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.
|Number of pages||17|
|State||Published - Jun 13 2023|
Bibliographical noteFunding 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.
© 2023 The Authors. Published by American Chemical Society.
ASJC Scopus subject areas
- Chemistry (all)
- Chemical Engineering (all)