A novel random mutagenesis approach using human mutagenic DNA polymerases to generate enzyme variant libraries.
The in vitro MutaGen 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 beta and pol eta. Three large libraries (>10(5) independent clones) were generated (one with pol beta 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 beta 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 beta 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, MutaGen thus appears as a very useful tool for gene and protein randomisation.
Emond
Stéphane
S
Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France.
Mondon
Philippe
P
Pizzut-Serin
Sandra
S
Douchy
Laurent
L
Crozet
Fabien
F
Bouayadi
Khalil
K
Kharrat
Hakim
H
Potocki-Véronèse
Gabrielle
G
Monsan
Pierre
P
Remaud-Simeon
Magali
M
D000595:Amino Acid Sequence; D019951:DNA Polymerase beta / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D015723:Gene Library; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D054643:INDEL Mutation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009343:Neisseria / Q000201:enzymology*; D011108:Polymers / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism; D000099270:Y-Family DNA Polymerases
D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase; D005964:Glucosyltransferases; D011108:Polymers; D013395:Sucrose; C019700:amylosucrase; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases