New Molecular Tools for Regulation and Improvement of A40926 Glycopeptide Antibiotic Production in Nonomuraea gerenzanensis ATCC 39727.

Oleksandr Yushchuk,Flavia Marinelli,Andres Andreo-Vidal,Giorgia Letizia Marcone,Mervyn Bibb,Elisa Binda

doi:10.3389/fmicb.2020.00008

Abstract

Genome sequencing has revealed that Nonomuraea spp. represent a still largely unexplored source of specialized metabolites. Nonomuraea gerenzanensis ATCC 39727 is the most studied representative species since it produces the glycopeptide antibiotic (GPA) A40926 – the precursor of the clinically relevant antibiotic dalbavancin, approved by the FDA in 2014 for the treatment of acute skin infections caused by multi-drug resistant Gram-positive pathogens. The clinical relevance of dalbavancin has prompted increased attention on A40926 biosynthesis and its regulation. In this paper, we investigated how to enhance the genetic toolkit for members of the Nonomuraea genus, which have proved quite recalcitrant to genetic manipulation. By constructing promoter-probe vectors, we tested the activity of 11 promoters (heterologous and native) using the GusA reporter system in N. gerenzanensis and in Nonomuraea coxensis; this latter species is phylogenetically distant from N. gerenzanesis and also possesses the genetic potential to produce A40926 or a very similar GPA. Finally, the strongest constitutive promoter analyzed in this study, aac(3)IVp, was used to overexpress the cluster-situated regulatory genes controlling A40926 biosynthesis (dbv3 and dbv4 from N. gerenzanensis and nocRI from N. coxensis) in N. gerenzanensis, and the growth and productivity of the best performing strains were assessed at bioreactor scale using an industrial production medium. Overexpression of positive pathway-specific regulatory genes resulted in a significant increase in the level of A40926 production in N. gerenzanensis, providing a new knowledge-based approach to strain improvement for this valuable glycopeptide antibiotic.

Highlights

Research on glycopeptide antibiotics (GPAs) – drugs of “last resort” for treating severe infections caused by multi-drug resistant Gram-positive pathogens – has experienced a “renaissance” over the last decade (Marcone et al, 2018)
ATCC 55076, previously classified as Actinomadura parvosata subsp. kistnae S382–8 (Kusserow and Gulder, 2017; Nazari et al, 2017), have confirmed the hidden potential of these uncommon actinomycetes as prolific producers of specialized metabolites. We report that another member of this genus, Nonomuraea coxensis DSM 45129, which was isolated in Bangladesh in 2007 (Ara et al, 2007), has the genetic potential to produce A40926 or a very similar GPA; its biosynthetic gene clusters (BGCs) contains two regulatory genes, nocRI and nocRII, which are close homologs of dbv3 and dbv4, respectively
(ASM37988v1), we found a close homolog of the N. gerenzanensis regulatory gene dbv3

Summary

Introduction

Research on glycopeptide antibiotics (GPAs) – drugs of “last resort” for treating severe infections caused by multi-drug resistant Gram-positive pathogens – has experienced a “renaissance” over the last decade (Marcone et al, 2018). Important GPAs include two natural products (vancomycin and teicoplanin) and three second generation antibiotics (telavancin, A40926 Production in Nonomuraea spp. dalbavancin, and oritavancin), which are semisynthetic derivatives of natural products endowed with an increased antimicrobial potency and superior pharmacokinetic properties. The urgent need for new potent antibiotics has driven much recent interest in GPAs. For example, chemical synthesis recently resulted in the generation of a plethora of vancomycin derivatives with novel modifications that show superior antimicrobial activities (Okano et al, 2017; Wu and Boger, 2019), while teicoplanin has been conjugated with nanoparticles resulting in increased activity against biofilm-forming pathogens (Armenia et al, 2018). Heterologous expression of enzymes involved in later stages of GPA biosynthesis (glycosylation, sulfation, acylation etc.) in known producers or in vitro has already generated novel GPA derivatives that could not be prepared by chemical synthesis (Banik and Brady, 2008; Banik et al, 2010; Yim et al, 2014)

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