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Applied and Environmental Microbiology, December 2002, p. 6146-6151, Vol. 68, No. 12
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.12.6146-6151.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase

Jae Kwang Song,{dagger} Bora Chung, Young Hak Oh, and Joon Shick Rhee*

Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejon 305-701, Korea

Received 14 June 2002/ Accepted 25 September 2002

A method of mutagenic and unidirectional reassembly (MURA) that can generate libraries of DNA-shuffled and randomly truncated proteins was developed. The method involved fragmenting the template gene(s) randomly by DNase I and reassembling the small fragments with a unidirectional primer by PCR. The MURA products were treated with T4 DNA polymerase and subsequently with a restriction enzyme whose site was located on the region of the MURA primer. The N-terminal-truncated and DNA-shuffled library of a Serratia sp. phospholipase A1 prepared by this method had an essentially random variation of truncated size and also showed point mutations associated with DNA shuffling. After high-throughput screening on triglyceride-emulsified plates, several mutants exhibiting absolute lipase activity (NPL variants) were obtained. The sequence analysis and the lipase activity assay on the NPL variants revealed that N-terminal truncations at a region beginning with amino acids 61 to 71, together with amino acid substitutions, resulted in the change of substrate specificity from a phospholipase to a lipase. We therefore suggest that the MURA method, which combines incremental truncation with DNA shuffling, can contribute to expanding the searchable sequence space in directed evolution experiments.

* Corresponding author. Mailing address: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejon 305-701, Korea. Phone: 82-42-869-2613. Fax: 82-42-869-2610. E-mail: jsrhee{at}mail.kaist.ac.kr.

{dagger} Present address: Applied and Engineering Chemistry Division, Korea Research Institute of Chemical Technology, Yuseong-gu, Daejon 305-600, Korea.

Applied and Environmental Microbiology, December 2002, p. 6146-6151, Vol. 68, No. 12
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.12.6146-6151.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Yuan, L., Kurek, I., English, J., Keenan, R. (2005). Laboratory-Directed Protein Evolution. Microbiol. Mol. Biol. Rev. 69: 373-392 [Abstract] [Full Text]  
  • Osuna, J., Yanez, J., Soberon, X., Gaytan, P. (2004). Protein evolution by codon-based random deletions. Nucleic Acids Res 32: e136-e136 [Abstract] [Full Text]  


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