Efficient library construction by in vivo recombination with a telomere-originated autonomously replicating sequence of Hansenula polymorpha

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Title
Efficient library construction by in vivo recombination with a telomere-originated autonomously replicating sequence of Hansenula polymorpha
Author(s)
So-Young Kim; Jung Hoon SohnJung Hoon Bae; Y R Pyun; M O Agaphonov; M D Ter-Avanesyan; Eui Sung Choi
Bibliographic Citation
Applied and Environmental Microbiology, vol. 69, no. 8, pp. 4448-4454
Publication Year
2003
Abstract
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.
ISSN
0099-2240
Publisher
Amer Soc Microb
DOI
http://dx.doi.org/10.1128/AEM.69.8.4448-4454.2003
Type
Article
Appears in Collections:
Synthetic Biology and Bioengineering Research Institute > Synthetic Biology Research Center > 1. Journal Articles
Division of Bio Technology Innovation > BioProcess Engineering Center > 1. Journal Articles
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