Engineering BioBricks for Deoxysugar Biosynthesis and Generation of New Tetracenomycins

Abstract

<p>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 andelloramycins are typified by a shared oxidatively modified linear decaketide core, yet they are distinguished by the extent of O-methylationand the presence of a 2 ',3 ',4 '-tri-O-methyl-alpha-l-rhamnose appended at the 8-position ofelloramycin. The transfer of the TDP-l-rhamnose donor tothe 8-demethyl-tetracenomycin C aglycone acceptor is catalyzed bythe promiscuous glycosyltransferase ElmGT. ElmGT exhibits remarkable flexibility toward transfer of many TDP-deoxysugar substrates to 8-demethyltetracenomycinC, including TDP-2,6-dideoxysugars, TDP-2,3,6-trideoxysugars, andmethyl-branched deoxysugars in both d- and l-configurations.Previously, we developed an improved host, <i>Streptomyces coelicolor</i> M1146::cos16F4iE, which is a stable integrant harboring the required genes for 8-demethyltetracenomycin C biosynthesisand expression of ElmGT. In this work, we developed BioBricks gene cassettes for the metabolic engineering of deoxysugar biosynthesisin <i>Streptomyces </i>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-tetracenomycinC. In addition, we generated four new tetracenomycins including onemodified 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 recycleintermediate constructs, for the rapid assembly of diverse carbohydratepathways and glycodiversification of a variety of natural products.<br></p>