that the enzymes involved in incorporating the starter unit have a moderate ability to introduce different types of β-amino acids. In conclusion, we have proved the substrate flexibility of key enzymes involved in sipanmycin Biosynthesis, especially the glycosyltransferases, which can be exploited in future experiments.
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Expanding the Chemical Diversity of the Antitumoral Compound Mithramycin by Combinatorial Biosynthesis and Biocatalysis: The Quest for Mithralogs with Improved Therapeutic Window
Planta medica, 2015Co-Authors: Carmen Méndez, Francisco Moris, Javier González-sabín, José A. SalasAbstract:Mithramycin is an antitumor compound of the aureolic acid family produced by Streptomyces ar- gillaceus. It has been used to treat several types of cancer including testicular carcinoma, chronic and acute myeloid leukemia as well as hypercal- cemias and Pagetʼs disease. Although the use of mithramycin in humans has been limited because its side effects, in recent years a renewed interest has arisen since new uses and activities have been ascribed to it. Chemically, mithramycin is charac- terized by a tricyclic aglycone bearing two ali- phatic side chains attached at C3 and C7, and di- saccharide and trisaccharide units attached at po- sitions 2 and 6, respectively. The mithramycin gene cluster has been characterized. This has al- lowed for the development of several mithramy- cin analogs ("mithralogs") by Combinatorial bio- synthesis and/or biocatalysis. The Combinatorial Biosynthesis strategies include gene inactivation and/or the use of sugar Biosynthesis plasmids for sugar modification. In addition, lipase-based bio- catalysis enabled selective modifications of the hydroxyl groups, providing further mithramycin analogs. As a result, new mithramycin analogs with higher antitumor activity and/or less toxicity have been generated. One, demycarosyl-3D-β-D- digitoxosyl-mithramycin SK (EC-8042), is being tested in regulatory preclinical assays, represent- ing an opportunity to open the therapeutic win- dow of this promising molecular scaffold.
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a novel mithramycin analogue with high antitumor activity and less toxicity generated by Combinatorial Biosynthesis
Journal of Medicinal Chemistry, 2012Co-Authors: Luz Elena Nunez, José A. Salas, Alejandro F. Braña, Francisco Moris, Maria Perez, Stephen Eric Nybo, Javier Gonzalezsabin, Nuria Menendez, Khaled A Shaaban, Jurgen RohrAbstract:Mithramycin is an antitumor compound produced by Streptomyces argillaceus that has been used for the treatment of several types of tumors and hypercalcaemia processes. However, its use in humans has been limited because of its side effects. Using Combinatorial Biosynthesis approaches, we have generated seven new mithramycin derivatives, which differ from the parental compound in the sugar profile or in both the sugar profile and the 3-side chain. From these studies three novel derivatives were identified, demycarosyl-3D-β-d-digitoxosylmithramycin SK, demycarosylmithramycin SDK, and demycarosyl-3D-β-d-digitoxosylmithramycin SDK, which show high antitumor activity. The first one, which combines two structural features previously found to improve pharmacological behavior, was generated following two different strategies, and it showed less toxicity than mithramycin. Preliminary in vivo evaluation of its antitumor activity through hollow fiber assays, and in subcutaneous colon and melanoma cancers xenografts ...
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post pks tailoring steps in natural product producing actinomycetes from the perspective of Combinatorial Biosynthesis
Natural Product Reports, 2010Co-Authors: Carlos Olano, Carmen Méndez, José A. SalasAbstract:Covering: 2002 to September 2009 In recent years, a number of gene clusters involved in the Biosynthesis of polyketide compounds have been characterized and the genes have been used for designing and developing novel chemical entities by Combinatorial Biosynthesis. This review covers the highlights of Combinatorial Biosynthesis using polyketide-modifying enzymes such as oxidoreductases, group transferases, halogenases, cyclases and deoxysugar Biosynthesis enzymes, focusing on those from actinomycetes (with 315 references cited).
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Post-PKS tailoring steps in natural product-producing actinomycetes from the perspective of Combinatorial Biosynthesis.
