A discrete intermediate for the biosynthesis of both the enediyne core and the anthraquinone moiety of enediyne natural products

Minakshi Bhardwaj; Zheng Cui; Erome Daniel Hankore; Faruk H. Moonschi; Hoda Saghaeiannejad Esfahani; Edward Kalkreuter; Chun Gui; Dong Yang; George N. Phillips; Jon S. Thorson; Ben Shen; Steven G. Van Lanena

doi:10.1073/pnas.2220468120

A discrete intermediate for the biosynthesis of both the enediyne core and the anthraquinone moiety of enediyne natural products

Minakshi Bhardwaj, Zheng Cui, Erome Daniel Hankore, Faruk H. Moonschi, Hoda Saghaeiannejad Esfahani, Edward Kalkreuter, Chun Gui, Dong Yang, George N. Phillips, Jon S. Thorson, Ben Shen, Steven G. Van Lanena

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

Abstract

The enediynes are structurally characterized by a 1,5-diyne-3-ene motif within a 9- or 10-membered enediyne core. The anthraquinone-fused enediynes (AFEs) are a subclass of 10-membered enediynes that contain an anthraquinone moiety fused to the enediyne core as exemplified by dynemicins and tiancimycins. A conserved iterative type I polyketide synthase (PKSE) is known to initiate the biosynthesis of all enediyne cores, and evidence has recently been reported to suggest that the anthraquinone moiety also originates from the PKSE product. However, the identity of the PKSE product that is converted to the enediyne core or anthraquinone moiety has not been established. Here, we report the utilization of recombinant E. coli coexpressing various combinations of genes that encode a PKSE and a thioesterase (TE) from either 9- or 10-membered enediyne biosynthetic gene clusters to chemically complement ΔPKSE mutant strains of the producers of dynemicins and tiancimycins. Additionally, ¹³C-labeling experiments were performed to track the fate of the PKSE/TE product in the ΔPKSE mutants. These studies reveal that 1,3,5,7,9,11,13-pentadecaheptaene is the nascent, discrete product of the PKSE/TE that is converted to the enediyne core. Furthermore, a second molecule of 1,3,5,7,9,11,13-pentadecaheptaene is demonstrated to serve as the precursor of the anthraquinone moiety. The results establish a unified biosynthetic paradigm for AFEs, solidify an unprecedented biosynthetic logic for aromatic polyketides, and have implications for the biosynthesis of not only AFEs but all enediynes.

Original language	English
Article number	e2220468120
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	120
Issue number	9
DOIs	https://doi.org/10.1073/pnas.2220468120
State	Published - Feb 28 2023

Bibliographical note

Funding Information:
ACKNOWLEDGMENTS. This work was supported by the National Cancer Institute grants CA217255 (J.S.T., G.N.P., and S.G.V.L.) and CA204484 (B.S.), the National Institute of General Medicine grant GM134954 (B.S.), the National Institute of General Medicine fellowship GM134688 (E.K.), the NIH Center of Biomedical Research Excellence (COBRE) in Pharmaceutical Research and Innovation grant P20GM130456 (J.S.T.), and the National Center for Advancing Translational Sciences grants UL1TR000117 and UL1TR001998 (J.S.T.), and the University of Kentucky College of Pharmacy. Some NMR data were acquired on a Bruker AVANCE NEO 600 MHz high-performance digital NMR spectrometer funded by NIH grant S10OD28690.We thank Vivekanandan Subramanian and the College of Pharmacy PharmNMR Center for analytical support.

