SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea

Abstract

Background: Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement.

Results: We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis.

Conclusions: SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence on erythromycin yield. Like bldD, SACE_5599 is involved in morphological development of S. erythraea, suggesting a very close relationship between secondary metabolite biosynthesis and morphological differentiation in this organism. While the mode of action of SACE_5599 remains to be elucidated, the manipulation of this gene clearly shows potential for improvement of erythromycin production in S. erythraea in industrial setting. We have also demonstrated the applicability of the comparative proteomics approach for identifying new regulatory elements involved in biosynthesis of secondary metabolites in industrial conditions.

Figure 1

Relative expression of SACE_5599 and erythromycin biosynthesis of the WT and industrial high-producing ABE1441 strains. Data from one representative fermentation process are shown, normalized to expression of 16S rRNA. Grey lines represent relative expression of SACE_5599 () and erythromycin concentration () in the ABE1441 strain. Black lines represent relative expression of SACE_5599 () and erythromycin concentration () in the WT strain.

Figure 2

Phenotypes of S. erythraea mutants with inactivated or in trans - overexpressed SACE_5599 on ABSM4 agar plates containing apramycin: A) NRRL23338/pSET152; B) ABE1441/pSET152; C) ABE1441/ΔSACE_5599 complemented with SACE_5599 (pABE110; pABE112); D) ABE1441/ΔSACE_5599 (pABE110); E) ABE1441/pSET152 + SACE_5599 (pABE104) F) NRRL23338/pSET152 + SACE_5599 (pABE104). Construction of plasmid constructs is presented in Table 1. Equivalent result was observed when plates without apramycin were used (not shown).

Figure 3

Genomic context and amino acid sequence of SACE_5599. A) Chromosomal locus of SACE_5599. Genes located close to SACE_5599 and their putative functions are indicated. SACE numbers are indicated above the arrows. SACE_5596 encodes a hypothetical protein with no similarity to other proteins in sequence databases. The position of SACE_5599 in S. erythraea genome is schematically represented. The »core« region is marked with bold grey line and the »non-core« region with thin black line. B) Sequence of the 219 amino acid ORF of SACE_5599 with peptides detected in proteomic analysis underlined, conserved tryptophan residues marked in bold and the position of alternative translation start (184 aa variant) is shaded in grey.

Figure 4

Yield of erythromycin produced by different mutant strains of S. erythraea. Bars encompass 95% of the sample population. Horizontal lines represent the median values and perpendicular lines indicate extreme values (min, max). Asterisks denote statistically significant differences between experimental group samples compared to control samples. The data were analyzed using SAS/STAT program as described in Methods. Panel A): Erythromycin production by NRRL23338 transformants, determined by the microbiological assay; WT: control 1 NRRL23338; A: control 2 NRRL23338 + pSET152; B: NRRL23338 + SACE_5599-219 aa (pABE104); C: NRRL23338 + SACE_5599-219 aa-HA (pABE106); D: NRRL23338 + BldD (pABE21). Panel B): Erythromycin production by industrial high-producing strain ABE1441 transformants, determined by the HPLC-UV method; ABE1441: control 1 ABE1441; E: control 2 ABE1441 + pSET152; F: ABE1441 ΔSACE_5599 (pABE110); G: ABE1441 ΔSACE_5599 + SACE_5599-219 aa-HA (pABE112) sporulating strains; H: ABE1441 + SACE_5599-219 aa (pABE104); I: ABE1441 + BldD (pABE21).

Figure 5

Evaluation of in-trans expression of SACE_5599 in S.erythraea. A) qPCR analysis of expression levels of SACE_5599 (normalized to expression of 16S rRNA) in WT and strains with additional copy of SACE_5599 (pABE53, pABE104), compared to the ABE1441 high-producing strain. B) Western blot analysis of in trans expression of 219 aa (lanes 2–5) and 184 aa (lanes 7–10) SACE_5599-HA and the control NRRL23338 strain with pSET152 (lane 1). Molecular mass markers were loaded in lane 6 and their positions are schematically presented on the right side of the blot. Bands of apparent molecular mass of 33 kDa are observed in 4 independent 219 aa transformants of the NRRL23338 strain, two containing the RBS sequence (pABE107) in the expression vector (lanes 2–3) and two without the RBS sequence (pABE106) (lanes 4–5). A very weak band of apparent molecular mass of 25 kDa was observed when shorter variant (184 aa) of SACE_5599 was expressed, either without RBS (plasmid pABE102 – lanes 7 and 8) or with RBS (plasmid pABE103 – lanes 9 and 10).