Engineering BioBricks for Deoxysugar Biosynthesis and Generation of New Tetracenomycins

Heli Tirkkonen; Katelyn V. Brown; Magdalena Niemczura; Zélie Faudemer; Courtney Brown; Larissa V. Ponomareva; Yosra A. Helmy; Jon S. Thorson; S. Eric Nybo; Mikko Metsä-Ketelä; Khaled A. Shaaban

doi:10.1021/acsomega.3c02460

Engineering BioBricks for Deoxysugar Biosynthesis and Generation of New Tetracenomycins

Heli Tirkkonen, Katelyn V. Brown, Magdalena Niemczura, Zélie Faudemer, Courtney Brown, Larissa V. Ponomareva, Yosra A. Helmy, Jon S. Thorson, S. Eric Nybo, Mikko Metsä-Ketelä, Khaled A. Shaaban

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

3 Scopus citations

Abstract

Tetracenomycins and elloramycins are polyketide natural products produced by several actinomycetes that exhibit antibacterial and anticancer activities. They inhibit ribosomal translation by binding in the polypeptide exit channel of the large ribosomal subunit. The tetracenomycins and elloramycins are typified by a shared oxidatively modified linear decaketide core, yet they are distinguished by the extent of O-methylation and the presence of a 2′,3′,4′-tri-O-methyl-α-l-rhamnose appended at the 8-position of elloramycin. The transfer of the TDP-l-rhamnose donor to the 8-demethyl-tetracenomycin C aglycone acceptor is catalyzed by the promiscuous glycosyltransferase ElmGT. ElmGT exhibits remarkable flexibility toward transfer of many TDP-deoxysugar substrates to 8-demethyltetracenomycin C, including TDP-2,6-dideoxysugars, TDP-2,3,6-trideoxysugars, and methyl-branched deoxysugars in both d- and l-configurations. Previously, we developed an improved host, Streptomyces coelicolor M1146::cos16F4iE, which is a stable integrant harboring the required genes for 8-demethyltetracenomycin C biosynthesis and expression of ElmGT. In this work, we developed BioBricks gene cassettes for the metabolic engineering of deoxysugar biosynthesis in Streptomyces spp. As a proof of concept, we used the BioBricks expression platform to engineer biosynthesis for d-configured TDP-deoxysugars, including known compounds 8-O-d-glucosyl-tetracenomycin C, 8-O-d-olivosyl-tetracenomycin C, 8-O-d-mycarosyl-tetracenomycin C, and 8-O-d-digitoxosyl-tetracenomycin C. In addition, we generated four new tetracenomycins including one modified with a ketosugar, 8-O-4′-keto-d-digitoxosyl-tetracenomycin C, and three modified with 6-deoxysugars, including 8-O-d-fucosyl-tetracenomycin C, 8-O-d-allosyl-tetracenomycin C, and 8-O-d-quinovosyl-tetracenomycin C. Our work demonstrates the feasibility of BioBricks cloning, with the ability to recycle intermediate constructs, for the rapid assembly of diverse carbohydrate pathways and glycodiversification of a variety of natural products.

Original language	English
Pages (from-to)	21237-21253
Number of pages	17
Journal	ACS Omega
Volume	8
Issue number	23
DOIs	https://doi.org/10.1021/acsomega.3c02460
State	Published - Jun 13 2023

Bibliographical note

Funding Information:
Research reported in this publication was supported by the National Science Foundation under Grant No. ENG-2015951 (S.E.N.), the National Cancer Institute of the National Institutes of Health under Award No. R15CA252830 (S.E.N.), the National Institutes of Health grant R37 AI052218, the 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), the 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.). The authors also acknowledge a grant from the Turku University Foundation. They thank the College of Pharmacy NMR Center (University of Kentucky) for NMR support and thank Prof. Dr. Mervyn Bibb (John Innes Centre, Norwich, U.K.) for the gift of strain S. coelicolor M1146. They also thank Dr. Isaac Brownell (Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD) for the gift of Merkel cells MKL1 and MCC26.

Publisher Copyright:
© 2023 The Authors. Published by American Chemical Society.

Funding

Research reported in this publication was supported by the National Science Foundation under Grant No. ENG-2015951 (S.E.N.), the National Cancer Institute of the National Institutes of Health under Award No. R15CA252830 (S.E.N.), the National Institutes of Health grant R37 AI052218, the 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), the 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.). The authors also acknowledge a grant from the Turku University Foundation. They thank the College of Pharmacy NMR Center (University of Kentucky) for NMR support and thank Prof. Dr. Mervyn Bibb (John Innes Centre, Norwich, U.K.) for the gift of strain S. coelicolor M1146. They also thank Dr. Isaac Brownell (Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD) for the gift of Merkel cells MKL1 and MCC26.

