A BioBricks Metabolic Engineering Platform for the Biosynthesis of Anthracyclinones in Streptomyces coelicolor

ACS Synthetic Biology, vol. 11, pp. 4193–4209

Abstract

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.

Authors 13

1. Rongbin Wang

   University of Turku

   Affiliation as printed

   Department of Life Technologies, University of Turku, FIN-20014 Turku, Finland

2. Jennifer T. Nguyen

   Ferris State University

   Affiliation as printed

   Department of Pharmaceutical Sciences, College of Pharmacy, Ferris State University, Big Rapids, Michigan 49307, United States

3. Jacob Hecht

   Ferris State University

   Affiliation as printed

   Department of Pharmaceutical Sciences, College of Pharmacy, Ferris State University, Big Rapids, Michigan 49307, United States

4. Nora Schwartz

   Ferris State University

   Affiliation as printed

   Department of Pharmaceutical Sciences, College of Pharmacy, Ferris State University, Big Rapids, Michigan 49307, United States

5. Katelyn Victoria Brown

   Ferris State University

   Affiliation as printed

   Department of Pharmaceutical Sciences, College of Pharmacy, Ferris State University, Big Rapids, Michigan 49307, United States

6. Larissa V. Ponomareva

   University of Kentucky · Ferris State University

   Affiliation as printed

   Department of Pharmaceutical Sciences , College of Pharmacy , Ferris State University , Big Rapids , Michigan 49307 , United States; orcid

   Center for Pharmaceutical Research and Innovation  and  Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States

7. Magdalena Niemczura

   University of Turku

   Affiliation as printed

   Department of Life Technologies, University of Turku, FIN-20014 Turku, Finland

8. Dino van Dissel Aachen Institute of Biology, Leiden University

   Leiden University · SINTEF

   Affiliation as printed

   Department of Biotechnology and Nanomedicine, SINTEF AS, P.O. Box 4760 Torgarden, NO-7465 Trondheim, Norway

   Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands

9. Gilles P. van Wezel Aachen Institute of Biology, Leiden University

   Leiden University

   Affiliation as printed

   Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands

10. Jon S. Thorson

    University of Kentucky

    Affiliation as printed

    Department of Pharmaceutical Sciences ,

    Center for Pharmaceutical Research and Innovation  and  Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States

11. Mikko Metsä‐Ketelä corresponding

    University of Turku

    Affiliation as printed

    Department of Life Technologies, University of Turku, FIN-20014 Turku, Finland

12. Khaled A. Shaaban corresponding

    University of Kentucky · Ferris State University

    Affiliation as printed

    Department of Pharmaceutical Sciences , College of Pharmacy , Ferris State University , Big Rapids , Michigan 49307 , United States; orcid

    Center for Pharmaceutical Research and Innovation  and  Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States

13. Stephen Eric Nybo corresponding

    Ferris State University

    Affiliation as printed

    Department of Pharmaceutical Sciences, College of Pharmacy, Ferris State University, Big Rapids, Michigan 49307, United States