Screening of a Genomic Library in Search of the Biosynthetic Gene Cluster of Epothilones in the myxobacterium Sorangium cellulosum

Résumé

The methods used to search for epothilone gene cluster modules in Sorangium cellulosum were surveyed. In this work, lambda genomic libraries of two closely related strains were constructed. The packaging efficiencies were 261860 pfu/ug and 987179.4pfu/ug respectively for So.9733-1 and So. 9881. Using the software Primer Premier, two probes corresponding to epophilone A and epothilone B were designed and used to detect signals for ksq and pcp during the screening by hybridization of the constructed lambda genomic library.

Among 24000 plaques tested with the KS probe of epothilone A, we obtained 78 positive clones, then these positive clones were tested with the PCP probe of epoB.

Only 4 plaques gave simultaneous signals with the two probes for epothilone A and epothilone B. DNA of the four recombinant phages was extracted, followed by SalI digestion. The restriction digested map of these clones was compared with the already reported information about the epothilone gene cluster. These results were in concordance with the reported partial upstream and downstream sequences of the epothilone gene cluster from strains SMP44 and So90. The results led to the isolation and identification of a fragment which may be sequenced and analyzed.

Keywords: Sorangium, lambda, epothilone, hybridization, probes