title: A high-throughput screen for directed evolution of the natural product sulfotransferase LipB.
abstract: In this article, the authors describe a colorimetric, high-throughput assay suitable for optimizing the activity of the recently discovered sulfotransferase LipB, by directed evolution. Crucially, LipB uses para-nitrophenol sulfate as donor in the sulfation of the nucleoside antibiotic liposidomycin B-I and other acceptor surrogates. Thus, using a robotic liquid-handling device, crude cell extracts were prepared from an Escherichia coli strain that overproduced LipB in wells of a microplate, and production of para-nitrophenol at 405 nm was monitored spectrophotometrically. Enzyme activity could be detected only in the presence of both LipB substrates and overexpressed LipB. The screen displays a suitable standard deviation for directed evolution and importantly is not limited to the natural desulfo-liposidomycin acceptor. The authors plan to use the screen to identify LipB variants with altered acceptor specificity and promiscuity for use in sulfation of natural products and other small-molecule therapeutics.
authors.affiliation: Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Koryakina
authors.forename: Jessica
authors.initials: J
authors.lastname: Neville
authors.forename: Koichi
authors.initials: K
authors.lastname: Nonaka
authors.forename: Steven G
authors.initials: SG
authors.lastname: Van Lanen
authors.forename: Gavin J
authors.initials: GJ
authors.lastname: Williams
mesh_terms: D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000617:Aminoglycosides / Q000378:metabolism; D055162:Biocatalysis; D001688:Biological Products / Q000737:chemistry / Q000378:metabolism*; D001711:Biotransformation; D003001:Cloning, Molecular; D003124:Colorimetry; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D057166:High-Throughput Screening Assays; D009578:Nitrobenzenes / Q000032:analysis / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000032:analysis / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D000617:Aminoglycosides; D001688:Biological Products; D029968:Escherichia coli Proteins; D009578:Nitrobenzenes; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; C047544:liposidomycins; D000217:Acyltransferases; C421483:LipB protein, E coli; C013154:4-nitrophenetol