title: Integrative genomic mining for enzyme function to enable engineering of a non-natural biosynthetic pathway.
abstract: The ability to biosynthetically produce chemicals beyond what is commonly found in Nature requires the discovery of novel enzyme function. Here we utilize two approaches to discover enzymes that enable specific production of longer-chain (C5-C8) alcohols from sugar. The first approach combines bioinformatics and molecular modelling to mine sequence databases, resulting in a diverse panel of enzymes capable of catalysing the targeted reaction. The median catalytic efficiency of the computationally selected enzymes is 75-fold greater than a panel of naively selected homologues. This integrative genomic mining approach establishes a unique avenue for enzyme function discovery in the rapidly expanding sequence databases. The second approach uses computational enzyme design to reprogramme specificity. Both approaches result in enzymes with >100-fold increase in specificity for the targeted reaction. When enzymes from either approach are integrated in vivo, longer-chain alcohol production increases over 10-fold and represents >95% of the total alcohol products.
authors.affiliation: Department of Chemistry, University of California Davis, One Shields Avenue, Davis, California 95616, USA.
authors.forename: Wai Shun
authors.initials: WS
authors.lastname: Mak
authors.affiliation: Department of Bioinformatics, University of California, Los Angeles, 172 Boyer Hall Box 951570, 611 Charles E. Young Drive East, Los Angeles, California 90095-1570, USA.
authors.forename: Stephen
authors.initials: S
authors.lastname: Tran
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California Los Angeles, 5531 Boelter Hall, Los Angeles, California 90095-1592, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Marcheschi
authors.affiliation: Department of Chemistry, University of California Davis, One Shields Avenue, Davis, California 95616, USA.
authors.forename: Steve
authors.initials: S
authors.lastname: Bertolani
authors.affiliation: Department of Biochemistry, University of Washington, 1705 NE Pacific St Seattle, Washington 98195-7350, USA.
authors.forename: James
authors.initials: J
authors.lastname: Thompson
authors.affiliation: Department of Biochemistry, University of Washington, 1705 NE Pacific St Seattle, Washington 98195-7350, USA.
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California Los Angeles, 5531 Boelter Hall, Los Angeles, California 90095-1592, USA.|Institute for Genomics and Proteomics, University of California Los Angeles, 611 Young Drive East, Los Angeles, California 90095, USA.
authors.forename: James C
authors.initials: JC
authors.lastname: Liao
authors.affiliation: Department of Chemistry, University of California Davis, One Shields Avenue, Davis, California 95616, USA.|Department of Biochemistry and Molecular Medicine, University of California Davis, 2700 Stockton Boulevard, Suite 2102, Sacramento, California 95817, USA.|Genome Center, University of California Davis, 451 Health Sciences Drive, Davis, California 95616, USA.
authors.forename: Justin B
authors.initials: JB
authors.lastname: Siegel
mesh_terms: D000438:Alcohols / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics*; D019295:Computational Biology; D004926:Escherichia coli; D023281:Genomics; D007651:Keto Acids / Q000378:metabolism; D008958:Models, Molecular
chemical_list: D000438:Alcohols; D007651:Keto Acids