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Erythromycin A (Er-A) is the most potent and clinically important member in the Er family produced by Saccharopolyspora erythraea. Er-B and Er-C, which are biologically much less active and cause greater side effects than Er-A, serve as the intermediates for Er-A biosynthesis and impurities in fermentation processes of many industrial strains. In this study, systematical modulation of the amounts of tailoring enzymes EryK (a P450 hydroxylase) and EryG (an S-adenosylmethionine-dependent O-methyltransferase) was carried out by genetic engineering in S. erythraea, including alterations of gene copy number ratio and organization and integrating the locus on the chromosome by homologous recombination. Introduction of additional eryK and eryG genes into S. erythraea showed significant impacts on their transcription levels and enhanced the biotransformation process from Er-D to Er-A with gene dose effects. At the eryK/eryG copy number ratio of 3:2 as well as their resultant transcript ratio of around 2.5:1 to 3.0:1, Er-B and Er-C were nearly completely eliminated and accordingly converted to Er-A, and the Er titer was improved by around 25% in the recombinant strain ZL1004 (genotype PermK*-K-K-G + PermE*-K + PermA*-G) and ZL1007 (genotype PermK*-K-G-K + PermE*-K + PermA*-G). This study may contribute to the continuous efforts toward further evaluation of the Er-producing system, with the aims of improving Er-A purity and production at the fermentation stage and lowering the production costs and environmental concerns in industry.

applied and environmental microbiology

Genetic Modulation of the Overexpression of Tailoring Genes eryK and eryG Leading to the Improvement of Erythromycin A Purity and Production in Saccharopolyspora erythraea Fermentation