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      A Serine-Substituted P450 Catalyzes Highly Efficient Carbene Transfer to Olefins In Vivo

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          Abstract

          Genetically encoded catalysts for non-natural chemical reactions will open new routes to sustainable production of chemicals. We designed a unique serine-heme ligated cytochrome “P411” that catalyzes efficient and selective carbene transfers from diazoesters to olefins in intact Escherichia coli cells. The mutation C400S in cytochrome P450 BM3 gives a signature ferrous-CO Soret peak at 411 nm, abolishes monooxygenation activity, raises the resting state Fe III/II reduction potential, and significantly improves NAD(P)H-driven cyclopropanation activity.

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          Most cited references29

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          Rapid planetesimal formation in turbulent circumstellar discs

          The initial stages of planet formation in circumstellar gas discs proceed via dust grains that collide and build up larger and larger bodies (Safronov 1969). How this process continues from metre-sized boulders to kilometre-scale planetesimals is a major unsolved problem (Dominik et al. 2007): boulders stick together poorly (Benz 2000), and spiral into the protostar in a few hundred orbits due to a head wind from the slower rotating gas (Weidenschilling 1977). Gravitational collapse of the solid component has been suggested to overcome this barrier (Safronov 1969, Goldreich & Ward 1973, Youdin & Shu 2002). Even low levels of turbulence, however, inhibit sedimentation of solids to a sufficiently dense midplane layer (Weidenschilling & Cuzzi 1993, Dominik et al. 2007), but turbulence must be present to explain observed gas accretion in protostellar discs (Hartmann 1998). Here we report the discovery of efficient gravitational collapse of boulders in locally overdense regions in the midplane. The boulders concentrate initially in transient high pressures in the turbulent gas (Johansen, Klahr, & Henning 2006), and these concentrations are augmented a further order of magnitude by a streaming instability (Youdin & Goodman 2005, Johansen, Henning, & Klahr 2006, Johansen & Youdin 2007) driven by the relative flow of gas and solids. We find that gravitationally bound clusters form with masses comparable to dwarf planets and containing a distribution of boulder sizes. Gravitational collapse happens much faster than radial drift, offering a possible path to planetesimal formation in accreting circumstellar discs.
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            Stereoselective cyclopropanation reactions.

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              Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin.

              Malaria, caused by Plasmodium sp, results in almost one million deaths and over 200 million new infections annually. The World Health Organization has recommended that artemisinin-based combination therapies be used for treatment of malaria. Artemisinin is a sesquiterpene lactone isolated from the plant Artemisia annua. However, the supply and price of artemisinin fluctuate greatly, and an alternative production method would be valuable to increase availability. We describe progress toward the goal of developing a supply of semisynthetic artemisinin based on production of the artemisinin precursor amorpha-4,11-diene by fermentation from engineered Saccharomyces cerevisiae, and its chemical conversion to dihydroartemisinic acid, which can be subsequently converted to artemisinin. Previous efforts to produce artemisinin precursors used S. cerevisiae S288C overexpressing selected genes of the mevalonate pathway [Ro et al. (2006) Nature 440:940-943]. We have now overexpressed every enzyme of the mevalonate pathway to ERG20 in S. cerevisiae CEN.PK2, and compared production to CEN.PK2 engineered identically to the previously engineered S288C strain. Overexpressing every enzyme of the mevalonate pathway doubled artemisinic acid production, however, amorpha-4,11-diene production was 10-fold higher than artemisinic acid. We therefore focused on amorpha-4,11-diene production. Development of fermentation processes for the reengineered CEN.PK2 amorpha-4,11-diene strain led to production of > 40 g/L product. A chemical process was developed to convert amorpha-4,11-diene to dihydroartemisinic acid, which could subsequently be converted to artemisinin. The strains and procedures described represent a complete process for production of semisynthetic artemisinin.
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                Author and article information

                Journal
                101231976
                32624
                Nat Chem Biol
                Nat. Chem. Biol.
                Nature chemical biology
                1552-4450
                1552-4469
                17 June 2013
                23 June 2013
                August 2013
                01 February 2014
                : 9
                : 8
                : 485-487
                Affiliations
                [1 ]Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA
                [2 ]Department of Chemistry and Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
                Author notes
                Article
                NIHMS482143
                10.1038/nchembio.1278
                3720782
                23792734
                eb2434f4-0b27-45e0-aa2e-fa4a9ae2e278

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                History
                Funding
                Funded by: National Institute of Diabetes and Digestive and Kidney Diseases : NIDDK
                Award ID: R37 DK019038 || DK
                Funded by: National Institute of Diabetes and Digestive and Kidney Diseases : NIDDK
                Award ID: R01 DK019038 || DK
                Categories
                Article

                Biochemistry
                Biochemistry

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