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Applied and Environmental Microbiology, August 2003, p. 4448-4454, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4448-4454.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Efficient Library Construction by In Vivo Recombination with a Telomere-Originated Autonomously Replicating Sequence of Hansenula polymorpha

So-Young Kim,1 Jung-Hoon Sohn,1 Jung-Hoon Bae,1 Yu-Ryang Pyun,2 Michael O. Agaphonov,3 Michael D. Ter-Avanesyan,3 and Eui-Sung Choi1*

Laboratory of Microbial Functions, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yusong, Taejon 305-333,1 Department of Biotechnology, Yonsei University, Seoul 120-749, Korea,2 Institute of Experimental Cardiology, Cardiology Research Center, 121552 Moscow, Russia3

Received 27 January 2003/ Accepted 8 May 2003

A high frequency of transformation and an equal gene dosage between transformants are generally required for activity-based selection of mutants from a library obtained by directed evolution. An efficient library construction method was developed by using in vivo recombination in Hansenula polymorpha. Various linear sets of vectors and insert fragments were transformed and analyzed to optimize the in vivo recombination system. A telomere-originated autonomously replicating sequence (ARS) of H. polymorpha, reported as a recombination hot spot, facilitates in vivo recombination between the linear transforming DNA and chromosomes. In vivo recombination of two linear DNA fragments containing the telomeric ARS drastically increases the transforming frequency, up to 10-fold, compared to the frequency of circular plasmids. Direct integration of the one-end-recombined linear fragment into chromosomes produced transformants with single-copy gene integration, resulting in the same expression level for the reporter protein between transformants. This newly developed in vivo recombination system of H. polymorpha provides a suitable library for activity-based selection of mutants after directed evolution.

* Corresponding author. Mailing address: Laboratory of Microbial Functions, Korea Research Institute of Bioscience and Biotechnology, 52 Oun-dong, Yusong, Taejon 305-333, Korea. Phone: 82-42-860-4453. Fax: 82-42-860-4594. E-mail: choi4162{at}kribb.re.kr.

Applied and Environmental Microbiology, August 2003, p. 4448-4454, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4448-4454.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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