The loading domain of the erythromycin polyketide synthase is not essential for erythromycin biosynthesis in Saccharopolyspora erythraea.
6-Deoxyerythronolide B synthase (DEBS) is a large multifunctional enzyme that catalyses the biosynthesis of the erythromycin polyketide aglycone. DEBS is organized into six modules, each containing the enzymic domains required for a single condensation of carboxylic acid residues which make up the growing polyketide chain. Module 1 is preceded by loading acyltransferase (AT-L) and acyl carrier protein (ACP-L) domains, hypothesized to initiate polyketide chain growth with a propionate-derived moiety. Using recombinant DNA technology several mutant strains of Saccharopolyspora erythraea were constructed that lack the initial AT-L domain or that lack both the AT-L and ACP-L domains. These strains were still able to produce erythromycin, although at much lower levels than that produced by the wild-type strain. In addition, the AT-L domain expressed as a monofunctional enzyme was able to complement the deletion of this domain from the PKS, resulting in increased levels of erythromycin production. These findings indicate that neither the initial AT-L nor the ACP-L domains are required to initiate erythromycin biosynthesis; however, without these domains the efficiency of erythromycin biosynthesis is decreased significantly. It is proposed that in these mutants the first step in erythromycin biosynthesis is the charging of KS1 with propionate directly from propionyl-CoA.
Pereda
Ana
A
Antibacterial Discovery Research, Abbott Laboratories, D-47P AP9A, 100 Abbott Park Rd, Abbott Park, IL 60064, USA.
Summers
Richard G
RG
Antibacterial Discovery Research, Abbott Laboratories, D-47P AP9A, 100 Abbott Park Rd, Abbott Park, IL 60064, USA.
Stassi
Diane L
DL
Antibacterial Discovery Research, Abbott Laboratories, D-47P AP9A, 100 Abbott Park Rd, Abbott Park, IL 60064, USA.
Ruan
Xiaoan
X
Antibacterial Discovery Research, Abbott Laboratories, D-47P AP9A, 100 Abbott Park Rd, Abbott Park, IL 60064, USA.
Katz
Leonard
L
Antibacterial Discovery Research, Abbott Laboratories, D-47P AP9A, 100 Abbott Park Rd, Abbott Park, IL 60064, USA.
D000213:Acyl Carrier Protein / Q000235:genetics* / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004917:Erythromycin / Q000737:chemistry / Q000378:metabolism*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D016930:Saccharopolyspora / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D017384:Sequence Deletion; D014170:Transformation, Genetic
D000213:Acyl Carrier Protein; D000214:Acyl Coenzyme A; D017931:DNA Primers; D004269:DNA, Bacterial; D036341:Intercellular Signaling Peptides and Proteins; C090508:KSI protein, Hydra vulgaris; D009097:Multienzyme Complexes; D011506:Proteins; C009061:propionyl-coenzyme A; D004917:Erythromycin; D000217:Acyltransferases