Engineering Biosynthetic Pathways for Deoxysugars: Branched-Chain Sugar Pathways and Derivatives from the Antitumor Tetracenomycin

Abstract

Sugar biosynthesis cassette genes have been used to construct plasmids directing the biosynthesis of branched-chain deoxysugars: pFL942 (NDP-L-mycarose), pFL947 (NDP-4-deacetyl-L-chromose B), and pFL946/pFL954 (NDP-2,3,4-tridemethyl-L-nogalose). Expression of pFL942 and pFL947 in S. lividans 16F4, which harbors genes for elloramycinone biosynthesis and the flexible ElmGT glycosyltransferase of the elloramycin biosynthetic pathway, led to the formation of two compounds: 8-alpha-L-mycarosyl-elloramycinone and 8-demethyl-8-(4-deacetyl)-alpha-L-chromosyl-tetracenomycin C, respectively. Expression of pFL946 or pFL954 failed to produce detectable amounts of a novel glycosylated tetracenomycin derivative. Formation of these two compounds represents examples of the sugar cosubstrate flexibility of the ElmGT glycosyltransferase. The use of these cassette plasmids also provided insights into the substrate flexibility of deoxysugar biosynthesis enzymes as the C-methyltransferases EryBIII and MtmC, the epimerases OleL and EryBVII, and the 4-ketoreductases EryBIV and OleU.

Combinatorial Biosynthesis of Antitumor Deoxysugar Pathways in Streptomyces griseus : Reconstitution of “Unnatural Natural Gene Clusters” for the Biosynthesis of Four 2,6- d -Dideoxyhexoses

“…This plasmid derives from pFL943, which has been shown to direct the biosynthesis of TDP-L-olivose and TDP-L-rhamnose. pFL1012 contains the same gene functions as pFL943, but it lacks the oleL 3,5-epimerase gene, which prevents it from directing the biosynthesis of L-DOH (21). Notably, cultures of S. lividans 16F4(pFL1012) produced a glycosylated tetracenomycin with the same mass as DOLV-TCMC or DDIG-TCMC but showing a different retention time.…”

“…After lyophilization, 7.9 mg of D-digitoxosyl-tetracenomycin C (DDIG-TCMC) and 25.7 mg of D-boivinosyl-tetracenomycin C (DBOV-TCMC) were obtained. HPLC-mass spectrometry (MS) analysis was carried out as previously described (21).…”

“…To clarify this situation, we decided to use a different 4-ketoreductase, OleU, which is involved in the biosynthesis of L-oleandrose in the oleandomycin pathway (3). A first indication that OleU could be suitable for the biosynthesis of D-boivinose arose from the analysis of cultures of S. lividans 16F4 expressing pFL1012 (21). This plasmid derives from pFL943, which has been shown to direct the biosynthesis of TDP-L-olivose and TDP-L-rhamnose.…”

Modulation of Deoxysugar Transfer by the Elloramycin Glycosyltransferase ElmGT through Site-Directed Mutagenesis

“…The results in this paper clearly show that the promiscuous GTF activity of ElmGT can be modulated by specific amino acid substitutions in residues located in a short region within the ␣/␤/␣ motif. The flexibility of ElmGT for the attachment of different deoxysugars to 8DMTC has been widely reported previously (9,(17)(18)(19)21). However, the efficiency of transfer varies depending upon the particular deoxysugar.…”

“…ElmGT is an interesting GTF since it possesses remarkable donor substrate flexibility. It has been shown to transfer at least 11 different sugars to 8DMTC, including both L-and D-isomeric forms of some sugars (4,9,18,19,21). This surprising flexibility of ElmGT prompted us to get further insight into the identification of some amino acid residues involved in the selection of the sugar donor.…”