Recombinant DNA method for producing erythromycin analogs

Claim 1

A method for directing the biosynthesis of a specific erythromycin analog by genetic manipulation of a gene encoding the deoxyerythronolide B synthase function of an erythromycin-producing microorganism, said method comprising the steps of: (a) isolating a genomic DNA segment comprising the eryA gene of Saccharopolyspora erythraea; (b) identifying discrete fragments of said genomic DNA each encoding a separate polypeptide domain, wherein each domain provides no more than one of the enzymatic activities associated with said deoxyerythronolide B synthase function, said enzymatic activities consisting of β-ketoreductase, dehydratase, acyl carrier protein, enoylreductase, β-ketoacyl ACP synthase and acyltransferase; (c) altering at least one region of said eryA gene by modifying said DNA fragments to produce at least one alteration of said deoxyerythronolide B synthase function, said alteration selected from the group consisting of (i) inactivation of one or more domains providing enzymatic activity affecting the processing of β-carbonyl groups of polyketide subunits, (ii) addition of one or more domains providing enzymatic activity affecting the processing of β-carbonyl groups of polyketide subunits, (iii) inactivation of one or more domains providing enzymatic activity affecting the condensation of carbon units to a nascent deoxyerythronolide B structure, (iv) addition of domains providing enzymatic activities affecting the length of said deoxyerythronolide B structure, (v) deletion of one or more domains providing one or more enzymatic activities affecting the length of said deoxyerythronolide B structure, and (vi) substitution of one acyltransferase domain with another isologous acyltransferase domain of different specificity; (d) transforming a cell of said erythromycin-producing microorganism with said altered region of said eryA gene under conditions suitable for the occurrence of a homologous recombination event replacing a corresponding region of said cell's eryA gene with said altered region; (e) growing a culture of said transformed cell under conditions suitable for the formation of said specific erythromycin analog; and (f) isolating said specific erythromycin analog from said culture.

Claim 2

A method for directing the biosynthesis of a specific erythromycin analog by genetic manipulation of a gene encoding the deoxyerythronolide B synthase function of an erythromycin-producing microorganism, said method comprising the steps of: (a) isolating a genomic DNA segment comprising the eryA gene of Saccharopolyspora erythraea; (b) identifying discrete fragments of said genomic DNA each encoding a separate polypeptide domain, wherein each domain provides no more than one of the enzymatic activities associated with said deoxyerythronolide B synthase function, said enzymatic activities consisting of β-ketoreductase, dehydratase, acyl carrier protein, enoylreductase, β-ketoacyl ACP synthase and acyltransferase; (c) altering at least one region of said eryA gene by modifying said DNA fragments to produce at least one alteration of said deoxyerythronolide B synthase function, said alteration selected from the group consisting of (i) inactivation of one or more domains providing enzymatic activity affecting the processing of β-carbonyl groups of polyketide subunits, (ii) addition of one or more domains providing enzymatic activity affecting the processing of β-carbonyl groups of polyketide subunits, (iii) inactivation of one or more domains providing enzymatic activity affecting the condensation of carbon units to a nascent deoxyerythronolide B structure, (iv) addition of domains providing enzymatic activities affecting the length of said deoxyerythronolide B structure, (v) deletion of one or more domains providing one or more enzymatic activities affecting the length of said deoxyerythronolide B structure, and (vi) substitution of one acyltransferase domain with another isologous acyltransferase domain of different specificity; (d) transforming a cell of said erythromycin-producing microorganism with said altered region of said eryA gene under conditions suitable for the occurrence of a homologous recombination event replacing a corresponding region of said cell's eryA gene with said altered region; (e) growing a culture of said transformed cell under conditions suitable for the formation of said specific erythromycin analog; and (f) isolating said specific erythromycin analog from said culture.