Engineering aromatic precursor supply and regulation in glycopeptide antibiotic producers

Abstract

Glycopeptide antibiotics (GPAs) are clinically indispensable natural products produced by actinomycetes. Exemplary are vancomycin and teicoplanin, which are used as last-resort antibiotics against multidrug-resistant Gram-positive pathogens. GPAs are classified into type I-V based on differences in peptide scaffold, crosslinking patterns and tailoring reactions. Their biosynthesis is exceptionally demanding, requiring tight coordination between biosynthetic gene cluster (BGC), specific regulatory networks, complex biosynthetic pathways and a sustained supply of aromatic amino acids, such as hydroxyphenylglycine (Hpg), β-hydroxytyrosine (Bht) and dihydroxyphenylglycine (Dpg), whose precursor 4-hydroxyphenylpyruvate (4-HPP) and tyrosine (Tyr) are derived from the shikimate pathway. Despite extensive industrial optimization, GPA production remains limited by feedback-regulated primary metabolism, regulatory constraints and the frequent transcriptional silence of BGCs. In this work, we combined metabolic pathway engineering and regulatory activation strategies to systematically address these limitations. We employed metabolic engineering in producers of canonical GPAs, whose respective BGCs encode duplicated isoenzymes of key enzymes of the shikimate pathway – 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (Dahp) and prephenate dehydrogenase (Pdh). These isoenzymes play a central role in sustaining precursor flux. Biochemical analyses and genetic manipulation of the producers revealed that feedback regulation, rather than catalytic efficiency alone, limits or promotes GPA biosynthesis. Consistently, feedback-aware overexpression of key enzymes substantially enhanced balhimycin, ristomycin and teicoplanin production in Amycolatopsis balhimycina, Amycolatopsis japonica and Actinoplanes teichomyceticus. The aim of the second part of this work was the activation of silent type V GPA BGCs that had been identified by genome mining in the genomes of three actinomycetes. To activate these BGCs, insights gained in the first part of the study were applied to ensure sufficient supply of the GPA biosynthetic precursors 4-HPP and Tyr. As no GPA production could be detected under standard laboratory conditions, activation strategies were pursued that combined heterologous and endogenous overexpression of StrR- and LuxR-like pathway-specific regulators with modular precursor supply systems for Hpg and Dpg. Despite these targeted regulatory and metabolic interventions, no production of type V GPAs could be detected. 9 Together, this work provides a systematic framework for the optimization of GPA biosynthesis. It demonstrates that efficient production requires careful alignment between feedback-regulated precursor supply and targeted activation of BGCs. These results highlight key factors governing biosynthetic control and provide valuable guiding principles for future approaches aimed at improving GPA yields in actinobacteria.