Funders	Funder number
Center of Biomedical Research Excellence
John Innes Centre, Norwich	M1146, MCC26
National Science Foundation (NSF)	ENG-2015951
National Institutes of Health (NIH)	R37 AI052218, P20 GM130456, R15CA252830
National Childhood Cancer Registry – National Cancer Institute
National Center for Advancing Translational Sciences (NCATS)	S10OD28690, UL1TR001998, UL1TR000117
University of Kentucky College of Pharmacy
Academy of Finland	340013
Turun Yliopisto
Novo Nordisk Fonden	NNF19OC0057511

ASJC Scopus subject areas

General Chemistry
General Chemical Engineering

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10.1021/acsomega.3c02460

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@article{39f59fc1bba94b97ab88d0a0765894f1,

title = "Engineering BioBricks for Deoxysugar Biosynthesis and Generation of New Tetracenomycins",

abstract = "Tetracenomycins and elloramycins are polyketide natural products produced by several actinomycetes that exhibit antibacterial and anticancer activities. They inhibit ribosomal translation by binding in the polypeptide exit channel of the large ribosomal subunit. The tetracenomycins and elloramycins are typified by a shared oxidatively modified linear decaketide core, yet they are distinguished by the extent of O-methylation and the presence of a 2′,3′,4′-tri-O-methyl-α-l-rhamnose appended at the 8-position of elloramycin. The transfer of the TDP-l-rhamnose donor to the 8-demethyl-tetracenomycin C aglycone acceptor is catalyzed by the promiscuous glycosyltransferase ElmGT. ElmGT exhibits remarkable flexibility toward transfer of many TDP-deoxysugar substrates to 8-demethyltetracenomycin C, including TDP-2,6-dideoxysugars, TDP-2,3,6-trideoxysugars, and methyl-branched deoxysugars in both d- and l-configurations. Previously, we developed an improved host, Streptomyces coelicolor M1146::cos16F4iE, which is a stable integrant harboring the required genes for 8-demethyltetracenomycin C biosynthesis and expression of ElmGT. In this work, we developed BioBricks gene cassettes for the metabolic engineering of deoxysugar biosynthesis in Streptomyces spp. As a proof of concept, we used the BioBricks expression platform to engineer biosynthesis for d-configured TDP-deoxysugars, including known compounds 8-O-d-glucosyl-tetracenomycin C, 8-O-d-olivosyl-tetracenomycin C, 8-O-d-mycarosyl-tetracenomycin C, and 8-O-d-digitoxosyl-tetracenomycin C. In addition, we generated four new tetracenomycins including one modified with a ketosugar, 8-O-4′-keto-d-digitoxosyl-tetracenomycin C, and three modified with 6-deoxysugars, including 8-O-d-fucosyl-tetracenomycin C, 8-O-d-allosyl-tetracenomycin C, and 8-O-d-quinovosyl-tetracenomycin C. Our work demonstrates the feasibility of BioBricks cloning, with the ability to recycle intermediate constructs, for the rapid assembly of diverse carbohydrate pathways and glycodiversification of a variety of natural products.",

author = "Heli Tirkkonen and Brown, {Katelyn V.} and Magdalena Niemczura and Z{\'e}lie Faudemer and Courtney Brown and Ponomareva, {Larissa V.} and Helmy, {Yosra A.} and Thorson, {Jon S.} and Nybo, {S. Eric} and Mikko Mets{\"a}-Ketel{\"a} and Shaaban, {Khaled A.}",

note = "Funding Information: Research reported in this publication was supported by the National Science Foundation under Grant No. ENG-2015951 (S.E.N.), the National Cancer Institute of the National Institutes of Health under Award No. R15CA252830 (S.E.N.), the National Institutes of Health grant R37 AI052218, the 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), the 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.). The authors also acknowledge a grant from the Turku University Foundation. They thank the College of Pharmacy NMR Center (University of Kentucky) for NMR support and thank Prof. Dr. Mervyn Bibb (John Innes Centre, Norwich, U.K.) for the gift of strain S. coelicolor M1146. They also thank Dr. Isaac Brownell (Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD) for the gift of Merkel cells MKL1 and MCC26. Publisher Copyright: {\textcopyright} 2023 The Authors. Published by American Chemical Society.",

year = "2023",

month = jun,

day = "13",

doi = "10.1021/acsomega.3c02460",

language = "English",

volume = "8",

pages = "21237--21253",

number = "23",

}

TY - JOUR

T1 - Engineering BioBricks for Deoxysugar Biosynthesis and Generation of New Tetracenomycins

AU - Tirkkonen, Heli

AU - Brown, Katelyn V.

AU - Niemczura, Magdalena

AU - Faudemer, Zélie

AU - Brown, Courtney

AU - Ponomareva, Larissa V.

AU - Helmy, Yosra A.

AU - Thorson, Jon S.

AU - Nybo, S. Eric

AU - Metsä-Ketelä, Mikko

AU - Shaaban, Khaled A.

