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

Rongbin Wang; Jennifer Nguyen; Jacob Hecht; Nora Schwartz; Katelyn V. Brown; Larissa V. Ponomareva; Magdalena Niemczura; Dino Van Dissel; Gilles P. Van Wezel; Jon S. Thorson; Mikko Metsä-Ketelä; Khaled A. Shaaban; S. Eric Nybo

doi:10.1021/acssynbio.2c00498

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

Rongbin Wang, Jennifer Nguyen, Jacob Hecht, Nora Schwartz, Katelyn V. Brown, Larissa V. Ponomareva, Magdalena Niemczura, Dino Van Dissel, Gilles P. Van Wezel, Jon S. Thorson, Mikko Metsä-Ketelä, Khaled A. Shaaban, S. Eric Nybo

Research output: Contribution to journal › Article › peer-review

9 Scopus citations

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.

Original language	English
Pages (from-to)	4193-4209
Number of pages	17
Journal	ACS Synthetic Biology
Volume	11
Issue number	12
DOIs	https://doi.org/10.1021/acssynbio.2c00498
State	Published - Dec 16 2022

Bibliographical note

Publisher Copyright:
© 2022 American Chemical Society.

Funding

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.

Funders	Funder number
Corporacion Nacional del Cobre
NIGMS-supported Center of Biomedical Research Excellence
National Science Foundation (NSF)	ENG-2015951
National Institutes of Health (NIH)	P20 GM130456
National Childhood Cancer Registry – National Cancer Institute	R15CA252830
National Center for Advancing Translational Sciences (NCATS)	S10OD28690, UL1TR001998, UL1TR000117
University of Kentucky College of Pharmacy
National Council for Eurasian and East European Research	101055020
Academy of Finland	340013
China Scholarship Council	202107960010
Turun Yliopisto
Novo Nordisk Fonden	NNF19OC0057511

Keywords

BioBricks
Streptomyces coelicolor
anthracyclinones
anticancer
natural product biosynthesis
synthetic biology

ASJC Scopus subject areas

Biomedical Engineering
Biochemistry, Genetics and Molecular Biology (miscellaneous)

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Access to Document

10.1021/acssynbio.2c00498

Cite this

Wang, R., Nguyen, J., Hecht, J., Schwartz, N., Brown, K. V., Ponomareva, L. V., Niemczura, M., Van Dissel, D., Van Wezel, G. P., Thorson, J. S., Metsä-Ketelä, M., Shaaban, K. A., & Eric Nybo, S. (2022). A BioBricks Metabolic Engineering Platform for the Biosynthesis of Anthracyclinones in Streptomyces coelicolor. ACS Synthetic Biology, 11(12), 4193-4209. https://doi.org/10.1021/acssynbio.2c00498

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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.",

keywords = "BioBricks, Streptomyces coelicolor, anthracyclinones, anticancer, natural product biosynthesis, synthetic biology",

author = "Rongbin Wang and Jennifer Nguyen and Jacob Hecht and Nora Schwartz and Brown, {Katelyn V.} and Ponomareva, {Larissa V.} and Magdalena Niemczura and {Van Dissel}, Dino and {Van Wezel}, {Gilles P.} and Thorson, {Jon S.} and Mikko Mets{\"a}-Ketel{\"a} and Shaaban, {Khaled A.} and {Eric Nybo}, S.",

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Wang, R, Nguyen, J, Hecht, J, Schwartz, N, Brown, KV, Ponomareva, LV, Niemczura, M, Van Dissel, D, Van Wezel, GP, Thorson, JS, Metsä-Ketelä, M, Shaaban, KA & Eric Nybo, S 2022, 'A BioBricks Metabolic Engineering Platform for the Biosynthesis of Anthracyclinones in Streptomyces coelicolor', ACS Synthetic Biology, vol. 11, no. 12, pp. 4193-4209. https://doi.org/10.1021/acssynbio.2c00498

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T1 - A BioBricks Metabolic Engineering Platform for the Biosynthesis of Anthracyclinones in Streptomyces coelicolor

AU - Wang, Rongbin

AU - Nguyen, Jennifer

AU - Hecht, Jacob

AU - Schwartz, Nora

AU - Brown, Katelyn V.

AU - Ponomareva, Larissa V.

AU - Niemczura, Magdalena

AU - Van Dissel, Dino

AU - Van Wezel, Gilles P.

AU - Thorson, Jon S.

AU - Metsä-Ketelä, Mikko

AU - Shaaban, Khaled A.

AU - Eric Nybo, S.

PY - 2022/12/16

Y1 - 2022/12/16

N2 - 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.

AB - 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.

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

Abstract

Bibliographical note

Funding

Keywords

ASJC Scopus subject areas

UN SDGs

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