Acyltransferase domain substitutions in erythromycin polyketide synthase yield novel erythromycin derivatives

Abstract

The methylmalonyl coenzyme A (methylmalonyl-CoA)-specific acyltransferase (AT) domains of modules 1 and 2 of the 6-deoxyerythronolide B synthase (DEBS1) of Saccharopolyspora erythraea ER720 were replaced with three heterologous AT domains that are believed, based on sequence comparisons, to be specific for malonyl-CoA. The three substituted AT domains were "Hyg" AT2 from module 2 of a type I polyketide synthase (PKS)-like gene cluster isolated from the rapamycin producer Streptomyces hygroscopicus ATCC 29253, "Ven" AT isolated from a PKS-like gene cluster of the pikromycin producer Streptomyces venezuelae ATCC 15439, and RAPS AT14 from module 14 of the rapamycin PKS gene cluster of S. hygroscopicus ATCC 29253. These changes led to the production of novel erythromycin derivatives by the engineered strains of S. erythraea ER720. Specifically, 12-desmethyl-12-deoxyerythromycin A, which lacks the methyl group at C-12 of the macrolactone ring, was produced by the strains in which the resident AT1 domain was replaced, and 10-desmethylerythromycin A and 10-desmethyl-12-deoxyerythromycin A, both of which lack the methyl group at C-10 of the macrolactone ring, were produced by the recombinant strains in which the resident AT2 domain was replaced. All of the novel erythromycin derivatives exhibited antibiotic activity against Staphylococcus aureus. The production of the erythromycin derivatives through AT replacements confirms the computer predicted substrate specificities of "Hyg" AT2 and "Ven" AT and the substrate specificity of RAPS AT14 deduced from the structure of rapamycin. Moreover, these experiments demonstrate that at least some AT domains of the complete 6-deoxyerythronolide B synthase of S. erythraea can be replaced by functionally related domains from different organisms to make novel, bioactive compounds.

Gene Replacement for the Generation of Designed Novel Avermectin Derivatives with Enhanced Acaricidal and Nematicidal Activities

“…PKS reassembly can be manipulated on the levels of domain, module, and PKS gene. To date, there are a number of successful precedents for structural modification through manipulation of domains (25)(26)(27)(28) and modules (29,30), but structural modifications through PKS gene replacement are little reported. In fact, domain/module swaps are often only marginally successful or entirely unsuccessful, leading to only very small amounts of the desired compound or no product at all (26-28, 31, 32).…”

Identification and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis

“…Domain replacements within the erythromycin PKS have been shown to result in the production of novel bioactive compounds (11,22,25). The ethylmalonyl AT domain (AT5) and the methoxy AT domain (AT6) of the niddamycin cluster could conceivably be used to replace the mmAT domains in the erythromycin PKS to generate erythromycin derivatives with novel polyketide backbone structures that would be difficult to produce by chemical methods.…”

Biosynthesis of Polyketides in Heterologous Hosts

“…For example, a single enzyme, malonyl-CoA synthetase, can be used to synthesize numerous ␣-carboxylated CoA thioesters from their corresponding exogenously supplied 1,3-dicarboxylic acids (N. Pohl and C. Khosla, unpublished data). Fourth, the modularity of many PKSs makes it feasible to tailor their substrate preferences to the available intracellular pool of CoA thioesters in a given host (48,65,73). A similar approach can also be used to constrain the substrate range of NRPSs, if desired.…”