Jared C Lewis
University Of Chicago
Project start date: 2010-02-01
Project end date: 2013-12-31
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Jared C Lewis
DIRECTED EVOLUTION OF A CYTOCHROME P450 FOR SYNTHESIS OF ARTEMISINIC ALCOHOL
Jared C Lewis, Nih Postdoctoral Fellow
California Institute Of Technology, Office Of Sponsored Research, Mail Code 201-15, Pasadena, Ca 91125
Grant 5F32GM079932-03 from National Institute Of General Medical Sciences
Abstract: This proposal describes the directed evolution of a novel cytochrome P450 capable of oxidizing amorphadiene to artemisinic alcohol or artemisinic aldehyde, both of which are key intermediates in the semi-synthesis of artemesinin, in E. coli. The project will commence with the design and validation of a screen to identify P450s capable of catalyzing the desired reaction and analysis of mutant P450 libraries maintained in the Arnold group for viable catalysts. Mutagenesis and Recombination techniques will be used to generate subsequent mutant libraries based on the hits obtained from this screen, and iteration of this process should provide a P450 capable of selectively catalyzing the desired reaction. Finally, the optimized enzyme will be over-expressed in a strain of E. coli engineered to overproduce amorphadiene in order to provide high yields of artemisinic alcohol or artemisinic aldehyde from simple sugars. A simple semi-synthetic route can then be used to convert this material to artemesinin, a highly important anti- malarial. Malaria claims the lives of over one million people each year and threatens approximately 300-500 million individuals located predominately in tropical environments due to increasing resistance to current anti- malarial medicines. Artemisinin, a natural product isolated from the East Asian shrub Artemesia annua, has been heralded as a breakthrough in the treatment of malaria, but short supply and high cost have hindered its widespread use. This proposal describes the directed evolution of a novel cytochrome P450 enzyme capable of oxidizing amorphadiene to artemisinic alcohol or artemisinic aldehyde, both of which are key intermediates in the semi-synthesis of artemesinin, in E. coli
Keywords: Adoption; Alcohols; Aldehydes; Anti-Malarials; Antimalarial Agents; Antimalarial Drugs; Antimalarials; Area; Artemisinins; Asians; Back; Biological Factors; Biotechnology; Cells; Chemical Class, Alcohol; Chromogenic Substrates; Collaborations; Combined Modality Therapy; Complex; Cytochrome P-450; Cytochrome P-450 Enzyme System; Cytochrome P450; DNA Recombination; DNA recombination (naturally occurring); Detection; Dorsum; Drug Costs; Drug resistance; Drugs; E coli; Economically Deprived; Economically Deprived Population; Engineering; Engineerings; Environment; Enzymes; Escherichia coli; Evolution; Factor, Biologic; Future; Genetic Recombination; Genetics-Mutagenesis; Habitats; High Throughput Assay; Individual; Investigators; Laboratories; Libraries; Life; Malaria; Medication; Medicine; Membrane; Methods and Techniques; Methods, Other; Molecular Biology, Mutagenesis; Multimodal Therapy; Multimodal Treatment; Multimodality Treatment; Mutagenesis; Mutate; Natural Products; P450; Paludism; Pharmaceutic Preparations; Pharmaceutical Preparations; Plants; Plants, General; Plasmodium Infections; Plasmodium falciparum; Process; Production; Reaction; Recombination; Recombination, Genetic; Relative; Relative (related person); Research Personnel; Researchers; Resistance; Route; Science of Medicine; Substrates, Chromogenic; Techniques; Validation; WHO; Work; World Health Organization; analog; arteannuin; artemisinin; artemisinine; base; catalyst; combination therapy; combined modality treatment; combined treatment; cost; cost effective; design; designing; directed evolution; drug resistant; drug/agent; high throughput screening; large scale production; membrane structure; multimodality therapy; mutant; novel; oriental; oxidation; qinghaosu; quing hau sau; quinghaosu; rapid detection; resistance to Drug; resistant; resistant to Drug; success; sugar
Project start date: 2007-07-23
Project end date: 2010-07-22
Budget start date: 23-JUL-2009
Budget end date: 22-JUL-2010
5F32GM079932-03 (2009): $50054
5F32GM079932-02 (2008): $46826
TRANSITION METAL CATALYSIS AND METABOLIC ENGINEERING USING ARTIFICIAL METALLOENZY
Jared C Lewis, Nih Postdoctoral Fellow
California Institute Of Technology, Office Of Sponsored Research, Mail Code 201-15, Pasadena, Ca 91125
Grant 1K99GM087551-01A1 from National Institute Of General Medical Sciences
Abstract: Practical application of new synthetic molecules for the betterment of human health depends directly on the efficiency with which these compounds can be synthesized, but this is frequently limited by poor reaction yields throughout long reaction sequences in which intermediate compounds must be isolated and purified. Metabolic engineers have demonstrated that novel biosynthetic pathways can be assembled in order to produce chemicals in vivo with no isolation of intermediates in an aqueous aerobic environment, but these sequences are limited to transformations catalyzed by natural enzymes. This proposal describes the design, preparation, and application of a new class of artificial metalloenzymes that combines the scope of chemical catalysis with the efficiency of biosynthesis in an unprecedented manner to produce molecules of exceptional biological importance. The proposed system offers a number of significant advantages over previous artificial metalloenzyme constructs, which enable its use for in vivo catalysis and metabolic engineering. This ambitious project will be conducted as part of the candidate´s long term goals of increasing the efficiency of organic synthesis, particularly for the production of biologically active molecules. In the mentored phase (K99) of the proposed research, amino acids with catalytically active palladacycle side chains will be