Funding Information:
This work was supported by the National Cancer Institute grants CA217255 (J.S.T., G.N.P., and S.G.V.L.) and CA204484 (B.S.), the National Institute of General Medicine grant GM134954 (B.S.), the National Institute of General Medicine fellowship GM134688 (E.K.), the NIH Center of Biomedical Research Excellence (COBRE) in Pharmaceutical Research and Innovation grant P20GM130456 (J.S.T.), and the National Center for Advancing Translational Sciences grants UL1TR000117 and UL1TR001998 (J.S.T.), and the University of Kentucky College of Pharmacy. Some NMR data were acquired on a Bruker AVANCE NEO 600 MHz high-performance digital NMR spectrometer funded by NIH grant S10OD28690. We thank Vivekanandan Subramanian and the College of Pharmacy PharmNMR Center for analytical support.

Publisher Copyright:
Copyright © 2023 the Author(s). Published by PNAS.

Keywords

biosynthesis
enediyne
natural products
polyene
polyketide

ASJC Scopus subject areas

General

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10.1073/pnas.2220468120

Cite this

Bhardwaj, M., Cui, Z., Hankore, E. D., Moonschi, F. H., Esfahani, H. S., Kalkreuter, E., Gui, C., Yang, D., Phillips, G. N., Thorson, J. S., Shen, B., & Van Lanena, S. G. (2023). A discrete intermediate for the biosynthesis of both the enediyne core and the anthraquinone moiety of enediyne natural products. Proceedings of the National Academy of Sciences of the United States of America, 120(9), [e2220468120]. https://doi.org/10.1073/pnas.2220468120

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title = "A discrete intermediate for the biosynthesis of both the enediyne core and the anthraquinone moiety of enediyne natural products",

abstract = "The enediynes are structurally characterized by a 1,5-diyne-3-ene motif within a 9- or 10-membered enediyne core. The anthraquinone-fused enediynes (AFEs) are a subclass of 10-membered enediynes that contain an anthraquinone moiety fused to the enediyne core as exemplified by dynemicins and tiancimycins. A conserved iterative type I polyketide synthase (PKSE) is known to initiate the biosynthesis of all enediyne cores, and evidence has recently been reported to suggest that the anthraquinone moiety also originates from the PKSE product. However, the identity of the PKSE product that is converted to the enediyne core or anthraquinone moiety has not been established. Here, we report the utilization of recombinant E. coli coexpressing various combinations of genes that encode a PKSE and a thioesterase (TE) from either 9- or 10-membered enediyne biosynthetic gene clusters to chemically complement ΔPKSE mutant strains of the producers of dynemicins and tiancimycins. Additionally, 13C-labeling experiments were performed to track the fate of the PKSE/TE product in the ΔPKSE mutants. These studies reveal that 1,3,5,7,9,11,13-pentadecaheptaene is the nascent, discrete product of the PKSE/TE that is converted to the enediyne core. Furthermore, a second molecule of 1,3,5,7,9,11,13-pentadecaheptaene is demonstrated to serve as the precursor of the anthraquinone moiety. The results establish a unified biosynthetic paradigm for AFEs, solidify an unprecedented biosynthetic logic for aromatic polyketides, and have implications for the biosynthesis of not only AFEs but all enediynes.",

keywords = "biosynthesis, enediyne, natural products, polyene, polyketide",

author = "Minakshi Bhardwaj and Zheng Cui and Hankore, {Erome Daniel} and Moonschi, {Faruk H.} and Esfahani, {Hoda Saghaeiannejad} and Edward Kalkreuter and Chun Gui and Dong Yang and Phillips, {George N.} and Thorson, {Jon S.} and Ben Shen and {Van Lanena}, {Steven G.}",