N1 - Funding Information: Research reported in this publication was supported by the National Science Foundation under Grant No. ENG-2015951 (S.E.N.), the National Cancer Institute of the National Institutes of Health under Award No. R15CA252830 (S.E.N.), the National Institutes of Health grant R37 AI052218, the 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), the 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.). The authors also acknowledge a grant from the Turku University Foundation. They thank the College of Pharmacy NMR Center (University of Kentucky) for NMR support and thank Prof. Dr. Mervyn Bibb (John Innes Centre, Norwich, U.K.) for the gift of strain S. coelicolor M1146. They also thank Dr. Isaac Brownell (Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD) for the gift of Merkel cells MKL1 and MCC26. Publisher Copyright: © 2023 The Authors. Published by American Chemical Society.

PY - 2023/6/13

Y1 - 2023/6/13

N2 - Tetracenomycins and elloramycins are polyketide natural products produced by several actinomycetes that exhibit antibacterial and anticancer activities. They inhibit ribosomal translation by binding in the polypeptide exit channel of the large ribosomal subunit. The tetracenomycins and elloramycins are typified by a shared oxidatively modified linear decaketide core, yet they are distinguished by the extent of O-methylation and the presence of a 2′,3′,4′-tri-O-methyl-α-l-rhamnose appended at the 8-position of elloramycin. The transfer of the TDP-l-rhamnose donor to the 8-demethyl-tetracenomycin C aglycone acceptor is catalyzed by the promiscuous glycosyltransferase ElmGT. ElmGT exhibits remarkable flexibility toward transfer of many TDP-deoxysugar substrates to 8-demethyltetracenomycin C, including TDP-2,6-dideoxysugars, TDP-2,3,6-trideoxysugars, and methyl-branched deoxysugars in both d- and l-configurations. Previously, we developed an improved host, Streptomyces coelicolor M1146::cos16F4iE, which is a stable integrant harboring the required genes for 8-demethyltetracenomycin C biosynthesis and expression of ElmGT. In this work, we developed BioBricks gene cassettes for the metabolic engineering of deoxysugar biosynthesis in Streptomyces spp. As a proof of concept, we used the BioBricks expression platform to engineer biosynthesis for d-configured TDP-deoxysugars, including known compounds 8-O-d-glucosyl-tetracenomycin C, 8-O-d-olivosyl-tetracenomycin C, 8-O-d-mycarosyl-tetracenomycin C, and 8-O-d-digitoxosyl-tetracenomycin C. In addition, we generated four new tetracenomycins including one modified with a ketosugar, 8-O-4′-keto-d-digitoxosyl-tetracenomycin C, and three modified with 6-deoxysugars, including 8-O-d-fucosyl-tetracenomycin C, 8-O-d-allosyl-tetracenomycin C, and 8-O-d-quinovosyl-tetracenomycin C. Our work demonstrates the feasibility of BioBricks cloning, with the ability to recycle intermediate constructs, for the rapid assembly of diverse carbohydrate pathways and glycodiversification of a variety of natural products.

AB - Tetracenomycins and elloramycins are polyketide natural products produced by several actinomycetes that exhibit antibacterial and anticancer activities. They inhibit ribosomal translation by binding in the polypeptide exit channel of the large ribosomal subunit. The tetracenomycins and elloramycins are typified by a shared oxidatively modified linear decaketide core, yet they are distinguished by the extent of O-methylation and the presence of a 2′,3′,4′-tri-O-methyl-α-l-rhamnose appended at the 8-position of elloramycin. The transfer of the TDP-l-rhamnose donor to the 8-demethyl-tetracenomycin C aglycone acceptor is catalyzed by the promiscuous glycosyltransferase ElmGT. ElmGT exhibits remarkable flexibility toward transfer of many TDP-deoxysugar substrates to 8-demethyltetracenomycin C, including TDP-2,6-dideoxysugars, TDP-2,3,6-trideoxysugars, and methyl-branched deoxysugars in both d- and l-configurations. Previously, we developed an improved host, Streptomyces coelicolor M1146::cos16F4iE, which is a stable integrant harboring the required genes for 8-demethyltetracenomycin C biosynthesis and expression of ElmGT. In this work, we developed BioBricks gene cassettes for the metabolic engineering of deoxysugar biosynthesis in Streptomyces spp. As a proof of concept, we used the BioBricks expression platform to engineer biosynthesis for d-configured TDP-deoxysugars, including known compounds 8-O-d-glucosyl-tetracenomycin C, 8-O-d-olivosyl-tetracenomycin C, 8-O-d-mycarosyl-tetracenomycin C, and 8-O-d-digitoxosyl-tetracenomycin C. In addition, we generated four new tetracenomycins including one modified with a ketosugar, 8-O-4′-keto-d-digitoxosyl-tetracenomycin C, and three modified with 6-deoxysugars, including 8-O-d-fucosyl-tetracenomycin C, 8-O-d-allosyl-tetracenomycin C, and 8-O-d-quinovosyl-tetracenomycin C. Our work demonstrates the feasibility of BioBricks cloning, with the ability to recycle intermediate constructs, for the rapid assembly of diverse carbohydrate pathways and glycodiversification of a variety of natural products.

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Engineering BioBricks for Deoxysugar Biosynthesis and Generation of New Tetracenomycins

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