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PEDS Advance Access originally published online on February 20, 2008
Protein Engineering Design and Selection 2008 21(4):267-274; doi:10.1093/protein/gzn004
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

A novel random mutagenesis approach using human mutagenic DNA polymerases to generate enzyme variant libraries

Stéphane Emond1,2,3, Philippe Mondon4, Sandra Pizzut-Serin1,2,3, Laurent Douchy4, Fabien Crozet4, Khalil Bouayadi4, Hakim Kharrat4, Gabrielle Potocki-Véronèse1,2,3, Pierre Monsan1,2,3 and Magali Remaud-Simeon1,2,3,5

1 Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France 2INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France 3CNRS, UMR5504, F-31400 Toulouse, France 4MilleGen SA, Immeuble BIOSTEP, Bâtiment A, rue Pierre et Marie Curie, BP 38183, 31681 Labège cedex, France

5 To whom correspondence should be addressed. E-mail: magali.remaud{at}insa-toulouse.fr

The in vitro MutaGenTM procedure is a new random mutagenesis method based on the use of low-fidelity DNA polymerases. In the present study, this technique was applied on a 2 kb gene encoding amylosucrase, an attractive enzyme for the industrial synthesis of amylose-like polymers. Mutations were first introduced during a single replicating step performed by mutagenic polymerases pol β and pol {eta}. Three large libraries (>105 independent clones) were generated (one with pol β and two with pol {eta}). The sequence analysis of randomly chosen clones confirmed the potential of this strategy for the generation of diversity. Variants generated by pol β were 4–7-fold less mutated than those created with pol {eta}, indicating that our approach enables mutation rate control following the DNA polymerase employed for mutagenesis. Moreover, pol β and pol {eta} provide different and complementary mutation spectra, allowing a wider sequence space exploration than error-prone PCR protocols employing Taq polymerase. Interestingly, some of the variants generated by pol {eta} displayed unusual modifications, including combinations of base substitutions and codon deletions which are rarely generated using other methods. By taking advantage of the mutation bias of naturally highly error-prone DNA polymerases, MutaGenTM thus appears as a very useful tool for gene and protein randomisation.

Keywords: amylosucrase/directed evolution/low-fidelity DNA polymerase/protein randomisation/random mutagenesis

Received October 1, 2007; revised January 13, 2008; accepted January 14, 2008.


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