Actinomycetes produce a variety of clinically indispensable molecules, such as antineoplastic anthracyclines. However, the actinomycetes are hindered in their further development as genetically engineered hosts for the synthesis of new anthracycline analogues due to their slow growth kinetics associated with their mycelial life cycle and the lack of a comprehensive genetic toolbox for combinatorial biosynthesis. In this report, we tackled both issues via the development of the BIOPOLYMER (BIOBricks POLYketide Metabolic EngineeRing) toolbox: a comprehensive synthetic biology toolbox consisting of engineered strains, promoters, vectors, and biosynthetic genes for the synthesis of anthracyclinones. An improved derivative of the production host Streptomyces coelicolor M1152 was created by deleting the matAB gene cluster that specifies extracellular poly-β-1,6-N-acetylglucosamine (PNAG). This resulted in a loss of mycelial aggregation, with improved biomass accumulation and anthracyclinone production. We then leveraged BIOPOLYMER to engineer four distinct anthracyclinone pathways, identifying optimal combinations of promoters, genes, and vectors to produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone at titers between 15-20 mg/L. Optimization of nogalamycinone production strains resulted in titers of 103 mg/L. We structurally characterized six anthracyclinone products from fermentations, including new compounds 9,10-seco-7-deoxy-nogalamycinone and 4-O-β-d-glucosyl-nogalamycinone. Lastly, we tested the antiproliferative activity of the anthracyclinones in a mammalian cancer cell viability assay, in which nogalamycinone, auramycinone, and aklavinone exhibited moderate cytotoxicity against several cancer cell lines. We envision that BIOPOLYMER will serve as a foundational platform technology for the synthesis of designer anthracycline analogues.
|Number of pages||17|
|Journal||ACS Synthetic Biology|
|State||Published - Dec 16 2022|
Bibliographical noteFunding Information:
Research reported in this publication was supported by the National Science Foundation under Grant No. ENG-2015951 (S.E.N.), by the National Cancer Institute of the National Institutes of Health under Award No. R15CA252830 (S.E.N.), the NIGMS-supported Center of Biomedical Research Excellence (COBRE) in Pharmaceutical Research and Innovation (CPRI, NIH P20 GM130456), the University of Kentucky College of Pharmacy, the National Center for Advancing Translational Sciences (UL1TR000117 and UL1TR001998) and a National Institutes of Health shared instrumentation Grant (S10OD28690), Novo Nordisk Foundation Grant No. NNF19OC0057511 (to M.M.-K) and Academy of Finland Grant No. 340013 (to M.M.-K.). We also acknowledge a grant from the Turku University Foundation and support from the China Scholarship Council 202107960010 (R.W.). G.v.W. was funded by ERC Advanced grant 101055020 from the European Research Council. We thank the College of Pharmacy NMR Center (University of Kentucky) for NMR support. We thank Dr. Lou Charkoudian for the gift of strain S. lividans K4-114. We thank Prof. Dr. Mervyn Bibb for the gift of strains S. coelicolor M1146 and M1152. We thank Dr. Isaac Brownell (Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA) for the gift of Merkel cells MKL1 and MCC26.
© 2022 American Chemical Society.
- Streptomyces coelicolor
- natural product biosynthesis
- synthetic biology
ASJC Scopus subject areas
- Biomedical Engineering
- Biochemistry, Genetics and Molecular Biology (miscellaneous)