title: Observing Biosynthetic Activity Utilizing Next Generation Sequencing and the DNA Linked Enzyme Coupled Assay.
abstract: Currently, the identification of new genes drastically outpaces current experimental methods for determining their enzymatic function. This disparity necessitates the development of high-throughput techniques that operate with the same scalability as modern gene synthesis and sequencing technologies. In this paper, we demonstrate the versatility of the recently reported DNA-Linked Enzyme-Coupled Assay (DLEnCA) and its ability to support high-throughput data acquisition through next-generation sequencing (NGS). Utilizing methyltransferases, we highlight DLEnCA's ability to rapidly profile an enzyme's substrate specificity, determine relative enzyme kinetics, detect biosynthetic formation of a target molecule, and its potential to benefit from the scales and standardization afforded by NGS. This improved methodology minimizes the effort in acquiring biosynthetic knowledge by tying biochemical techniques to the rapidly evolving abilities in sequencing and synthesizing DNA.
authors.affiliation: Department of Biological Engineering, Synthetic Biology Institute, University of California, Berkeley , Berkeley, California 94704, United States.
authors.forename: Markus de
authors.identifier: 0000-0001-8263-9198
authors.initials: M
authors.lastname: Raad
authors.affiliation: Department of Biological Engineering, Synthetic Biology Institute, University of California, Berkeley , Berkeley, California 94704, United States.
authors.forename: Cyrus
authors.initials: C
authors.lastname: Modavi
authors.affiliation: Department of Biological Engineering, Synthetic Biology Institute, University of California, Berkeley , Berkeley, California 94704, United States.
authors.forename: David J
authors.initials: DJ
authors.lastname: Sukovich
authors.affiliation: Department of Biological Engineering, Synthetic Biology Institute, University of California, Berkeley , Berkeley, California 94704, United States.
authors.forename: J Christopher
authors.initials: JC
authors.lastname: Anderson
mesh_terms: D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics; D002394:Catechol O-Methyltransferase / Q000737:chemistry; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D029681:Arabidopsis Proteins; C507996:IAMT1 protein, Arabidopsis; D008780:Methyltransferases; D002394:Catechol O-Methyltransferase; C019608:caffeate O-methyltransferase