synthesized, characterized, and incorporated into a suitable scaffold protein. The catalytic activity of the resulting metalloenzymes will be evaluated using a variety of C-C bond forming reactions. The proposed amino acids catalysts could prove highly useful for a variety of applications in their own right, and their incorporation into proteins would mark a significant achievement in the fields of UAA incorporation and biocatalysis with potential applications well beyond the scope of this application. This research will be conducted in the laboratory of Professor Frances Arnold, a leader in the field of protein engineering, at the California Institute of Technology, a world-renowned research institution. Professor Arnold has a strong record as a mentor of successful members of industry and academia, and she and the candidate have outlined a career development plan focusing on mentorship, writing, and research to ensure the candidate continues this trend. The facilities, faculty, and staff at Caltech are ideal for completion of the proposed research and will contribute greatly to the candidate´s overall development as an independent scientist. Independent (R00) research will focus on directed evolution of artificial metalloenzymes for in vivo palladium catalysis of pharmaceutically important cross-coupling reactions with potential applications in organic synthesis and bio-orthogonal diagnostics. Optimized metalloenzymes will also be expressed with additional enzymes in E. coli in order to biosynthesize biologically active molecules, including indolocarbazole natural product derivatives. Success in this venture would greatly expand the scope of molecules available via metabolic engineering and simplify the production of new compounds for the betterment of human health. This work will build directly on the candidate´s experiences in the Arnold lab, and should foster the development of an exciting and collaborative research environment in the candidate´s independent laboratory focusing on the development and application of enzymes for sustainable organic synthesis. The research outlined in this proposal has the potential to greatly improve public health by creating a new class of artificial metalloenzymes for the synthesis biologically active molecules. This platform will enable inclusion of powerful transition metal catalysts in metabolic pathways in unprecedented fashion in order to efficiently produce chemicals in vivo
Keywords: Academia; Achievement; Achievement Attainment; Aerobic; Alkenes; Amaze; Amino Acids; Amino Acyl T RNA Synthetases; Amino Acyl-tRNA Ligases; Amino Acyl-tRNA Synthetases; Aminoacyl Transfer RNA Synthetase; Aminoacyl-tRNA Synthetase; Anabolism; Binding; Binding (Molecular Function); Biological; Biological Factors; Biology; Boronic Acids; California; Catalysis; Chemicals; Collection; Complex; Crude Oil; Development; Development Plans; Diagnostic; Discipline; Drug Industry; E coli; Engineering; Engineerings; Ensure; Environment; Enzymes; Escherichia coli; Factor, Biologic; Faculty; Fostering; Genetic Engineering of Proteins; Goals; Health; Human; Human, General; Individual; Industry; Industry, Pharmaceutic; Institutes; Institution; Ions; L-Tryptophan; Laboratories; Levotryptophan; Life; Man (Taxonomy); Man, Modern; Medical; Mentors; Mentorship; Metabolic; Metabolic Pathway; Metals; Molecular Configuration; Molecular Conformation; Molecular Interaction; Molecular Stereochemistry; Natural Products; Nature; Olefins; Organic Solvents; Organic Synthesis; Organic solvent product; Organism; Palladium; Pathway interactions; Pd element; Peptides; Petroleum; Pharmaceutical Agent; Pharmaceutical Industry; Pharmaceuticals; Pharmacologic Substance; Pharmacological Substance; Phase; Physiologic; Physiological; Plans, Development; Preparation; Procedures; Production; Protein Engineering; Proteins; Public Health; Reaction; Reagent; Research; Ribonucleic acids, transfer; Scaffolding Protein; Science; Scientist; Side; Site; Specificity; Speed; Speed (motion); System; System, LOINC Axis 4; Technology; Transfer RNA; Transfer RNA Synthetase; Transition Elements; Triplet Codon-Amino Acid Adaptor; Tryptophan; Work; Writing; aminoacid; aminoacid tRNA ligase; application in practice; aqueous; aryl halide; biosynthesis; career development; catalyst; chemical reaction; chemical synthesis; conformation; conformational state; design; designing; directed evolution; experience; gene product; improved; in vivo; living system; member; metalloenzyme; novel; pathway; petroleum oil; practical application; professor; public health medicine (field); public health relevance; success; tRNA; tRNA Synthetase; transition metal; trend
Relevance: The research outlined in this proposal has the potential to greatly improve public health by creating a new class of artificial metalloenzymes for the synthesis biologically active molecules. This platform will enable inclusion of powerful transition metal catalysts in metabolic pathways in unprecedented fashion in order to efficiently produce chemicals in vivo
Project start date: 2010-02-01
Project end date: 2012-01-31
Budget start date: 1-FEB-2010
Budget end date: 31-JAN-2011
PFA/PA: PA-09-036
1K99GM087551-01A1 (2010): $90000
Directed Evolution Of A Cytochrome P450 For Synthesis Of Artemisinic Alcohol
Jared C Lewis
California Institute Of Technology
office Of Sponsored Research, Mail Code 201-15
pasadena, Ca 91125
Grant 1F32GM079932-01 from National Institute Of General Medical Sciences IRG: ZRG1
Keywords: alcohol, cytochrome P450, cytochrome oxidase, directed evolution, enzyme Asian, Plasmodium falciparum, aldehyde, analog, back, base, biological product, biotechnology, catalyst, cell, combination therapy, environment, evolution, health, high throughput technology, library, malaria, material, medicine, membrane, mutant, oxidation, plant, reduction, success
Project start date: 2007-07-23
Project end date: 2010-07-22
1F32GM079932-01 (2007): $44846