Cloning of genes involved in erythromycin biosynthesis from Saccharopolyspora erythraea using a novel actinomycete-Escherichia coli cosmid.
Two plasmids were constructed that replicate in Saccharopolyspora (Sac.) erythraea, Escherichia coli and Streptomyces (S.) lividans, and used for the cloning of a locus involved in the synthesis of the macrolide antibiotic erythromycin (Er). Plasmid pAL7002 contains the thiostrepton-resistance gene (tsr), a replicon-containing fragment from pJVI and pUC9. Plasmid pNJI contains the lambda cos site but is otherwise similar to pAL7002. A library of total DNA from Sac. erythraea was constructed in pNJI and probed in colony hybridizations with a DNA fragment containing ermE, the Sac. erythraea ErR-encoding gene. Plasmids obtained were subsequently introduced into EryA mutants of Sac. erythraea blocked in synthesis of Er (Ery-) and transformants were screened for restoration of Er production (Ery+). Several plasmids were found to convert two mutants to Ery+, but a third EryA strain could not be restored to Ery+ by any of the plasmids employed. A 5-kb segment, designated eryAI, responsible for restoring the Ery+ phenotype in the EryA strains, was identified and mapped in the segment 12 to 17 kb downstream from ermE. Gene disruption experiments indicated that the 5-kb length of eryAI is fully internal to an eryAI-containing transcript. In Southern blots it was shown that one of the EryA strains carried a small deletion in eryAI and that, in at least some of the transformants restored to Ery+, the deletion had been replaced by the wild-type eryAI allele.(ABSTRACT TRUNCATED AT 250 WORDS)
Tuan
J S
JS
Corporate Molecular Biology, Abbott Laboratories, North Chicago, IL 60064.
Weber
J M
JM
Staver
M J
MJ
Leung
J O
JO
Donadio
S
S
Katz
L
L
D000191:Actinomycetaceae / Q000235:genetics*; D000483:Alleles; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003360:Cosmids; D004252:DNA Mutational Analysis; D004271:DNA, Fungal / Q000235:genetics; D004917:Erythromycin / Q000096:biosynthesis*; D004926:Escherichia coli; D005800:Genes, Fungal; D005822:Genetic Vectors; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D004271:DNA, Fungal; D004917:Erythromycin