A split-gene transketolase essential for phosphonothrixin biosynthesis

Phosphonothrixin (PTX) is a herbicidal natural product characterized by the presence of an acetyl group and a phosphonic acid moiety. It is produced by the actinobacterium Saccharothrix sp. ST-888. The biosynthesis of PTX has been elucidated, and the split-gene transketolase PtxB5/6 has been proposed to mediate the key acetyltransferase reaction in a thiamine diphosphate-dependent manner. In this chapter, we describe the identification of the PTX biosynthetic gene cluster in Saccharothrix sp. ST-888 and detail the experimental procedures used to characterize PTX biosynthetic enzymes. In vitro assays with purified recombinant PtxB5/6 demonstrated that the enzyme recognizes hydroxyethyl-thiamine diphosphate (provided by the acetohydroxyacid synthase homolog PtxB7) as a substrate and catalyzes the stereoselective acetylation of (3-hydroxy-2-oxopropyl)phosphonic acid at the C2 position, ultimately resulting in PTX formation. These experiments collectively highlighted a previously unrecognized reaction mechanism driven by the cooperative actions of the split-gene transketolase PtxB5/6 and the acetohydroxyacid synthase homolog PtxB7.