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 remark... More
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 TDPdeoxysugar 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-quinovosyltetracenomycin 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.
