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. (Journal of Biomolecular Screening. 2011;16:845-851)

A robust protocol for directed aryl sulfotransferase evolution toward the carbohydrate building block GlcNAc

“…ASTs use phenolic sulfate esters (e.g., p ‐nitrophenyl sulfate ( p NPS), 4‐methylumbelliferyl sulfate (4‐MUS) and 1‐naphthyl sulfate) as sulfuryl donors (Kobashi, Kim, & Morikawa, ; Malojcić & Glockshuber, ) that are more cost‐efficient than PAPS. Prokaryotic sulfotransferase have usually a broad substrate scope (Kim, Yoon, Koizumi, & Kobashi, ; van der Horst et al, ) and bacterial expression systems can be used for enzyme production (Kim, Hyun, Lee, Kobashi, & Kim, ) and reengineering (Koryakina, Neville, Nonaka, Van Lanen, & Williams, ). For instance, it has been reported that ASTs are able to sulfurylate phenolic antibiotics (Kim et al, ), steroids (van der Horst, van Lieshout, Bury, Hartog, & Wever, ), flavonoids (Roubalova et al, ; van der Horst et al, ), and, as recently reported, large molecules like lignin (Prinsen, Narani, Hartog, Wever, & Rothenberg, ).…”

“…Nevertheless, in one manuscript a directed evolution attempt was reported in which a colorimetric endpoint assay in 96‐well microtiter plate (MTP)‐format was evaluated for a bacterial sulfotransferase LipB (improved uridine sulfurylation). Screening of 200 variants (generated by error prone PCR; epPCR) did not result in a reported LipB variant with an improved property (Koryakina et al, ).…”