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Applied and Environmental Microbiology, November 2004, p. 6789-6799, Vol. 70, No. 11
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.11.6789-6799.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Engineering of Chimeric Class II Polyhydroxyalkanoate Synthases

Nuttawee Niamsiri,^1,2 Soazig C. Delamarre,¹ Young-Rok Kim,^1, and Carl A. Batt^1*

Department of Food Science, Cornell University, Ithaca, New York,¹ Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand²

Received 17 February 2004/ Accepted 20 June 2004

PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs). Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties. To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1_Po) that was devoid of an internal 540-bp fragment. Randomly amplified PCR products (created with primers based on conserved phaC sequences flanking the deleted internal fragment) were generated using genomic DNA isolated from soil and were substituted for the 540-bp internal region. The chimeric genes were expressed in a PHA-negative strain of Ralstonia eutropha, PHB^–4 (DSM 541). Out of 1,478 recombinant clones screened for PHA production, we obtained five different chimeric phaC1_Po genes that produced more PHA than the native phaC1_Po. Chimeras S1-71, S4-8, S5-58, S3-69, and S3-44 exhibited 1.3-, 1.4-, 2.0-, 2.1-, and 3.0-fold-increased levels of in vivo activity, respectively. All of the mutants mediated the synthesis of PHAs with a slightly increased molar fraction of 3-hydroxyoctanoate; however, the weight-average molecular weights (M_w) of the PHAs in all cases remained almost the same. Based upon DNA sequence analyses, the various phaC fragments appear to have originated from Pseudomonas fluorescens and Pseudomonas aureofaciens. The amino acid sequence analyses showed that the chimeric proteins had 17 to 20 amino acid differences from the wild-type phaC1_Po, and these differences were clustered in the same positions in the five chimeric clones. A threading model of PhaC1_Po, developed based on homology of the enzyme to the Burkholderia glumae lipase, suggested that the amino acid substitutions found in the active chimeras were located mostly on the protein model surface. Thus, our combinatorial genetic engineering strategy proved to be broadly useful for improving the catalytic activities of PHA synthase enzymes.

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* Corresponding author. Mailing address: Department of Food Science, Stocking Hall, Cornell University, Ithaca, NY 14853. Phone: (607) 254-5371. Fax: (607) 255-8741. E-mail: cab10{at}cornell.edu.

Present address: Department of Molecular and Cell Biology, Harvard University, Cambridge, MA 02138.

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Applied and Environmental Microbiology, November 2004, p. 6789-6799, Vol. 70, No. 11
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.11.6789-6799.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.