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

ACS Omega. 2023 Jun 1;8(23):21237-21253. doi: 10.1021/acsomega.3c02460. eCollection 2023 Jun 13.

Authors

Affiliations

¹ Department of Life Technologies, University of Turku, FIN-20014 Turku, Finland.
² Department of Pharmaceutical Sciences, College of Pharmacy, Ferris State University, Big Rapids, Michigan 49307, United States.
³ Chemistry and Chemical Engineering Department, SIGMA Clermont, 63170 Aubière, France.
⁴ Center for Pharmaceutical Research and Innovation, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States.
⁵ Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States.
⁶ Department of Veterinary Science, College of Agriculture, Food, and Environment, University of Kentucky, Lexington, Kentucky 40546, United States.

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.

Abstract

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