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

Area of Science:

• Biocatalysis and Enzyme Engineering
• Natural Product Biosynthesis
• Medicinal Chemistry

Background:

• Amide bond formation is crucial for natural products and pharmaceuticals.
• Chemical amide synthesis is often inefficient and wasteful.
• Enzymatic biocatalysis offers a sustainable alternative for amide synthesis.

Purpose of the Study:

• To characterize two novel amide bond synthetases (ABSs) from rubromycin biosynthesis.
• To investigate the substrate tolerance and biocatalytic potential of these ABSs.
• To generate novel rubromycin analogs for potential therapeutic applications.

Main Methods:

• Isolation and characterization of two distinct ABS enzymes (ShRaABS and LtRaABS) from Streptomyces hyaluromycini and Lentzea tibetensis.
• Enzyme assays to determine substrate specificity and tolerance for amino- and carboxy-donors.
• Biocatalytic generation of unnatural rubromycin analogs using LtRaABS.

Main Results:

• Characterized ShRaABS and LtRaABS, involved in attaching substituents to the rubromycin backbone.
• LtRaABS demonstrated broad substrate tolerance, enabling the synthesis of numerous unnatural rubromycins.
• Generated a novel rubromycin analog with Gram-negative antibiotic activity and azide-functionalized rubromycins for click chemistry.

Conclusions:

• LtRaABS is a promising biocatalyst for generating diverse rubromycin analogs.
• Novel rubromycins exhibit potential as antibiotics against Gram-negative bacteria.
• Azide-functionalized rubromycins pave the way for bioorthogonal chemistry applications, including antibody-drug conjugates.