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Journal of Bacteriology, July 2004, p. 4177-4184, Vol. 186, No. 13
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.13.4177-4184.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Altering the Substrate Specificity of Polyhydroxyalkanoate Synthase 1 Derived from Pseudomonas putida GPo1 by Localized Semirandom Mutagenesis

Der-Shyan Sheu and Chia-Yin Lee*

Graduate Institute of Agricultural Chemistry, National Taiwan University, Taipei 106, Taiwan

Received 8 January 2004/ Accepted 23 March 2004

The substrate specificity of polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Pp, class II) from Pseudomonas putida GPo1 (formerly known as Pseudomonas oleovorans GPo1) was successfully altered by localized semirandom mutagenesis. The enzyme evolution system introduces multiple point mutations, designed on the basis of the conserved regions of the PHA synthase family, by using PCR-based gene fragmentation with degenerate primers and a reassembly PCR. According to the opaqueness of the colony, indicating the accumulation of large amounts of PHA granules in the cells, 13 PHA-accumulating candidates were screened from a mutant library, with Pseudomonas putida GPp104 PHA– as the host. The in vivo substrate specificity of five candidates, L1-6, D7-47, PS-A2, PS-C2, and PS-E1, was evaluated by the heterologous expression in Ralstonia eutropha PHB–4 supplemented with octanoate. Notably, the amount of 3-hydroxybutyrate (short-chain-length [SCL] 3-hydroxyalkanoate [3-HA] unit) was drastically increased in recombinants that expressed evolved mutant enzymes L1-6, PS-A2, PS-C2, and PS-E1 (up to 60, 36, 50, and 49 mol%, respectively), relative to the amount in the wild type (12 mol%). Evolved enzyme PS-E1, in which 14 amino acids had been changed and which was heterologously expressed in R. eutropha PHB–4, not only exhibited broad substrate specificity (49 mol% SCL 3-HA and 51 mol% medium-chain-length [MCL] 3-HA) but also conferred the highest PHA production (45% dry weight) among the candidates. The 3-HA and MCL 3-HA units of the PHA produced by R. eutropha PHB–4/pPS-E1 were randomly copolymerized in a single polymer chain, as analytically confirmed by acetone fractionation and the 13C nuclear magnetic resonance spectrum.

* Corresponding author. Mailing address: Graduate Institute of Agricultural Chemistry, National Taiwan University, 1, Sec. 4, Roosevelt Rd., Taipei 106, Taiwan. Phone: 886 2 23630231, ext. 2816. Fax: 886 2 23660581. E-mail: m477{at}ntu.edu.tw.

Journal of Bacteriology, July 2004, p. 4177-4184, Vol. 186, No. 13
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.13.4177-4184.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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