Evolving thermostability in mutant libraries of ligninolytic oxidoreductases expressed in yeast

Microbial Cell Factories

Research

Angel T Martinez¹  Antonio Ballesteros²  Miguel Alcalde²  Diana Maté²  Eva García-Ruiz³ 

[1] Centro de Investigaciones Biológicas, CSIC, 28040, Madrid, Spain;Department of Biocatalysis, Institute of Catalysis, CSIC, 28049, Madrid, Spain;Department of Biocatalysis, Institute of Catalysis, CSIC, 28049, Madrid, Spain;Centro de Investigaciones Biológicas, CSIC, 28040, Madrid, Spain;

关键词: Directed Evolution;    Crossover Event;    Mutant Library;    Versatile Peroxidase;    Mutation G330R;   

DOI  :  10.1186/1475-2859-9-17

 received in 2009-12-03, accepted in 2010-03-18,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundIn the picture of a laboratory evolution experiment, to improve the thermostability whilst maintaining the activity requires of suitable procedures to generate diversity in combination with robust high-throughput protocols. The current work describes how to achieve this goal by engineering ligninolytic oxidoreductases (a high-redox potential laccase -HRPL- and a versatile peroxidase, -VP-) functionally expressed in Saccharomyces cerevisiae.ResultsTaking advantage of the eukaryotic machinery, complex mutant libraries were constructed by different in vivo recombination approaches and explored for improved stabilities and activities. A reliable high-throughput assay based on the analysis of T50 was employed for discovering thermostable oxidases from mutant libraries in yeast. Both VP and HRPL libraries contained variants with shifts in the T50 values. Stabilizing mutations were found at the surface of the protein establishing new interactions with the surrounding residues.ConclusionsThe existing tradeoff between activity and stability determined from many point mutations discovered by directed evolution and other protein engineering means can be circumvented combining different tools of in vitro evolution.

【 授权许可】

Unknown   
© García-Ruiz et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.