title: A new substrate specificity for acyl transferase domains of the ascomycin polyketide synthase in Streptomyces hygroscopicus.
abstract: Ascomycin (FK520) is a structurally complex macrolide with immunosuppressant activity produced by Streptomyces hygroscopicus. The biosynthetic origin of C12-C15 and the two methoxy groups at C13 and C15 has been unclear. It was previously shown that acetate is not incorporated into C12-C15 of the macrolactone ring. Here, the acyl transferase (AT) of domain 8 in the ascomycin polyketide synthase was replaced with heterologous ATs by double homologous recombination. When AT8 was replaced with methylmalonyl-CoA-specific AT domains, the strains produced 13-methyl-13-desmethoxyascomycin, whereas when AT8 was replaced with a malonyl-specific domain, the strains produced 13-desmethoxyascomycin. These data show that ascomycin AT8 does not use malonyl- or methylmalonyl-CoA as a substrate in its native context. Therefore, AT8 must be specific for a substrate bearing oxygen on the alpha carbon. Feeding experiments showed that [(13)C]glycerol is incorporated into C12-C15 of ascomycin, indicating that both modules 7 and 8 of the polyketide synthase use an extender unit that can be derived from glycerol. When AT6 of the 6-deoxyerythronolide B synthase gene was replaced with ascomycin AT8 and the engineered gene was expressed in Streptomyces lividans, the strain produced 6-deoxyerythronolide B and 2-demethyl-6-deoxyerythronolide B. Therefore, although neither malonyl-CoA nor methylmalonyl-CoA is a substrate for ascomycin AT8 in its native context, both are substrates in the foreign context of the 6-deoxyerythronolide B synthase. Thus, we have demonstrated a new specificity for an AT domain in the ascomycin polyketide synthase and present evidence that specificity can be affected by context.
authors.affiliation: Kosan Biosciences, Inc., Hayward, California 94545, USA. reeves@kosan.com
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Reeves
authors.forename: Loleta M
authors.initials: LM
authors.lastname: Chung
authors.forename: Yaoquan
authors.initials: Y
authors.lastname: Liu
authors.forename: Qun
authors.initials: Q
authors.lastname: Xue
authors.forename: John R
authors.initials: JR
authors.lastname: Carney
authors.forename: W Peter
authors.initials: WP
authors.lastname: Revill
authors.forename: Leonard
authors.initials: L
authors.lastname: Katz
mesh_terms: D000217:Acyltransferases / Q000737:chemistry*; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*
chemical_list: D009097:Multienzyme Complexes; C058028:immunomycin; D000217:Acyltransferases; D016559:Tacrolimus