Natural product reports, 2010Co-Authors: Carlos Olano, Carmen Méndez, José A. SalasAbstract:In recent years, a number of gene clusters involved in the Biosynthesis of polyketide compounds have been characterized and the genes have been used for designing and developing novel chemical entities by Combinatorial Biosynthesis. This review covers the highlights of Combinatorial Biosynthesis using polyketide-modifying enzymes such as oxidoreductases, group transferases, halogenases, cyclases and deoxysugar Biosynthesis enzymes, focusing on those from actinomycetes (with 315 references cited).
Carmen Méndez - One of the best experts on this subject based on the ideXlab platform.
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New Sipanmycin Analogues Generated by Combinatorial Biosynthesis and Mutasynthesis Approaches Relying on the Substrate Flexibility of Key Enzymes in the Biosynthetic Pathway
Applied and environmental microbiology, 2020Co-Authors: Mónica G. Malmierca, José A. Salas, Carmen Méndez, Ignacio Pérez-victoria, Jesús Martín, Fernando Reyes, Carlos OlanoAbstract:The appearance of new infectious diseases, the increase in multidrug-resistant bacteria, and the need for more effective chemotherapeutic agents have oriented the interests of researchers toward the search for metabolites with novel or improved bioactivities. Sipanmycins are disaccharyl glycosylated macrolactams that exert antibiotic and cytotoxic activities. By applying Combinatorial Biosynthesis and mutasynthesis approaches, we have generated eight new members of the sipanmycin family. The introduction of plasmids harboring genes responsible for the Biosynthesis of several deoxysugars into sipanmycin-producing Streptomyces sp. strain CS149 led to the production of six derivatives with altered glycosylation patterns. After structural elucidation of these new metabolites, we conclude that some of these sugars are the result of the combination of the enzymatic machinery encoded by the introduced plasmids and the native enzymes of the d-sipanose biosynthetic pathway of the wild-type CS149 strain. In addition, two analogues of the parental compounds with a modified polyketide backbone were generated by a mutasynthesis approach, feeding cultures of a mutant strain defective in sipanmycin Biosynthesis with 3-aminopentanoic acid. The generation of new sipanmycin analogues shown in this work relied on the substrate flexibility of key enzymes involved in sipanmycin Biosynthesis, particularly the glycosyltransferase pair SipS9/SipS14 and enzymes SipL3, SipL1, SipL7, and SipL2, which are involved in the incorporation of the polyketide synthase starting unit.IMPORTANCE Combinatorial Biosynthesis has proved its usefulness in generating derivatives of already known compounds with novel or improved pharmacological properties. Sipanmycins are a family of glycosylated macrolactams produced by Streptomyces sp. strain CS149, whose antiproliferative activity is dependent on the sugar moieties attached to the aglycone. In this work, we report the generation of several sipanmycin analogues with different deoxysugars, showing the high degree of flexibility exerted by the glycosyltransferase machinery with respect to the recognition of diverse nucleotide-activated sugars. In addition, modifications in the macrolactam ring were introduced by mutasynthesis approaches, indicating that the enzymes involved in incorporating the starter unit have a moderate ability to introduce different types of β-amino acids. In conclusion, we have proved the substrate flexibility of key enzymes involved in sipanmycin Biosynthesis, especially the glycosyltransferases, which can be exploited in future experiments.
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Expanding the Chemical Diversity of the Antitumoral Compound Mithramycin by Combinatorial Biosynthesis and Biocatalysis: The Quest for Mithralogs with Improved Therapeutic Window
Planta medica, 2015Co-Authors: Carmen Méndez, Francisco Moris, Javier González-sabín, José A. SalasAbstract:Mithramycin is an antitumor compound of the aureolic acid family produced by Streptomyces ar- gillaceus. It has been used to treat several types of cancer including testicular carcinoma, chronic and acute myeloid leukemia as well as hypercal- cemias and Pagetʼs disease. Although the use of mithramycin in humans has been limited because its side effects, in recent years a renewed interest has arisen since new uses and activities have been ascribed to it. Chemically, mithramycin is charac- terized by a tricyclic aglycone bearing two ali- phatic side chains attached at C3 and C7, and di- saccharide and trisaccharide units attached at po- sitions 2 and 6, respectively. The mithramycin gene cluster has been characterized. This has al- lowed for the development of several mithramy- cin analogs ("mithralogs") by Combinatorial bio- synthesis and/or biocatalysis. The Combinatorial Biosynthesis strategies include gene inactivation and/or the use of sugar Biosynthesis plasmids for sugar modification. In addition, lipase-based bio- catalysis enabled selective modifications of the hydroxyl groups, providing further mithramycin analogs. As a result, new mithramycin analogs with higher antitumor activity and/or less toxicity have been generated. One, demycarosyl-3D-β-D- digitoxosyl-mithramycin SK (EC-8042), is being tested in regulatory preclinical assays, represent- ing an opportunity to open the therapeutic win- dow of this promising molecular scaffold.
