Construction and functional screening of a metagenomic library using a T7 RNA polymerase-based expression cosmid vector.
The metagenomic approach has greatly accelerated the discovery of new enzymes by giving access to the genetic potential of microorganisms from various environments. Function-based screening depends on adequate expression of the foreign genes in the heterologous host, which can be challenging in large-insert libraries. In this study, the shuttle cosmid vector pFX583 was used for the construction and screening of a metagenomic library. This vector allows T7 RNA polymerase-directed transcription of the cloned DNA and can be used in Escherichia coli and Streptomyces lividans. The DNA used for the library construction was obtained from an enriched biomass. The library was screened for lipolytic and proteolytic activities using E. coli and S. lividans as hosts. Numerous E. coli clones with lipolytic activity were detected. Unfortunately, proteases could not be detected in both hosts. From the lipolytic activity screen, a gene coding for a new lipase was isolated, and partial characterization was conducted.
INRS-Institut Armand-Frappier, 531 boul. des Prairies, Laval, QC, H7V 1B7, Canada. francois-xavier.lussier@iaf.inrs.ca
François-Xavier
FX
Lussier
Olivier
O
Chambenoit
Amélie
A
Côté
Jean-François
JF
Hupé
François
F
Denis
Pierre
P
Juteau
Réjean
R
Beaudet
François
F
Shareck
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003360:Cosmids; D012321:DNA-Directed RNA Polymerases; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015698:Genomic Library; D057166:High-Throughput Screening Assays; D008049:Lipase / Q000737:chemistry / Q000235:genetics; D056186:Metagenomics; D008969:Molecular Sequence Data; D048170:Streptomyces lividans / Q000201:enzymology / Q000235:genetics; D014764:Viral Proteins
D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D008049:Lipase