Prospecting metagenomic enzyme subfamily genes for DNA family shuffling by a novel PCR-based approach.
DNA family shuffling is a powerful method for enzyme engineering, which utilizes recombination of naturally occurring functional diversity to accelerate laboratory-directed evolution. However, the use of this technique has been hindered by the scarcity of family genes with the required level of sequence identity in the genome database. We describe here a strategy for collecting metagenomic homologous genes for DNA shuffling from environmental samples by truncated metagenomic gene-specific PCR (TMGS-PCR). Using identified metagenomic gene-specific primers, twenty-three 921-bp truncated lipase gene fragments, which shared 64-99% identity with each other and formed a distinct subfamily of lipases, were retrieved from 60 metagenomic samples. These lipase genes were shuffled, and selected active clones were characterized. The chimeric clones show extensive functional and genetic diversity, as demonstrated by functional characterization and sequence analysis. Our results indicate that homologous sequences of genes captured by TMGS-PCR can be used as suitable genetic material for DNA family shuffling with broad applications in enzyme engineering.
Wang
Qiuyan
Q
Center for Biomedicine and Health, Hangzhou Normal University, Hangzhou 310012, China.
Wu
Huili
H
Wang
Anming
A
Du
Pengfei
P
Pei
Xiaolin
X
Li
Haifeng
H
Yin
Xiaopu
X
Huang
Lifeng
L
Xiong
Xiaolong
X
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods; D023281:Genomics; D008049:Lipase / Q000235:genetics; D056186:Metagenomics / Q000379:methods*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004247:DNA; D008049:Lipase