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post pks tailoring steps in natural product producing actinomycetes from the perspective of Combinatorial Biosynthesis
Natural Product Reports, 2010Co-Authors: Carlos Olano, Carmen Méndez, José A. SalasAbstract:Covering: 2002 to September 2009 In recent years, a number of gene clusters involved in the Biosynthesis of polyketide compounds have been characterized and the genes have been used for designing and developing novel chemical entities by Combinatorial Biosynthesis. This review covers the highlights of Combinatorial Biosynthesis using polyketide-modifying enzymes such as oxidoreductases, group transferases, halogenases, cyclases and deoxysugar Biosynthesis enzymes, focusing on those from actinomycetes (with 315 references cited).
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Post-PKS tailoring steps in natural product-producing actinomycetes from the perspective of Combinatorial Biosynthesis.
Natural product reports, 2010Co-Authors: Carlos Olano, Carmen Méndez, José A. SalasAbstract:In recent years, a number of gene clusters involved in the Biosynthesis of polyketide compounds have been characterized and the genes have been used for designing and developing novel chemical entities by Combinatorial Biosynthesis. This review covers the highlights of Combinatorial Biosynthesis using polyketide-modifying enzymes such as oxidoreductases, group transferases, halogenases, cyclases and deoxysugar Biosynthesis enzymes, focusing on those from actinomycetes (with 315 references cited).
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Generation of potent and selective kinase inhibitors by Combinatorial Biosynthesis of glycosylated indolocarbazoles
Chemical communications (Cambridge England), 2009Co-Authors: Cesar Sanchez, Carmen Méndez, Alejandro F. Braña, Francisco Moris, Aaroa Pérez Salas, Martina Palomino, Antonio Pineda-lucena, Rodrigo J. Carbajo, José A. SalasAbstract:We report the generation of novel glycosylated indolocarbazoles by Combinatorial Biosynthesis, and the identification of two novel potent and selective compounds inhibitors of JAK2 and Ikkb kinases.
Gavin J. Williams - One of the best experts on this subject based on the ideXlab platform.
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synthetic biology Combinatorial Biosynthesis and chemo enzymatic synthesis of isoprenoids
Journal of Industrial Microbiology & Biotechnology, 2020Co-Authors: Alexandra A. Malico, Miles A. Calzini, Anuran K. Gayen, Gavin J. WilliamsAbstract:Isoprenoids are a large class of natural products with myriad applications as bioactive and commercial compounds. Their diverse structures are derived from the biosynthetic assembly and tailoring of their scaffolds, ultimately constructed from two C5 hemiterpene building blocks. The modular logic of these platforms can be harnessed to improve titers of valuable isoprenoids in diverse hosts and to produce new-to-nature compounds. Often, this process is facilitated by the substrate or product promiscuity of the component enzymes, which can be leveraged to produce novel isoprenoids. To complement rational enhancements and even re-programming of isoprenoid Biosynthesis, high-throughput approaches that rely on searching through large enzymatic libraries are being developed. This review summarizes recent advances and strategies related to isoprenoid synthetic biology, Combinatorial Biosynthesis, and chemo-enzymatic synthesis, focusing on the past 5 years. Emerging applications of cell-free Biosynthesis and high-throughput tools are included that culminate in a discussion of the future outlook and perspective of isoprenoid biosynthetic engineering.
