Erythromycin, a medically important antibiotic, is produced by Saccharopolyspora erythraea. Unusually, the erythromycin biosynthetic gene cluster lacks a regulatory gene, and the regulation of its biosynthesis remains largely unknown. In this study, through gene deletion, complementation and overexpression experiments, we identified a novel TetR family transcriptional regulator SACE_3986 negatively regulating erythromycin biosynthesis in S. erythraea A226. When SACE_3986 was further inactivated in an industrial strain WB, erythromycin A yield of the mutant was increased by 54.2 % in average compared with that of its parent strain, displaying the universality of SACE_3986 as a repressor for erythromycin production in S. erythraea. qRT-PCR analysis indicated that SACE_3986 repressed the transcription of its adjacent gene SACE_3985 (which encodes a short-chain dehydrogenase/reductase), erythromycin biosynthetic gene eryAI and the resistance gene ermE. As determined by EMSA analysis, purified SACE_3986 protein specifically bound to the intergenic region between SACE_3985 and SACE_3986, whereas it did not bind to the promoter regions of eryAI and ermE. Furthermore, overexpression of SACE_3985 in A226 led to enhanced erythromycin A yield by at least 32.6 %. These findings indicate that SACE_3986 is a negative regulator of erythromycin biosynthesis, and the adjacent gene SACE_3985 is one of its target genes. The present study provides a basis to increase erythromycin production by engineering of SACE_3986 and SACE_3985 in S. erythraea.
Glycosyltransferase DesVII and its auxiliary partner DesVIII from Streptomyces venezulae, homologs of EryCIII and EryCII in Saccharopolyspora erythraea, have previously been demonstrated to be flexible on their substrates in vitro. Herein, we investigated their in vivo function by interspecies complementation in the mutant strains of Sac. erythraea A226. As desVII and desVIII were concomitantly expressed in the ΔeryCIII mutant, the erythromycin A (Er-A) production was restored. Interestingly, co-expression of desVII and desVIII in the ΔeryBV mutant exhibited an increased Er-A yield by 15 % in comparison to A226. Hence, DesVII/DesVIII not only replaced EryCIII to upload D-desosamine to C5 position of 3-O-mycarosyl erythronolide B (MEB) but also in vivo attached L-mycarose, not D-desosamine to C3 position of erythronolide B (EB) with a higher activity than EryBV. Furthermore, expression of desVII in ΔeryCIII and ΔeryBV-CIII partially restored the Er-A production; however, no Er-A was detected while desVII was expressed in ΔeryBV. It was implicated that DesVII coupled with EryCII to form the DesVII/EryCII complex for attaching above two deoxysugars in the absence of EryCIII in Sac. erythraea. In addition, when desVII and desVIII were co-expressed in ΔeryBV-CII, Er-A was recovered with a lower yield than ΔeryBV-CIII. Our study presents an opportunity with Sac. erythraea as a cell factory for macrolide glycodiversification.
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