note = "Funding Information: ACKNOWLEDGMENTS. This work was supported by the National Cancer Institute grants CA217255 (J.S.T., G.N.P., and S.G.V.L.) and CA204484 (B.S.), the National Institute of General Medicine grant GM134954 (B.S.), the National Institute of General Medicine fellowship GM134688 (E.K.), the NIH Center of Biomedical Research Excellence (COBRE) in Pharmaceutical Research and Innovation grant P20GM130456 (J.S.T.), and the National Center for Advancing Translational Sciences grants UL1TR000117 and UL1TR001998 (J.S.T.), and the University of Kentucky College of Pharmacy. Some NMR data were acquired on a Bruker AVANCE NEO 600 MHz high-performance digital NMR spectrometer funded by NIH grant S10OD28690.We thank Vivekanandan Subramanian and the College of Pharmacy PharmNMR Center for analytical support. Funding Information: This work was supported by the National Cancer Institute grants CA217255 (J.S.T., G.N.P., and S.G.V.L.) and CA204484 (B.S.), the National Institute of General Medicine grant GM134954 (B.S.), the National Institute of General Medicine fellowship GM134688 (E.K.), the NIH Center of Biomedical Research Excellence (COBRE) in Pharmaceutical Research and Innovation grant P20GM130456 (J.S.T.), and the National Center for Advancing Translational Sciences grants UL1TR000117 and UL1TR001998 (J.S.T.), and the University of Kentucky College of Pharmacy. Some NMR data were acquired on a Bruker AVANCE NEO 600 MHz high-performance digital NMR spectrometer funded by NIH grant S10OD28690. We thank Vivekanandan Subramanian and the College of Pharmacy PharmNMR Center for analytical support. Publisher Copyright: Copyright {\textcopyright} 2023 the Author(s). Published by PNAS.",

year = "2023",

month = feb,

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doi = "10.1073/pnas.2220468120",

language = "English",

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Bhardwaj, M, Cui, Z, Hankore, ED, Moonschi, FH, Esfahani, HS, Kalkreuter, E, Gui, C, Yang, D, Phillips, GN, Thorson, JS, Shen, B & Van Lanena, SG 2023, 'A discrete intermediate for the biosynthesis of both the enediyne core and the anthraquinone moiety of enediyne natural products', Proceedings of the National Academy of Sciences of the United States of America, vol. 120, no. 9, e2220468120. https://doi.org/10.1073/pnas.2220468120

A discrete intermediate for the biosynthesis of both the enediyne core and the anthraquinone moiety of enediyne natural products. / Bhardwaj, Minakshi; Cui, Zheng; Hankore, Erome Daniel et al.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 120, No. 9, e2220468120, 28.02.2023.

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

TY - JOUR

T1 - A discrete intermediate for the biosynthesis of both the enediyne core and the anthraquinone moiety of enediyne natural products

AU - Bhardwaj, Minakshi

AU - Cui, Zheng

AU - Hankore, Erome Daniel

AU - Moonschi, Faruk H.

AU - Esfahani, Hoda Saghaeiannejad

AU - Kalkreuter, Edward

AU - Gui, Chun

AU - Yang, Dong

AU - Phillips, George N.

AU - Thorson, Jon S.

AU - Shen, Ben

AU - Van Lanena, Steven G.

N1 - Funding Information: ACKNOWLEDGMENTS. This work was supported by the National Cancer Institute grants CA217255 (J.S.T., G.N.P., and S.G.V.L.) and CA204484 (B.S.), the National Institute of General Medicine grant GM134954 (B.S.), the National Institute of General Medicine fellowship GM134688 (E.K.), the NIH Center of Biomedical Research Excellence (COBRE) in Pharmaceutical Research and Innovation grant P20GM130456 (J.S.T.), and the National Center for Advancing Translational Sciences grants UL1TR000117 and UL1TR001998 (J.S.T.), and the University of Kentucky College of Pharmacy. Some NMR data were acquired on a Bruker AVANCE NEO 600 MHz high-performance digital NMR spectrometer funded by NIH grant S10OD28690.We thank Vivekanandan Subramanian and the College of Pharmacy PharmNMR Center for analytical support. Funding Information: This work was supported by the National Cancer Institute grants CA217255 (J.S.T., G.N.P., and S.G.V.L.) and CA204484 (B.S.), the National Institute of General Medicine grant GM134954 (B.S.), the National Institute of General Medicine fellowship GM134688 (E.K.), the NIH Center of Biomedical Research Excellence (COBRE) in Pharmaceutical Research and Innovation grant P20GM130456 (J.S.T.), and the National Center for Advancing Translational Sciences grants UL1TR000117 and UL1TR001998 (J.S.T.), and the University of Kentucky College of Pharmacy. Some NMR data were acquired on a Bruker AVANCE NEO 600 MHz high-performance digital NMR spectrometer funded by NIH grant S10OD28690. We thank Vivekanandan Subramanian and the College of Pharmacy PharmNMR Center for analytical support. Publisher Copyright: Copyright © 2023 the Author(s). Published by PNAS.

