Bioengineering and Functionalization of Rubromycin Polyketides by Pathway-Derived Amide Bond Synthetases

The formation of amide bonds is key to the biosynthesis of numerous natural products as well as to the industrial production of pharmaceuticals and valuable chemicals. As an alternative to the often inefficient and wasteful chemical syntheses of amides, emerging sustainable biocatalytic strategies rely on enzymes, such as amide bond synthetases (ABSs). Here we report the characterization of two distinct types of ABSs from the biosynthesis of the typically antibacterial and cytotoxic rubromycin polyketides, hyaluromycin and coumarubrin, which are produced by Streptomyces hyaluromycini and Lentzea tibetensis, respectively. These enzymes, ShRaABS and LtRaABS, are presumed to regiospecifically attach the aminocyclopentenone and aminocoumarin substituents (or their biosynthetic precursors) to the rubromycin backbone. Both ABSs were scrutinized for their substrate tolerance toward amino- and carboxy-donors, establishing LtRaABS as the more promising biocatalyst that allowed the generation of numerous unnatural rubromycins. These compounds include a "Trojan Horse" dopamine congener as the first known rubromycin with antibiotic activity against a Gram-negative Pseudomonas sp. by exploiting the strain's siderophore-uptake machinery. In addition, LtRaABS allowed the azide functionalization of rubromycins, thereby setting the stage for bioorthogonal click chemistry that can be employed in the future, for example, for the generation of antibody-drug conjugates.