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A Novel Semi-biosynthetic Route for Artemisinin Production Using Engineered Substrate-Promiscuous P450BM3 Jeffrey A. Dietrich†,¶,‡‡, Yasuo Yoshikuni†,¶,¿,ő ,‡‡,§§, Karl J. Fisher#, Frank X. Woolard#, Denise Ockey#, Derek J. McPhee#, Neil S. Renninger#, Michelle C. Y. Chang¶,‡, , David Baker¿,ő , and Jay D. Keasling†,¶,‡,§,††,* †UCSF/UCB Joint Graduate Group in Bioengineering, ‡California Institute for Quantitative Biomedical Research (QB3), !Department of Chemistry, and §Department of Chemical Engineering, University of California at Berkeley, Berkeley, California 94720, ¶Synthetic Biology Department, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94710, ¿Department of Biochemistry, University of Washington, Seattle, Washington 98195, ő Howard Hughes Medical Institute, Seattle, Washington 98195, #Amyris Biotechnologies Inc., Emeryville, California 94208, and ††Joint BioEnergy Institute, Emeryville, California 94208. ‡‡These authors contributed equally to this work. §§Present address: Bio Architecture Lab, Inc., Seattle, Washington 98103. ABSTRACT Production of fine heterologus pathways in microbial hosts is frequently hindered by insufficient knowledge of the native metabolic pathway and its cognate enzymes; often the pathway is unresolved and enzymes lack detailed characterization. An alternative paradigm to using native pathways is de novo pathway design using well-characterized, substrate-promiscuous enzymes. We demonstrate this concept using P450BM3 from Bacillus megaterium . Using a computer model, we illustrate how key P450BM3 activ site mutations enable binding of non-native substrate amorphadiene, incorporating these mutations into P450BM3 enabled the selective oxidation of amorphadiene arteminsinic-11s,12-epoxide, at titers of 250 mg L1 in E. coli . We also demonstrate high-yeilding, selective transformations to dihydroartemisinic acid, the immediate precursor to the high value anti-mala