Characterization of the Postaglycone Modifications in Ristomycin Biosynthesis

Area of Science:

• Microbiology
• Biochemistry
• Synthetic Biology

Background:

• Ristomycin (ristocetin) is a type III glycopeptide antibiotic (GPA) with potent activity against Gram-positive pathogens.
• The post-assembly tailoring steps in ristomycin biosynthesis are not fully understood, hindering the development of novel analogs.

Purpose of the Study:

• To delineate the postaglycone tailoring steps for ristomycin maturation.
• To propose a comprehensive model for ristomycin biosynthesis, including glycosylation and methylation.
• To identify ristomycin glycoforms with improved antimicrobial activity.

Main Methods:

• Generation and characterization of *Amycolatopsis* sp. TNS106 mutants.
• In vitro biochemical assays of key enzymes (RgtfB, RgtfC, MtfA).
• Integration of new findings with existing literature to build a biosynthetic model.

Main Results:

• Confirmed the function and timing of glycosyltransferases (RgtfB, RgtfC) and methyltransferase (MtfA).
• Proposed a sequential model for ristomycin glycosylation and C-terminal methylation.
• Identified ristomycin glycoforms with enhanced antimicrobial potency.
• Demonstrated that specific glycosyl modifications (e.g., ristosamine removal, rhamnose addition) impact activity, especially against vancomycin-resistant *Enterococcus*.

Conclusions:

• The study provides a detailed model for ristomycin post-assembly modifications.
• Engineered strains offer a platform for combinatorial biosynthesis of novel GPAs.
• Identified glycoforms with improved activity, advancing GPA development.