A high-throughput screen for directed evolution of the natural product sulfotransferase LipB.

Irina Koryakina, Jessica Neville, Koichi Nonaka, Steven G Van Lanen, Gavin J Williams

Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA.

Journal of biomolecular screening 2011 Sep

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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.

Citation

Irina Koryakina, Jessica Neville, Koichi Nonaka, Steven G Van Lanen, Gavin J Williams. A high-throughput screen for directed evolution of the natural product sulfotransferase LipB. Journal of biomolecular screening. 2011 Sep;16(8):845-51