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Applied and Environmental Microbiology, May 2002, p. 2411-2419, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2411-2419.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Enhanced Accumulation and Changed Monomer Composition in Polyhydroxyalkanoate (PHA) Copolyester by In Vitro Evolution of Aeromonas caviae PHA Synthase

Polymer Chemistry Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198,¹ Department of Biological and Environmental Sciences, Saitama University, 255 Simo-Okubo, Saitama, Saitama 338-8570,² Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan³

Received 28 September 2001/ Accepted 2 January 2002

By in vitro evolution experiment, we have first succeeded in acquiring higher active mutants of a synthase that is a key enzyme essential for bacterial synthesis of biodegradable polyester, polyhydroxyalkanoate (PHA). Aeromonas caviae FA440 synthase, termed PhaC_Ac, was chosen as a good target for evolution, since it can synthesize a PHA random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] that is a tough and flexible material compared to polyhydroxybutyrate (PHB) homopolyester. The in vitro enzyme evolution system consists of PCR-mediated random mutagenesis targeted to a limited region of the phaC_Ac gene and screening mutant enzymes with higher activities based on two types of polyester accumulation system by using Escherichia coli for the synthesis of PHB (by JM109 strain) (S. Taguchi, A. Maehara, K. Takase, M. Nakahara, H. Nakamura, and Y. Doi, FEMS Microbiol. Lett. 198:65-71, 2001) and of P(3HB-co-3HHx) {by LS5218 [fadR601 atoC(Con)] strain}. The expression vector for the phaC_Ac gene, together with monomer-supplying enzyme genes, was designed to synthesize PHB homopolyester from glucose and P(3HB-co-3HHx) copolyester from dodecanoate. Two evolved mutant enzymes, termed E2-50 and T3-11, screened through the evolution system exhibited 56 and 21% increases in activity toward 3HB-coenzyme A, respectively, and consequently led to enhanced accumulation (up to 6.5-fold content) of P(3HB-co-3HHx) in the recombinant LS5218 strains. Two single mutations in the mutants, N149S for E2-50 and D171G for T3-11, occurred at positions that are not highly conserved among the PHA synthase family. It should be noted that increases in the 3HHx fraction (up to 16 to 18 mol%) were observed for both mutants compared to the wild type (10 mol%).

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