Ethyl-Substituted Erythromycin Derivatives Produced by Directed Metabolic Engineering

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 95; no. 13; pp. 7305 - 7309
• Main Authors: Stassi, D. L., Kakavas, S. J., Reynolds, K. A., Gunawardana, G., Swanson, S., Zeidner, D., Jackson, M., Liu, H., Buko, A., Katz, L.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 23.06.1998; National Acad Sciences; National Academy of Sciences; The National Academy of Sciences
• Subjects: Acyl Coenzyme A - metabolism; Amino Acid Sequence; Anti-Bacterial Agents - chemistry; Biological Sciences; Biosynthesis; Chromosomes; DNA; Enzymes; Erythromycin - analogs & derivatives; Erythromycin - pharmacology; Genes; Genetics; Macrolides; Microbial Sensitivity Tests; Models, Chemical; Molecular Sequence Data; Molecules; Nucleotides; Plasmids; Polyketides; Protein Engineering; Restriction Mapping; Saccharopolyspora - genetics; Saccharopolyspora - metabolism; Saccharopolyspora erythraea; Structure-Activity Relationship; Yeast
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.95.13.7305

A previously unknown chemical structure, 6-desmethyl-6-ethylerythromycin A (6-ethylErA), was produced through directed genetic manipulation of the erythromycin (Er)-producing organism Saccharopolyspora erythraea. In an attempt to replace the methyl side chain at the C-6 position of the Er polyketide backbone with an ethyl moiety, the methylmalonate-specific acyltransferase (AT) domain of the Er polyketide synthase was replaced with an ethylmalonate-specific AT domain from the polyketide synthase involved in the synthesis of the 16-member macrolide niddamycin. The genetically altered strain was found to produce ErA, however, and not the ethyl-substituted derivative. When the strain was provided with precursors of ethylmalonate, a small quantity of a macrolide with the mass of 6-ethylErA was produced in addition to ErA. Because substrate for the heterologous AT seemed to be limiting, crotonyl-CoA reductase, a primary metabolic enzyme involved in butyryl-CoA production in streptomycetes, was expressed in the strain. The primary macrolide produced by the reengineered strain was 6-ethylErA.