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Synthetic biology, Combinatorial Biosynthesis, and chemo-enzymatic synthesis of isoprenoids
Journal of Industrial Microbiology & Biotechnology, 2020Co-Authors: Alexandra A. Malico, Miles A. Calzini, Anuran K. Gayen, Gavin J. WilliamsAbstract:Isoprenoids are a large class of natural products with myriad applications as bioactive and commercial compounds. Their diverse structures are derived from the biosynthetic assembly and tailoring of their scaffolds, ultimately constructed from two C5 hemiterpene building blocks. The modular logic of these platforms can be harnessed to improve titers of valuable isoprenoids in diverse hosts and to produce new-to-nature compounds. Often, this process is facilitated by the substrate or product promiscuity of the component enzymes, which can be leveraged to produce novel isoprenoids. To complement rational enhancements and even re-programming of isoprenoid Biosynthesis, high-throughput approaches that rely on searching through large enzymatic libraries are being developed. This review summarizes recent advances and strategies related to isoprenoid synthetic biology, Combinatorial Biosynthesis, and chemo-enzymatic synthesis, focusing on the past 5 years. Emerging applications of cell-free Biosynthesis and high-throughput tools are included that culminate in a discussion of the future outlook and perspective of isoprenoid biosynthetic engineering.
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Synthetic biology, Combinatorial Biosynthesis, and chemo‑enzymatic synthesis of isoprenoids
Journal of Industrial Microbiology & Biotechnology, 2020Co-Authors: Alexandra A. Malico, Miles A. Calzini, Anuran K. Gayen, Gavin J. WilliamsAbstract:Isoprenoids are a large class of natural products with myriad applications as bioactive and commercial compounds. Their diverse structures are derived from the biosynthetic assembly and tailoring of their scaffolds, ultimately constructed from two C5 hemiterpene building blocks. The modular logic of these platforms can be harnessed to improve titers of valuable isoprenoids in diverse hosts and to produce new-to-nature compounds. Often, this process is facilitated by the substrate or product promiscuity of the component enzymes, which can be leveraged to produce novel isoprenoids. To complement rational enhancements and even re-programming of isoprenoid Biosynthesis, high-throughput approaches that rely on searching through large enzymatic libraries are being developed. This review summarizes recent advances and strategies related to isoprenoid synthetic biology, Combinatorial Biosynthesis, and chemo-enzymatic synthesis, focusing on the past 5 years. Emerging applications of cell-free Biosynthesis and high-throughput tools are included that culminate in a discussion of the future outlook and perspective of isoprenoid biosynthetic engineering.
Florian Hollfelder - One of the best experts on this subject based on the ideXlab platform.
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an optimized atp ppi exchange assay in 96 well format for screening of adenylation domains for applications in Combinatorial Biosynthesis
Biotechnology Journal, 2007Co-Authors: Linda G. Otten, Michelle L. Schaffer, Benoit Villiers, Torsten Stachelhaus, Florian HollfelderAbstract:We report a new format for measuring ATP/[32P]pyrophosphate exchange in a higher throughput assay of adenylation domains (A-domains) of non-ribosomal peptide synthetases. These enzymes are key specificity determinants in the assembly line Biosynthesis of non-ribosomal peptides, an important class of natural products with an activity spectrum ranging from antibiotic to antitumor activities. Our assay in 96-well format allows the rapid measurement of approximately 1000 data points per week as a basis for precise assessment of the kinetics of A-domains. The assay also allows quantitative high-throughput screening of the substrate specificity of A-domains identifying alternative, promiscuous substrates. We show that our assay is able to give high quality data for the T278A mutant of the A-domain of the tyrocidine synthetase module TycA with a 330-fold lower kcat/KM. The large dynamic range of this assay will be useful for the screening of libraries of mutant A-domains. Finally we describe and evaluate a procedure for the high-throughput purification of A-domains in 96-well format for the latter purpose. Our approach will be of utility for mechanistic analysis, substrate profiling and directed evolution of the A-domains, to ultimately enable the Combinatorial Biosynthesis of non-natural analogues of non-ribosomal peptides that may have potential as alternative drug candidates.
