44 research outputs found
High-titer production of lathyrane diterpenoids from sugar by engineered Saccharomyces cerevisiae
Euphorbiaceae are an important source of medically important diterpenoids, such as the anticancer drug ingenol-3-angelate and the antiretroviral drug prostratin. However, extraction from the genetically intractable natural producers is often limited by the small quantities produced, while the organic synthesis of terpene-derived drugs is challenging and similarly low-yielding. While transplanting the biosynthetic pathway into a heterologous host has proven successful for some drugs, it has been largely unsuccessful for diterpenoids due to their elaborate biosynthetic pathways and lack of genetic resources and tools for gene discovery. We engineered casbene precursor production in S. cerevisiae, verified the ability of six Euphorbia lathyris and Jatropha curcas cytochrome P450s to oxidize casbene, and optimized the expression of these P450s and an alcohol dehydrogenase to generate jolkinol C, achieving ~800mg/L of jolkinol C and over 1g/L total oxidized casbanes in millititer plates, the highest titer of oxidized diterpenes in yeast reported to date. This strain enables the semisynthesis of biologically active jolkinol C derivatives and will be an important tool in the elucidation of the biosynthetic pathways for ingenanes, tiglianes, and lathyranes. These findings demonstrate the ability of S. cerevisiae to produce oxidized drug precursors in quantities that are sufficient for drug development and pathway discovery
A community resource for paired genomic and metabolomic data mining
Genomics and metabolomics are widely used to explore specialized metabolite diversity. The Paired Omics Data Platform is a community initiative to systematically document links between metabolome and (meta)genome data, aiding identification of natural product biosynthetic origins and metabolite structures.Peer reviewe
Oxidative cyclization of prodigiosin by an alkylglycerol monooxygenase-like enzyme
Prodiginines, which are tripyrrole alkaloids displaying a wide array of bioactivities, occur as linear and cyclic congeners. Identification of an unclustered biosynthetic gene led to the discovery of the enzyme responsible for catalyzing the regiospecific C-H activation and cyclization of prodigiosin to cycloprodigiosin in Pseudoalteromonas rubra. This enzyme is related to alkylglycerol monooxygenase and unrelated to RedG, the Rieske oxygenase that produces cyclized prodiginines in Streptomyces, implying convergent evolution
A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts
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Biotransformation discovery enabled by high-throughput mass-spectrometric enzyme activity screening and comparative genomics
The potential of replacing unsustainable petroleum feedstocks with the biological production of small molecules is stipulated on access to a wide array of enzyme-catalyzed transformations. In this dissertation, we have taken two distinct approaches to expanding the biocatalytic toolbox. In one case, we develop a high-throughput mass spectrometry-based assay for detecting enzyme activity. We lay out in detail the chemical synthesis, software development, assay optimization and benchmarking that has allowed the assay to become a robust method. We then showcase our technology to the screening of a cytochrome P450 mutant library, allowing us to identify enzyme variants with desired altered substrate specificity. In a complimentary enzyme discovery approach, we turn to the diversity of fascinating reactions already present in nature. Through comparative genomics of the marine bacterium Pseudoalteromonas rubra, we identify an enzyme responsible for catalyzing the impressive feat of regioselective C–H activation and C–C bond-formation in the biosynthesis of cycloprodigiosin
Recommended from our members
Biotransformation discovery enabled by high-throughput mass-spectrometric enzyme activity screening and comparative genomics
The potential of replacing unsustainable petroleum feedstocks with the biological production of small molecules is stipulated on access to a wide array of enzyme-catalyzed transformations. In this dissertation, we have taken two distinct approaches to expanding the biocatalytic toolbox. In one case, we develop a high-throughput mass spectrometry-based assay for detecting enzyme activity. We lay out in detail the chemical synthesis, software development, assay optimization and benchmarking that has allowed the assay to become a robust method. We then showcase our technology to the screening of a cytochrome P450 mutant library, allowing us to identify enzyme variants with desired altered substrate specificity. In a complimentary enzyme discovery approach, we turn to the diversity of fascinating reactions already present in nature. Through comparative genomics of the marine bacterium Pseudoalteromonas rubra, we identify an enzyme responsible for catalyzing the impressive feat of regioselective C–H activation and C–C bond-formation in the biosynthesis of cycloprodigiosin
Co-occurrence of enzyme domains guides the discovery of an oxazolone synthetase
Multidomain enzymes orchestrate two or more catalytic activities to carry out metabolic transformations with increased control and speed. Here, we report the design and development of a genome-mining approach for targeted discovery of biochemical transformations through the analysis of co-occurring enzyme domains (CO-ED) in a single protein. CO-ED was designed to identify unannotated multifunctional enzymes for functional characterization and discovery based on the premise that linked enzyme domains have evolved to function collaboratively. Guided by CO-ED, we targeted an unannotated predicted ThiF-nitroreductase di-domain enzyme found in more than 50 proteobacteria. Through heterologous expression and biochemical reconstitution, we discovered a series of natural products containing the rare oxazolone heterocycle and characterized their biosynthesis. Notably, we identified the di-domain enzyme as an oxazolone synthetase, validating CO-ED-guided genome mining as a methodology with potential broad utility for both the discovery of unusual enzymatic transformations and the functional annotation of multidomain enzymes
Mining genomes to illuminate the specialized chemistry of life
All organisms produce specialized organic molecules, ranging from small volatile chemicals to large gene-encoded peptides, that have evolved to provide them with diverse cellular and ecological functions. As natural products, they are broadly applied in medicine, agriculture and nutrition. The rapid accumulation of genomic information has revealed that the metabolic capacity of virtually all organisms is vastly underappreciated. Pioneered mainly in bacteria and fungi, genome mining technologies are accelerating metabolite discovery. Recent efforts are now being expanded to all life forms, including protists, plants and animals, and new integrative omics technologies are enabling the increasingly effective mining of this molecular diversity