DNA-shuffling

Family shuffling uses genes that have evolved in different microbial species from a common ancestral protein, therefore possessing sequences with a high degree of identity. This is a requirement for the process 50% sequence identity is needed. Proteolytic enzymes like subtilisin have been shuffled, using a large family of 26 

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Crameri, A., Raillard, S.-A., Bermudez, E., Stemmer, W.P.C. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391 288-291, 1998. 

Stemmer, W.P.C. DNA shuffling by random fragmentation and reassembly In vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91 10747-10751, 1994. 

Figure 2.5 Scheme for DNA shuffling illustrated for the case in which the parent genes originate from the WT by some form of mutagenesis [7e],... 

Efforts were also made to invert the sense of enantioselectivity in the hydrolytic kinetic resolution of ester (1) using PAL with preferential formation of (R)-2 [40,411-Using epPCR and DNA shuffling, an (R)-selective mutant showing an E value of 30 was evolved by screening about 45 000 clones for the (R) enantiomer. The best mutant is characterized by 11 mutations, which are different from those of the best (S)-selective variant X [41]. 

In another study that appeared prior to the advent of CASTing, the traditional combination of epPCR and DNA shuffling was used to enhance the enantioselectivity of the hydrolytic kinetic resolution of p-nitro phenyl glycidyl ether and other epoxides catalyzed by the EH from Agrobacterium radiobacter [59]. Several mutants were obtained with up to 13-fold improved enantioselectivity. The amino acid exchanges took place around the active site. 

Initial approaches to directed evolution of enzymes rested upon the introduction of random mutations in random sites of the enzyme by the use of the error-prone PCR technique [92] or on the DNA-shuffling method [93]. Extensive research has also been reported in which every amino acid site in an enzyme was systematically subjected to saturation mutagenesis [94]. 

Pseudomonas aeruginosa lipase-catalyzed hydrolysis of racemic ester 23 proceeds with very low enantioselectivity E = 1.1). Sequential use of error-prone PCR, saturation mutagenesis at chosen spots and DNA shuffling resulted in the formation of a mutant whose enantioselectivity was over 50. 

Crameri A, Whitehom EA, Tate E, Stemmer WP (1996) Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat Biotechnol 14 315-319... 

Kagami, O., Baik, S.-H. and Harayama, S. (2004) Effective DNA shuffling methods for enzyme evolution, in Enzyme Functionality (ed. A. Svendsen), Marcel Dekker, Inc., New York, pp. 425-441. 

Figure 3.2 Examples of gene shuffling methods used for DNA library creation in directed evolution, (a) Homology-dependent primer-independent DNA shuffling (b) homology-dependent primer-dependent StEP (c) homology-independent SHIPREC...

Stutzman-Engwall, K., Conlon, S., Fedechko, R. et al. (2005) Semi-synthetic DNA shuffling of aveC leads to improved industrial scale production of doramectin by Streptomyces avermitilis. Metabolic Engineering, 7,... 

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