Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening

Abstract

An efficient ␤-fucosidase was evolved by DNA shuff ling from the Escherichia coli lacZ ␤-galactosidase. Seven rounds of DNA shuff ling and colony screening on chromogenic fucose substrates were performed, using 10,000 colonies per round. Compared with native ␤-galactosidase, the evolved enzyme purified from cells from the final round showed a 1,000-fold increased substrate specificity for onitrophenyl fucopyranoside versus o-nitrophenyl galactopyranoside and a 300-fold increased substrate specificity for p-nitrophenyl fucopyranoside versus p-nitrophenyl galactopyranoside. The evolved cell line showed a 66-fold increase in p-nitrophenyl fucosidase specific activity. The evolved fucosidase has a 10-to 20-fold increased k cat ͞K m for the fucose substrates compared with the native enzyme. The DNA sequence of the evolved fucosidase gene showed 13 base changes, resulting in six amino acid changes from the native enzyme. This effort shows that the library size that is required to obtain significant enhancements in specificity and activity by reiterative DNA shuff ling and screening, even for an enzyme of 109 kDa, is within range of existing high-throughput technology. Reiterative generation of libraries and stepwise accumulation of improvements based on addition of beneficial mutations appears to be a promising alternative to rational design.Proteins and enzymes with novel functions and properties can be obtained either by searching the largely unknown natural species or by improving upon currently known natural proteins or enzymes. The latter approach may be more suitable for creating properties for which natural evolutionary processes are unlikely to have been selected.One promising strategy to create such novel properties is by directed molecular evolution. Starting with known natural protein(s), multiple rounds of mutagenesis, functional screening, and amplification can be carried out. When the mutation rate, library size, and selection pressures are properly balanced, the desired phenotype of a protein generally increases with each round (1-8). The advantage of such a process is that it can be used to rapidly evolve any protein, without any knowledge of its structure.

Effects of Recombination on Complex Regulatory Circuits

“…The authors estimate that mutating the same number of positions as are changed in a recombination event among these proteins would abolish protein function (Chang et al 1999). Such weak deleterious effects may contribute to the promise recombination shows in engineering new proteins (Stemmer 1994;Crameri et al 1997Crameri et al , 1998Zhang et al 1997;Chang et al 1999;Kolkman and Stemmer 2001;Leong et al 2003;Raillard et al 2001a,b). Because recombination can reorganize genetic systems on a much larger scale than point mutations, these weak effects on different levels of organization are intriguing.…”

Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

“…Chromogenic substrates have been used successfully in a number of studies utilizing a 96-well plate reader or visual screening of the colored colonies (48,49). With VAO, the end product vanillin formed upon the two-step oxidation of creosol could be measured directly in whole cells by absorption spectroscopy at 340 nm.…”

Perspective: Transposable Elements, Parasitic Dna, and Genome Evolution

“…Although simple DNA base substitutions are well suited for the generation, diversification, and optimization of local protein space (Maeshiro and Kimura 1998), a hierarchy of natural mutational events is required for the rapid generation of protein diversity. Experimental evolution (Moore et al 1997;Zhang et al 1997;Crameri et al 1998) and computer simulation experiments (Bogarad and Deem 1999) showed that sequence shuffling has the potential to improve protein function significantly better than does point mutation alone. Because they are uniquely competent to reshuffle DNA sequences, TEs and viruses are important generators of the more complex types of mutations in the mutational hierarchy.…”