Metabolic engineering of Streptomyces coelicolor for enhanced prodigiosins (RED) production
- 112 Downloads
Bacterial prodigiosins are red-colored secondary metabolites with multiple activities, such as anticancer, antimalarial and immunosuppressive, which hold great potential for medical applications. In this study, dramatically enhanced prodigiosins (RED) production in Streptomyces coelicolor was achieved by combinatorial metabolic engineering, including inactivation of the repressor gene ohkA, deletion of the actinorhodin (ACT) and calcium-dependent antibiotic (CDA) biosynthetic gene clusters (BGCs) and multi-copy chromosomal integration of the RED BGC. The results showed that ohkA deletion led to a 1-fold increase of RED production over the wild-type strain M145. Then, the ACT and CDA BGCs were deleted successively based on the ΔohkA mutant (SBJ101). To achieve multi-copy RED BGC integration, artificial ΦC31 attB site(s) were inserted simultaneously at the position where the ACT and CDA BGCs were deleted. The resulting strains SBJ102 (with a single deletion of the ACT BGC and insertion of one artificial attB site) and SBJ103 (with the deletion of both BGCs and insertion of two artificial attB sites) produced 1.9- and 6-fold higher RED titers than M145, respectively. Finally, the entire RED BGC was introduced into mutants from SBJ101 to SBJ103, generating three mutants (from SBJ104 to SBJ106) with chromosomal integration of one to three copies of the RED BGC. The highest RED yield was from SBJ106, which produced a maximum level of 96.8 mg g−1 cell dry weight, showing a 12-fold increase relative to M145. Collectively, the metabolic engineering strategies employed in this study are very efficient for the construction of high prodigiosin-producing strains.
KeywordsStreptomyces coelicolor prodigiosins metabolic engineering multi-copy integration
Unable to display preview. Download preview PDF.
This work was supported by the National Natural Science Foundation of China (31430004, 31421061, 31630003, 31370081 and 31570072) and the Science and Technology Commission of Shanghai Municipality (16490712100).
- Guthrie, E.P., Flaxman, C.S., White, J., Hodgson, D.A., Bibb, M.J., and Chater, K.F. (1998). A response-regulator-like activator of antibiotic synthesis from Streptomyces coelicolor A3(2) with an amino-terminal domain that lacks a phosphorylation pocket. Microbiology 144 (Pt 3), 727–738.CrossRefPubMedGoogle Scholar
- Kieser, T., Bibb M., Buttner M., and Chater K. (2000). Practical Streptomyces genetics. John Innes Foundation, Norwich, England.Google Scholar
- Lu, Y., He, J., Zhu, H., Yu, Z., Wang, R., Chen, Y., Dang, F., Zhang, W., Yang, S., and Jiang, W. (2011). An orphan histidine kinase, OhkA, regulates both secondary metabolism and morphological differentiation in Streptomyces coelicolor. J Bacteriol 193, 3020–3032.CrossRefPubMedPubMedCentralGoogle Scholar
- Mo, S.J., Sydor, P.K., Corre, C., Alhamadsheh, M.M., Stanley, A.E., Haynes, S.W., Song, L., Reynolds, K.A., and Challis, G.L. (2008). Elucidation of the Streptomyces coelicolor pathway to 2-undecylpyrrole, a key intermediate in undecylprodiginine and streptorubin B biosynthesis. Chem Biol 15, 137–148.CrossRefPubMedGoogle Scholar
- Stanley, A.E., Walton, L.J., Kourdi Zerikly, M., Corre, C., and Challis, G.L. (2006). Elucidation of the Streptomyces coelicolor pathway to 4-methoxy-2,2’-bipyrrole-5-carboxaldehyde, an intermediate in prodiginine biosynthesis. Chem Commun (Camb), 3981–3983.Google Scholar
- Swiatek, M.A., Gubbens, J., Bucca, G., Song, E., Yang, Y.H., Laing, E., Kim, B.G., Smith, C.P., and van Wezel, G.P. (2013). The ROK family regulator Rok7B7 pleiotropically affects xylose utilization, carbon catabolite repression, and antibiotic production in Streptomyces coelicolor. J Bacteriol 195, 1236–1248.CrossRefPubMedPubMedCentralGoogle Scholar
- Takano, E., Gramajo, H.C., Strauch, E., Andres, N., White, J., and Bibb, M.J. (1992). Transcriptional regulation of the redD transcriptional activator gene accounts for growth-phase-dependent production of the antibiotic undecylprodigiosin in Streptomyces coelicolor A3(2). Mol Microbiol 6, 2797–2804.CrossRefPubMedGoogle Scholar
- Williamson, N.R., Simonsen, H.T., Ahmed, R.A.A., Goldet, G., Slater, H., Woodley, L., Leeper, F.J., and Salmond, G.P.C. (2005). Biosynthesis of the red antibiotic, prodigiosin, in Serratia: identification of a novel 2-methyl-3-n-amyl-pyrrole (MAP) assembly pathway, definition of the terminal condensing enzyme, and implications for undecylprodigiosin biosynthesis in Streptomyces. Mol Microbiol 56, 971–989.CrossRefPubMedGoogle Scholar
- Yamanaka, K., Reynolds, K.A., Kersten, R.D., Ryan, K.S., Gonzalez, D.J., Nizet, V., Dorrestein, P.C., and Moore, B.S. (2014). Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A. Proc Natl Acad Sci USA 111, 1957–1962.CrossRefPubMedPubMedCentralGoogle Scholar