Ansamycins such as rifamycin, ansamitocin and geldanamycin are an important class of polyketide natural products. Their biosynthetic pathways are especially complex since they involve the formation of 3-amino-5-hydroxybenzoic acid (AHBA) followed by backbone assembly by a hybrid nonribosomal peptide synthetase (NRPS) / polyketide synthase (PKS). We have reconstituted the ability to synthesize P8/1-OG. an intermediate in rifamycin biosynthesis, in an extensively manipulated strain of E. coli. The parent strain, BAP1, contains the sfp phosphopantetheinyl transferase gene from Bacillus subtilis, which post-translationally modifies NRPS and PKS modules. AHBA biosynthesis in this host required introduction of seven genes from Amycolatopsis mediterranei (which produces rifamycin) and Actinosynnema pretiosum (which produces ansamitocin). Since the 4-module RifA protein (530kDa) from the rifamycin synthetase could not be efficiently produced in an intact form in E. coli, it was genetically split into two bimodular proteins separated by matched linker pairs to facilitate efficient inter-polypeptide transfer of a biosynthetic intermediate. A derivative of BAP1 was engineered that harbors the AHBA biosynthetic operon, the bicistronic RifA construct and the pccB and accAl genes from Streptomyces coelicolor, which enable methylmalonyl-CoA biosynthesis. Fermentation of this strain of E. coli yielded P8/1-OG and an N-acetyl P8/1-OG analog. In addition to providing a fundamentally new route to shikimate and ansamycin-type compounds, this result enables further genetic manipulation of AHBA-derived polyketide natural products with unprecedented power.
