Mutagenesis of the dehydratase active site in the erythromycin-producing polyketide synthase.

Eqthromycin A is a clinically-useful macrolide antibiotic produced by the Gram-positive bacterium Succhuropolyspora erythrueu [1,2]. The biosynthesis of erythromycin begins with the construction of a polyketide chain from one molecule of propionyl-CoA and six molecules of methylmalonyl-CoA, by a mechanism that resembles the biosynthesis of saturated fatty acids [ 1,3]. The polyketide chain is then cyclised to form the 14membered lactone 6deoxyerythronoli B , the first isolable intermediate in the pathway to erythromycin A [1,2]. The genes encoding the 6deoxyerythronolide B synthase (DEBS) are located in the eryA region of the ery biosynthetic gene cluster of S. erythueu, which extends at least 30 kbp along the chromosome [4]. Nucleic acid sequence analysis [5,6,7] has revealed the presence of 3 ORFs, encoding 3 large multifunctional polypeptides containing regions significantly homologous to previously identified active sites involved in fatty acid and polyketide biosynthesis. It is proposed that during macrolactone biosynthesis a different set of activities is requid to cany out each of the six cycles of chain extension and (where appropriate) reduction, with each polypeptide housing the activities necessary for 2 cycles of polyketide chain extension. Alignments of the amino acid sequences of the N-terminal and C-terminal halves of DEBS 2 and DEBS 3 with each other, with similar sequences from 6-methylsalicylate synthase from Ppatulwn, rat fatty acid synthase and with components of E.coli fatty acid synthase [7] allowed assignment of probable domain boundaries within the DEBS polyketides. The proposed boundaries correspond to points at which there is a sharp loss of homology between the compared sequences. Further alignments of the putative dehydratase domain of DEBS 2, portions of other polyketide synthases and fatty acid synthases and the active site sequence of E.coli 3hydroxydecanoyl dehydratase (Fig. 1) led to identification of a conserved histidine residue (DEBS 2 His2409) corresponding to His70 in the E.coli enzyme. The E.coli His70 residue has been shown by inhibition with 3decynoyl-N-acetylcysteamine [8,9] to be located in the dehydratase active site, where it probably acts as a general proton donor/acceptor in the reaction mechanism. It is therefore likely that DEBS 2 His2409 plays a similar role in the dehydration step in cycle 4 of the polyketide chain extension. In order to confirm this, and with the hope of producing a novel product, we sought to eliminate the dehydratase activity by replacing His2409 with a phenylalanine.Previously Donadio et ul. [6] deleted the entire keto-reductase domain required in cycle 5 of DEBS 3 and an appropriately altered macrolide was obtained. The mutated His2409 codon (lTC replacing CAC) was produced in vitro by PCR mutagenesis. The altered DNA fragment was cloned in pGM420 [ 101 and introduced into the wild type S.erythuea -2338 by gene replacement in vivo as described by Weber et ul.[ 101. The 2 base changes made also created a new EcoRI site, enabling detection of the mutation by Southem analysis of chromosomal DNA. A single mutant, 17.43, canying the engineered EcoRI site and the mutated codon was thus identified Western blot analysis showed that 17.43 produces DEBS 1,2 and 3 polypeptides of the expected size. Mutant 17.43 was grown in liquid medium and ethyl acetate extracts of the broth were analysed by bioassay, thin layer chromatography and electrospray mass spectrometry. No erythromycin or known erythromycin precursors were identified. Feeding with radiolabelled propionic acid, followed by analysis of ethyl acetate extracts of the culture medium by TLC, led to the detection of small amounts of several radiolabelled products. Similar minor metabolites could be detected in extracts of the wild type organism but were not produced when the disruption mutant 17.0, from which 17.43 was derived, was grown under the same conditions, indicating that they are products of the altered polyketide synthase. *