Functional Application of the Single-Module NRPS-like D-Alanyltransferase in Maytansinol Biosynthesis

Single-module nonribosomal peptide synthetases (NRPSs) have nonclassical domain compositions and play versatile roles in the biosynthesis of natural products. AstC, a prototypical single-module NRPS-like d-alanyltransferase with the architecture of adenylation-thiolation-thioesterase (A-T-TE) tridomain, was identified as the catalyst in the d-alanylation process during the post-polyketide synthase (PKS) modifications of ansatrienin biosynthesis. In this study, the function of the TE domain of AstC was elucidated in intermolecular esterification, and its substrate promiscuity was revealed for both acyl donors and polyketide acceptors. Through genome mining, a newly characterized AstC homolog was identified, SmAstC, and it was shown that SmAstC exhibited the highest catalytic activity for d-alanylation of N-desmethyl-4,5-desepoxymaytansinol (DDM), a precursor of the antitumor agent ansamitocins, obtained from Actinosynnema pretiosum HGF052. Harnessing the broad substrate selectivity of the TE domains and the spontaneous hydrolysis propensity of the d-alanylated products, the post-PKS modification of ansamitocin biosynthesis was reprogrammed through introducing the gene encoding SmAstC into A. pretiosum HGF052. The ansamitocin biosynthetic pathway in this engineered strain was switched toward the production of maytansinol that was a critical intermediate of maytansine-derived antibody-drug conjugates. The SmAstC-catalyzed d-alanylation of DDM interrupted the stringent post-PKS modification steps within the original biosynthesis of ansamitocins, which produced 3-O-d-alanyl maytansinol that readily underwent spontaneous hydrolysis to afford maytansinol. The innovative application of the single-module NRPS-like d-alanyltransferases combined with rational strain engineering has surmounted the longtime hurdle in converting DDM to maytansinol in vivo, enabling the biomanufacturing of maytansinol without precedent.