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An optimized ATP/PPi‐exchange assay in 96‐well format for screening of adenylation domains for applications in Combinatorial Biosynthesis
Biotechnology journal, 2007Co-Authors: Linda G. Otten, Michelle L. Schaffer, Benoit Villiers, Torsten Stachelhaus, Florian HollfelderAbstract:We report a new format for measuring ATP/[32P]pyrophosphate exchange in a higher throughput assay of adenylation domains (A-domains) of non-ribosomal peptide synthetases. These enzymes are key specificity determinants in the assembly line Biosynthesis of non-ribosomal peptides, an important class of natural products with an activity spectrum ranging from antibiotic to antitumor activities. Our assay in 96-well format allows the rapid measurement of approximately 1000 data points per week as a basis for precise assessment of the kinetics of A-domains. The assay also allows quantitative high-throughput screening of the substrate specificity of A-domains identifying alternative, promiscuous substrates. We show that our assay is able to give high quality data for the T278A mutant of the A-domain of the tyrocidine synthetase module TycA with a 330-fold lower kcat/KM. The large dynamic range of this assay will be useful for the screening of libraries of mutant A-domains. Finally we describe and evaluate a procedure for the high-throughput purification of A-domains in 96-well format for the latter purpose. Our approach will be of utility for mechanistic analysis, substrate profiling and directed evolution of the A-domains, to ultimately enable the Combinatorial Biosynthesis of non-natural analogues of non-ribosomal peptides that may have potential as alternative drug candidates.
Richard H Baltz - One of the best experts on this subject based on the ideXlab platform.
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Synthetic biology, genome mining, and Combinatorial Biosynthesis of NRPS-derived antibiotics: a perspective.
Journal of industrial microbiology & biotechnology, 2018Co-Authors: Richard H BaltzAbstract:Combinatorial Biosynthesis of novel secondary metabolites derived from nonribosomal peptide synthetases (NRPSs) has been in slow development for about a quarter of a century. Progress has been hampered by the complexity of the giant multimodular multienzymes. More recently, advances have been made on understanding the chemical and structural biology of these complex megaenzymes, and on learning the design rules for engineering functional hybrid enzymes. In this perspective, I address what has been learned about successful engineering of complex lipopeptides related to daptomycin, and discuss how synthetic biology and microbial genome mining can converge to broaden the scope and enhance the speed and robustness of Combinatorial Biosynthesis of NRPS-derived natural products for drug discovery.
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Combinatorial Biosynthesis of cyclic lipopeptide antibiotics a model for synthetic biology to accelerate the evolution of secondary metabolite biosynthetic pathways
ACS Synthetic Biology, 2014Co-Authors: Richard H BaltzAbstract:Nonribosomal peptide synthetases (NRPSs) are giant multi-enzymes that carry out sequencial assembly line couplings of amino acids to generate linear or cyclic peptides. NRPSs are composed of repeating enzyme domains with modular organization to activate and couple specific amino acids in a particular order. From a synthetic biology perspective, they can be considered as peptide assembly machines composed of devices to couple fatty acids to l-amino acids, l-amino acids to l-amino acids, and d-amino acids to l-amino acids. The coupling devices are composed of specific parts that contain two or more enzyme domains that can be exchanged Combinatorially to generate novel peptide assembly machines to produce novel peptides. The potent lipopeptide antibiotics daptomycin and A54145E have identical cyclic depsipeptide ring structures and stereochemistry but have divergent amino acid sequences. As their biosynthetic gene clusters are derived from an ancient ancestral lipopetide pathway, these lipopeptides provided an attractive model to develop Combinatorial Biosynthesis to generate antibiotics superior to daptomycin. These studies on Combinatorial Biosynthesis have helped generate guidelines for the successful assembly of NRPS parts and devices that can be used to generate novel lipopeptide structures and have established a basis for future synthetic biology studies to further develop Combinatorial Biosynthesis as a robust approach to natural product drug discovery.