PY - 2023/2/28

Y1 - 2023/2/28

N2 - The enediynes are structurally characterized by a 1,5-diyne-3-ene motif within a 9- or 10-membered enediyne core. The anthraquinone-fused enediynes (AFEs) are a subclass of 10-membered enediynes that contain an anthraquinone moiety fused to the enediyne core as exemplified by dynemicins and tiancimycins. A conserved iterative type I polyketide synthase (PKSE) is known to initiate the biosynthesis of all enediyne cores, and evidence has recently been reported to suggest that the anthraquinone moiety also originates from the PKSE product. However, the identity of the PKSE product that is converted to the enediyne core or anthraquinone moiety has not been established. Here, we report the utilization of recombinant E. coli coexpressing various combinations of genes that encode a PKSE and a thioesterase (TE) from either 9- or 10-membered enediyne biosynthetic gene clusters to chemically complement ΔPKSE mutant strains of the producers of dynemicins and tiancimycins. Additionally, 13C-labeling experiments were performed to track the fate of the PKSE/TE product in the ΔPKSE mutants. These studies reveal that 1,3,5,7,9,11,13-pentadecaheptaene is the nascent, discrete product of the PKSE/TE that is converted to the enediyne core. Furthermore, a second molecule of 1,3,5,7,9,11,13-pentadecaheptaene is demonstrated to serve as the precursor of the anthraquinone moiety. The results establish a unified biosynthetic paradigm for AFEs, solidify an unprecedented biosynthetic logic for aromatic polyketides, and have implications for the biosynthesis of not only AFEs but all enediynes.

AB - The enediynes are structurally characterized by a 1,5-diyne-3-ene motif within a 9- or 10-membered enediyne core. The anthraquinone-fused enediynes (AFEs) are a subclass of 10-membered enediynes that contain an anthraquinone moiety fused to the enediyne core as exemplified by dynemicins and tiancimycins. A conserved iterative type I polyketide synthase (PKSE) is known to initiate the biosynthesis of all enediyne cores, and evidence has recently been reported to suggest that the anthraquinone moiety also originates from the PKSE product. However, the identity of the PKSE product that is converted to the enediyne core or anthraquinone moiety has not been established. Here, we report the utilization of recombinant E. coli coexpressing various combinations of genes that encode a PKSE and a thioesterase (TE) from either 9- or 10-membered enediyne biosynthetic gene clusters to chemically complement ΔPKSE mutant strains of the producers of dynemicins and tiancimycins. Additionally, 13C-labeling experiments were performed to track the fate of the PKSE/TE product in the ΔPKSE mutants. These studies reveal that 1,3,5,7,9,11,13-pentadecaheptaene is the nascent, discrete product of the PKSE/TE that is converted to the enediyne core. Furthermore, a second molecule of 1,3,5,7,9,11,13-pentadecaheptaene is demonstrated to serve as the precursor of the anthraquinone moiety. The results establish a unified biosynthetic paradigm for AFEs, solidify an unprecedented biosynthetic logic for aromatic polyketides, and have implications for the biosynthesis of not only AFEs but all enediynes.

KW - biosynthesis

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KW - natural products

KW - polyene

KW - polyketide

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A discrete intermediate for the biosynthesis of both the enediyne core and the anthraquinone moiety of enediyne natural products

Abstract

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Keywords

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