Exploring shikimate pathway enzyme diversity for engineering of glycopeptide antibiotic biosynthesis

Supply of pathway precursors often limits the biosynthesis of specialized metabolites. Type I-IV glycopeptide antibiotics (GPAs) contain non-proteinogenic aromatic amino acids, requiring a sustained flux through the shikimate pathway. To secure precursor provision, many GPA producers encode secondary copies of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (Dahp_sec) and prephenate dehydrogenase (Pdh_sec) within their biosynthetic gene clusters (BGCs). The functional diversity and regulatory logic of these isoenzymes across taxa remains insufficiently understood. Phylogenetic analysis of Dahp_sec and Pdh_sec from characterized and uncharacterized GPA BGCs revealed substantial evolutionary diversification that does not correlate with GPA type or producer genus. We biochemically characterized Dahp_sec and Pdh_sec from Amycolatopsis coloradensis DSM 4425 (avoparcin producer) and Actinoplanes teichomyceticus NRRL B-16726 (teicoplanin producer), representing distinct evolutionary relatives. The analysis revealed pronounced differences in kinetic efficiency and allosteric regulation between the two enzymes. In A. coloradensis, Dahp_{ACOL_sec} and Pdh_{ACOL_sec} are strongly inhibited by Tyr, and Dahp_{ACOL_sec} is additionally inhibited by Phe and Trp, restricting shikimate-pathway flux and avoparcin production. In contrast, Dahp_{ATE_sec} and Pdh_{ATE_sec} from Act. teichomyceticus are primarily inhibited by Tyr, while Phe antagonizes this inhibition for Dahp_{ATE_sec}, supporting sustained precursor flux. Exploiting these regulatory features through targeted overexpression of dahp_{ATE_sec}, as well as pdh_{ATE_sec}, significantly increased teicoplanin production. Together, our results demonstrate that feedback regulation, rather than catalytic efficiency alone, makes shikimate-pathway enzymes suitable for sustained precursor supply, and that feedback-aware enzyme selection provides a powerful strategy for rational metabolic engineering of GPA production.