Pseudonocardia strain improvement for stimulation of the di-sugar heptaene Nystatin-like Pseudonocardia polyene B1 biosynthesis

Pseudonocardia autotrophica was previously identified to produce a toxicity-reduced and solubility-improved disaccharide-containing anti-fungal compound belonging to the tetraene-family, Nystatin-like Pseudonocardia Polyene A1 (NPP A1). Subsequently NPP B1, a novel derivative harboring a heptaene core structure, was produced by a pathway-engineered Pseudonocardia strain through inactivation of the specific enoly reductase gene domain in the NPP biosynthetic gene cluster. Although in vitro and in vivo efficacy and toxicity studies indicate that NPP B1 is a promising lead antifungal compound, further improvement is required to increase the extremely low production yield in the pathway-engineered strain. To overcome this challenge, we performed the N-methyl-N′-nitro-N-nitrosoguanidine (NTG) iterative random mutagenesis, followed by zone-of-inhibition agar plug assay. After three rounds of the mutagenesis-and-screening protocol, the production yield of NPP B1 increased to 6.25 mg/L, which is more than an eightfold increase compared to the parental strain. The qRT-PCR analysis revealed that transcripts of the NPP B1 biosynthetic genes were increased in the mutant strain. Interestingly, an endogenous 125-kb plasmid was found to be eliminated through this mutagenesis. To further improve the NPP B1 production yield, the 32-kb NPP-specific regulatory gene cluster was cloned and overexpressed in the mutant strain. The chromosomal integration of the extra copy of the six NPP-specific regulatory genes led to an additional increase of NPP B1 yield to 31.6 mg/L, which is the highest production level of NPP B1 ever achieved by P. autotrophica strains. These results suggest that a synergistic combination of both the traditional and genetic strain improvement approaches is a very efficient strategy to stimulate the production of an extremely low-level metabolite (such as NPP B1) in a pathway-engineered rare actinomycetes strain.