The loading domain of the erythromycin polyketide synthase is not essential for erythromycin biosynthesis in Saccharopolyspora erythraea

Abstract

6-Deoxyerythronol ide 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 AT4 and ACP-L domains. These strains were still able to produce erythromycin, although a t 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 KSI with propionate directly from propionyl-CoA.

Site-specific Mutagenesis and Domain Substitutions in the Loading Module of the Nystatin Polyketide Synthase, and Their Effects on Nystatin Biosynthesis in Streptomyces noursei

“…By constructing a nysA in-frame deletion mutant, we have demonstrated that NysA is essential for in vivo production of nystatin in S. noursei. This result suggests that the first extension module of NysB cannot be loaded directly by a starter carboxylic acid, as was apparently the case for erythromycin PKS, where inactivation of the 6-deoxyerythronolide B synthase loading module did not abolish erythromycin production completely (2,18). In ery-PKS, the direct loading of the KS domain of the first extension module was proposed as an alternative mechanism for chain initiation (3).…”

“…All the resulting constructs were verified by DNA sequencing. 2 O. Sekurova, personal communication. …”

Cloning, Sequencing, and Functional Analysis of an Iterative Type I Polyketide Synthase Gene Cluster for Biosynthesis of the Antitumor Chlorinated Polyenone Neocarzilin in “ Streptomyces carzinostaticus ”

“…LM is characterized by an AT and ACP set (AT L and ACP L , respectively); an example of the use of this LM is in erythromycin biosynthesis, in which propionyl-CoA is transferred (29,39). Another type of LM (KSQ/AT L /ACP L ) contains an extra KSQ domain, which is a mutated KS that acts as chain initiation factor (4).…”

Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase

“…Along with their semi-synthetic derivatives they now play a vital role as human and veterinary medicine therapeutic agents and agricultural agents including antibiotic, anticancer, antiparasitic, immunosuppressant and insecticide compounds (O'Hagan, 1991;Hopwood, 1997;Pereda et al, 1998;Peiru et al, 2005). Other functions include serving as defensive molecules in microbes as well as having a role in organism-to-organism signaling (Rawlings, 1997;Pfeifer and Khosla, 2001;Staunton and Weissman, 2001).…”