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Daptomycin and Related Lipopeptides Produced by Fermentation, Chemical Modification, and Combinatorial Biosynthesis
2014Co-Authors: Richard H BaltzAbstract:Among the structurally related ten-membered ring acidic cyclic lipopeptide antibiotics, daptomycin was the first to gain FDA approval in the USA. Daptomycin and related lipopeptides require Ca2+ for activity, and Ca2+-bound daptomycin acts as a cationic peptide and interacts with the negatively charged phosphotidylglycerol (PG) in the cytoplasmic membrane to trigger its antibacterial effects. Mutants of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, and Bacillus subtilis, which display incremental increases in resistance to daptomycin, have mutations in genes that cause reductions in the negative charge on the membrane or thickening of cell walls. Daptomycin was approved to treat skin and skin structure infections caused by Gram-positive pathogens, and bacteremia including right-sided endocarditis caused by S. aureus, but has not been approved to treat Streptococcus pneumoniae pneumonia. Many derivatives of A21978C (containing the core peptide of daptomycin) and A54145 have been generated by chemical modification or by Combinatorial Biosynthesis to identify antibiotics superior to daptomycin for the treatment of community-acquired pneumonia (CAP). Several compounds had antibacterial activity superior to daptomycin, but were not as active as vancomycin in a mouse model for pneumonia. Lipopeptide CB-813,315, however, was more active than daptomycin and vancomycin against Clostridium difficile in vitro, and is currently undergoing clinical trials to treat C. difficile-associated diarrhea (CDAD).
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Development of a Genetic System for Combinatorial Biosynthesis of Lipopeptides in Streptomyces fradiae and Heterologous Expression of the A54145 Biosynthesis Gene Cluster
Applied and environmental microbiology, 2010Co-Authors: Dylan C. Alexander, Vivian Miao, Paul Brian, Jessica Rock, Richard H BaltzAbstract:A54145 factors are calcium-dependent lipopeptide antibiotics produced by Streptomyces fradiae NRRL 18160. A54145 is structurally related to the clinically important daptomycin, and as such may be a useful scaffold for the development of a novel lipopeptide antibiotic. We developed methods to genetically manipulate S. fradiae by deletion mutagenesis and conjugal transfer of plasmids from Escherichia coli. Cloning the complete pathway on a bacterial artificial chromosome (BAC) vector and the construction of ectopic trans-complementation with plasmids utilizing the φC31 or φBT1 site-specific integration system allowed manipulation of A54145 Biosynthesis. The BAC clone pDA2002 was shown to harbor the complete A54145 Biosynthesis gene cluster by heterologous expression in Streptomyces ambofaciens and Streptomyces roseosporus strains in yields of >100 mg/liter. S. fradiae mutants defective in LptI methyltransferase function were constructed, and they produced only A54145 factors containing glutamic acid (Glu₁₂), at the expense of factors containing 3-methyl-glutamic acid (3mGlu₁₂). This provided a practical route to produce high levels of pure Glu₁₂-containing lipopeptides. A suite of mutant strains and plasmids was created for Combinatorial Biosynthesis efforts focused on modifying the A54145 peptide backbone to generate a compound with daptomycin antibacterial activity and activity in Streptococcus pneumoniae pulmonary infections.
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Combinatorial Biosynthesis of novel antibiotics related to daptomycin
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Kien T. Nguyen, Vivian Miao, Paul Brian, Min Chu, Daniel Ritz, Dylan C. Alexander, Richard H BaltzAbstract:Daptomycin, a cyclic lipopeptide produced by Streptomyces roseosporus, is the active ingredient of Cubicin (daptomycin-for-injection), a first-in-class antibiotic approved for treatment of skin and skin-structure infections caused by Gram-positive pathogens and bacteremia and endocarditis caused by Staphylococcus aureus, including methicillin-resistant strains. Genetic engineering of the nonribosomal peptide synthetase (NRPS) in the daptomycin biosynthetic pathway was exploited for the Biosynthesis of novel active antibiotics. λ-Red-mediated recombination was used to exchange single or multiple modules in the DptBC subunit of the NRPS to modify the daptomycin cyclic peptide core. We combined module exchanges, NRPS subunit exchanges, inactivation of the tailoring enzyme glutamic acid 3-methyltransferase, and natural variations of the lipid tail to generate a library of novel lipopeptides, some of which were as active as daptomycin against Gram-positive bacteria. One compound was more potent against an Escherichia coli imp mutant that has increased outer membrane permeability. This study established a robust Combinatorial Biosynthesis platform to produce novel peptide antibiotics in sufficient quantities for antimicrobial screening and drug development.
