Metabolic Engineering Strategies Based on Secondary Messengers (p)ppGpp and C‑di-GMP To Increase Erythromycin Yield in <i>Saccharopolyspora erythraea</i>

Secondary messengers (such as (p)­ppGpp and c-di-GMP) were proved to play important roles in antibiotic biosynthesis in actinobacteria. In this study, we found that transcription levels of erythromycin-biosynthetic (ery) genes were upregulated in nutrient limitation, which depended on (p)­ppGpp in Saccharopolyspora erythraea. Further study demonstrated that the expression of ery genes and intracellular concentrations of (p)­ppGpp showed synchronization during culture process. The erythromycin yield was significantly improved (about 200%) by increasing intracellular concentration of (p)­ppGpp through introduction of C-terminally truncated (p)­ppGpp synthetase RelA (1.43 kb of the N-terminal segment) from Streptomyces coelicolor into S. erythraea strain NRRL2338 (named as WT/pIB-PBAD-relA1–489). As the intracellular concentration of (p)­ppGpp in an industrial erythromycin-high-producing strain E3 was greatly higher (about 10- to 100-fold) than WT strain, the applications of the above-described strategy did not work in E3 strain. Further research revealed that low concentration of 2-oxoglutarate in E3 strain exerted a “nitrogen-rich” pseudosignal, leading to the downregulation of nitrogen metabolism genes, which limited the use of nitrogen sources and thus the high intracellular (p)­ppGpp concentration. Furthermore, the secondary messenger, c-di-GMP, was proved to be able to activate ery genes transcription by enhancing binding of BldD to promoters of ery genes. Overexpressing the diguanylate cyclase CdgB from S. coelicolor in S. erythraea increased the intracellular c-di-GMP concentration, and improved erythromycin production. These findings demonstrated that increasing the concentration of intracellular secondary messengers can activate ery genes transcription, and provided new strategies for designing metabolic engineering based on secondary messengers to improve antibiotics yield in actinobacteria