US 5,824,513 AGranted

Recombinant DNA method for producing erythromycin analogs

AssigneeAbbott Laboratories

InventorsLeonard Katz, Stefano Donadio, James B. McAlpine

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Abstract

A method to produce novel polyketide structures by designing and introducing specified changes in the DNA governing the synthesis of the polyketide is disclosed. The biosynthesis of specific polyketide analogs is accomplished by genetic manipulation of a polyketide-producing microorganism by isolating a polyketide biosynthetic gene-containing DNA sequence, identifying enzymatic activities associated within the DNA sequence, introducing one or more specified changes into the DNA sequence which codes for one of the enzymatic activities which results in an altered DNA sequence, introducing the altered DNA sequence into the polyketide-producing microorganism to replace the original sequence, growing a culture of the altered microorganism under conditions suitable for the formation of the specific polyketide analog, and isolating the specific polyketide analog from the culture. The method is most useful when the segment of the chromosome modified is involved in an enzymatic activity associated with polyketide biosynthesis, particularly for manipulating polyketide synthase genes from Saccarharopolyspora or Streptomyces.

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 .beta.-ketoreductase, dehydratase, acyl carrier protein, enoylreductase, .beta.-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 .beta.-carbonyl groups of polyketide subunits, (ii) addition of one or more domains providing enzymatic activity affecting the processing of .beta.-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.

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Facts

Application

07/642,734Filed 17 Jan 1991

Granted

20 Oct 1998

Term ends

Not estimatedThe source records don’t support an estimate.

Classification6 codes

C07H 17/08
Heterocyclic radicals containing only oxygen as ring hetero atoms: hetero rings containing eight or more ring members, e.g. erythromycins
C12N 15/52
DNA or RNA fragments; Modified forms thereof ; Non-coding nucleic acids having a biological activity: genes encoding for enzymes or proenzymes
C12P 19/62
Having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a…: the…
C07H
Sugars

Abstract

Claims28