Toward Improvement of Erythromycin A Production in an IndustrialSaccharopolyspora erythraea Strain via Facilitation of Genetic Manipulation with an Artificial attB Site for Specific Recombination

Large-scale production of erythromycin A (Er-A) relies on the organismSaccharopolyspora erythraea , in which lack of a typicalattB site largely impedes the application of phage ΦC31 integrase-mediated recombination into site-specific engineering. We herein report construction of an artificialattB site in an industrialS. erythraea strain, HL3168 E3, in an effort to break the bottleneck previously encountered during genetic manipulation mainly from homologous or unpredictable nonspecific integration. Replacement of a cryptic gene,nrps1-1 , with a cassette containing eightattB DNA sequences did not affect the high Er-producing ability, setting the stage for precisely engineering the industrial Er-producing strain for foreign DNA introduction with a reliable conjugation frequency. Transfer of either exogenous or endogenous genes of importance to Er-A biosynthesis, including theS -adenosylmethionine synthetase gene for positive regulation,vhb for increasing the oxygen supply, and two tailoring genes,eryK anderyG , for optimizing the biotransformation at the late stage, was achieved by taking advantage of this facility, allowing systematic improvement of Er-A production as well as elimination of the by-products Er-B and Er-C in fermentation. The strategy developed here can generally be applicable to other strains that lack theattB site.