Site-saturation Mutagenesis is more Efficient than DNA Shuffling for the Directed Evolution of β-Fucosidase from β-Galactosidase

• Type of Material: Article
• Authors: Monal R. Parikh, Emory University; Ichiro Matsumura, Emory University
• Language: English
• Date: 2005-09-23
• Publisher: Elsevier
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2005 Elsevier Ltd. All rights reserved.
• License: Creative Commons Attribution-NonCommercial-NoDerivs 2.5 Generic
• Final Published Version (URL): http://dx.doi.org/10.1016/j.jmb.2005.07.020
• Title of Journal or Parent Work: Journal of Molecular Biology
• ISSN: 0022-2836
• Volume: 352
• Issue: 3
• Start Page: 621
• End Page: 628
• Grant/Funding Information: I.M. was supported by the NIH/NIAID (1 R21AI054602-01).; M.P. was supported by NSF BIO/MCB (#0109668).; The FAME Center is supported by NSF/DBI (#0320786).
• Keywords: molecular recognition; random mutagenesis; substrate specificity; in vitro evolution; high-throughput screen
• Research Categories: Chemistry, Biochemistry

Abstract

Protein engineers use a variety of mutagenic strategies to adapt enzymes to novel substrates. Directed evolution techniques (random mutagenesis and high-throughput screening) offer a systematic approach to the management of protein complexity. This sub-discipline was galvanized by the invention of DNA shuffling, a procedure that randomly recombines point mutations in vitro. In one influential study, Escherichia coli β-galactosidase (BGAL) variants with enhanced β-fucosidase activity (tenfold increase in kcat/KM in reactions with the novel para-nitrophenyl-β-d-fucopyranoside substrate; 39-fold decrease in reactivity with the “native” para-nitrophenyl-β-d-galactopyranoside substrate) were evolved in seven rounds of DNA shuffling and screening. Here, we show that a single round of site-saturation mutagenesis and screening enabled the identification of β-fucosidases that are significantly more active (180-fold increase in kcat/KM in reactions with the novel substrate) and specific (700,000-fold inversion of specificity) than the best variants in the previous study. Site-saturation mutagenesis thus proved faster, less resource-intensive and more effective than DNA shuffling for this particular evolutionary pathway.