Production improvement of dalbavancin precursor A40926B0 in Nonomuraea gerenzanensis through strain reconstruction and fermentation optimization

Abstract

Dalbavancin is a potent next-generation lipoglycopeptide antibiotic, but the biosynthesis of its essential precursor, A40926B0, is severely hindered in its native producer Nonomuraea gerenzanensis. Complex competing metabolic pathways and strict transcriptional repression in this strain lead to critical industrial bottlenecks, including extreme product heterogeneity and notoriously low yields. To address these challenges, this study presents a systematic strategy to construct a high-producing strain and optimize the fermentation process for A40926B0. First, random mutagenesis of the strain N. gerenzanensis IPB-9 was performed using a 10 MeV high-energy electron linear accelerator. Screening identified mutant M0318, achieving a 43.6% increase in A40926B0 titer (808.8 mg/L). Concurrently, RNA-Seq analysis of N. gerenzanensis L70 was processed and seven endogenous strong promoters were quantitatively characterized and validated by using an eGFP reporter system and RT-qPCR to expand the genetic toolbox. Applying this toolkit, the strongest identified promoter (orf1597*p) was engineered to overexpress dbv3, the core pathway-specific positive regulator, in the M0318 chassis. This rational intervention further boosted A40926B0 production to 1.03 g/L. Furthermore, a comprehensive bioprocess optimization was executed. Following single-factor evaluations of shake-flask parameters, a Box-Behnken design (BBD)-based response surface methodology (RSM) was employed for systematic medium formulation, successfully elevating the titer to 1.41 g/L. Finally, this optimized process was successfully scaled to a 15 L bioreactor and the final A40926B0 titer reached 2.27 g/L, representing a 303.2% increase over the initial process, laying a foundation for the industrial-scale production of A40926B0.