Abstract
BackgroundThere is evidence from previous works that bacterial secondary metabolism may be stimulated by genetic manipulation of RNA polymerase (RNAP). In this study we have used rifampicin selection as a strategy to genetically improve the erythromycin producer Saccharopolyspora erythraea.ResultsSpontaneous rifampicin-resistant (rif) mutants were isolated from the parental strain NRRL2338 and two rif mutations mapping within rpoB, S444F and Q426R, were characterized. With respect to the parental strain, S444F mutants exhibited higher respiratory performance and up to four-fold higher final erythromycin yields; in contrast, Q426R mutants were slow-growing, developmental-defective and severely impaired in erythromycin production. DNA microarray analysis demonstrated that these rif mutations deeply changed the transcriptional profile of S. erythraea. The expression of genes coding for key enzymes of carbon (and energy) and nitrogen central metabolism was dramatically altered in turn affecting the flux of metabolites through erythromycin feeder pathways. In particular, the valine catabolic pathway that supplies propionyl-CoA for biosynthesis of the erythromycin precursor 6-deoxyerythronolide B was strongly up-regulated in the S444F mutants, while the expression of the biosynthetic gene cluster of erythromycin (ery) was not significantly affected. In contrast, the ery cluster was down-regulated (<2-fold) in the Q426R mutants. These strains also exhibited an impressive stimulation of the nitrogen regulon, which may contribute to lower erythromycin yields as erythromycin production was strongly inhibited by ammonium.ConclusionRifampicin selection is a simple and reliable tool to investigate novel links between primary and secondary metabolism and morphological differentiation in S. erythraea and to improve erythromycin production. At the same time genome-wide analysis of expression profiles using DNA microarrays allowed information to be gained about the mechanisms underlying the stimulatory/inhibitory effects of the rif mutations on erythromycin production.
Highlights
There is evidence from previous works that bacterial secondary metabolism may be stimulated by genetic manipulation of RNA polymerase (RNAP)
For further analysis and discussion, we focused our attention on the 198 differentially expressed genes (DEG) of the growth phase a when expression of erythromycin biosynthetic genes was maximal in the wild type strain (Figure 4A, right panel)
This study demonstrates the usefulness of the rif screening as a tool to search for higher-producer strains and provides new information about the molecular mechanisms underlying the stimulatory effect of several rif mutations on bacterial secondary metabolism
Summary
There is evidence from previous works that bacterial secondary metabolism may be stimulated by genetic manipulation of RNA polymerase (RNAP). Since the late 1970s, the availability of molecular genetics tools and useful information about the biosynthetic pathways and genetic control for most of secondary metabolites of commercial interest has opened the way for improving strains by rational engineering [2,3]. These rational strain improvement strategies benefit of the support of genomic, transcriptomic, proteomic, and metabolomic technologies [4,5,6,7,8,9,10,11]. Erythromycin biosynthesis in the mycelial actinomycete, Saccharopolyspora erythraea, has been widely studied as a model system for antibiotic production [12,13,14,15], and erythromycin and its semi-synthetic derivatives are widely used in the clinic; improved producers are still highly sought after
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