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

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) A40926the 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.

Genomic-Led Discovery of a Novel Glycopeptide Antibiotic by Nonomuraea coxensis DSM 45129

“… 23 Given all these similarities, we presume that NocRI/NocRII have functions identical to Dbv3/Dbv4 and both regulatory pairs might cross-talk between these species. Our previous results, 6 where heterologous expression of nocRI in N. gerenzanensis improved A40926 production, support this assumption. The single GPA resistance determinant encoded within noc is Noc27, a close (87%) orthologue of Dbv7 (VanY n ), which is a d , d -carboxypeptidase involved in A40926 self-resistance.…”

“…Two master regulators of A40926 biosynthesis—LuxR-like Dbv3 and StrR-like Dbv4—have orthologues coded within noc —NocRI (94% aa sequence identity) and NocRII (86% aa sequence identity), respectively. 6 In N. gerenzanensis , both Dbv3 and Dbv4 are crucial for biosynthesis activation. 23 Dbv4 was shown to bind the promoter regions of operons dbv30-35 (mainly coding for Dpg biosynthesis enzymes) and dbv14-8 (including the genes coding for cross-linking monooxygenases), and its binding sites were identified.…”

“… 22 , 37 In these conditions (namely a vegetative preculture in E26 medium and a GPA production step in FM2 medium using baffled flasks), N. coxensis tended to grow poorly, and no antimicrobial activity was detectable throughout the 168h cultivation from inoculum. Thus, we further screened different media and fermentation conditions previously used for growing other GPA producing strains, such as TM1 used for teicoplanin production by A. teichomyceticus ( 38 ) and R5 adopted for balhimycin production in Amycolatopsis balhimycina , 39 as well as VM0.1 and ISP2l previously employed for the vegetative cultivation of N. coxensis ( 6 ) (media composition detailed in the Supporting Information ). The production of antimicrobial activity toward Bacillus subtilis ATCC 6633 was observed only in TM1 and ISP2l media when glass beads were added to favor dispersed growth ( Figure S9 ).…”

“…To achieve this, we used the pSET152A expression platform, which has proven to be very effective for gene overexpression in both N. coxensis and N. gerenzanensis . 6 dbv29 was cloned into pSET152A to generate pSAD29, which was then introduced into N. coxensis by conjugation from Escherichia coli . N. coxensis pSAD29 + was grown in ISP2lm medium for 168 h, and the resulting GPA complex was purified using d -Ala- d -Ala affinity resin.…”

“… 6 , 16 Following the sequencing of the A40926 BGC ( dbv ) almost two decades ago, 18 multiple aspects of A40926 biosynthesis were investigated, including nonribosomal aglycone assembly and tailoring steps, 15 , 19 , 20 self-resistance, 21 , 22 and pathway-specific regulation of its production. 6 , 23 , 24 N. gerenzanensis was also engineered to produce A40926 derivatives that are better suited for downstream chemical modification to dalbavancin. 16 Another GPA produced by a Nonomuraea species is the type V GPA kistamicin ( Figure 1 ) from Nonomuraea sp.…”