Enhanced amphotericin B production by genetically engineered Streptomyces nodosus

Area of Science:

• Microbial Biotechnology
• Synthetic Biology
• Natural Product Biosynthesis

Background:

• Amphotericin B (AmB), a vital antifungal polyketide, is produced by Streptomyces nodosus.
• Low AmB production is often linked to competing metabolic pathways, such as type II polyketide synthases (PKS).
• Understanding the regulatory mechanisms and precursor competition is crucial for optimizing AmB industrial yields.

Purpose of the Study:

• To investigate the impact of PKS5 competition on AmB biosynthesis in S. nodosus.
• To enhance AmB production through genetic modification by deleting PKS5 and overexpressing key biosynthetic genes.
• To explore the relationship between intracellular reducibility and by-product formation (Amphotericin A).

Main Methods:

• Genome sequencing and transcriptome analysis of wild-type and mutant S. nodosus strains.
• Gene deletion (ΔPKS5) and gene overexpression (PKS amphA, acc1, mme, mcm) strategies.
• Fermentation in shake flasks and bioreactors to quantify AmB and Amphotericin A (AmA) yields.
• Analysis of intracellular reducibility in different S. nodosus genetic types.

Main Results:

• Deletion of the PKS5 gene increased AmB titer from 5.01 g/L to 6.32 g/L and reduced AmA from 0.51 g/L to 0.12 g/L.
• Overexpression of specific genes in a ΔPKS5 mutant further improved AmB production to 7.06 g/L (shake flask) and 15.6 g/L (bioreactor).
• A novel correlation was established between intracellular reducibility and AmA by-product formation, potentially linked to NADPH consumption.

Conclusions:

• Targeting competing PKS pathways, like PKS5, is an effective strategy to enhance AmB production.
• Combinatorial genetic engineering, including gene deletion and overexpression, significantly boosts AmB yields.
• The findings provide a foundation for improving the industrial production of AmB and other polyketides.