Comparative metabolomics analysis of amphotericin B high-yield mechanism for metabolic engineering.

BACKGROUND: The polyene macrocyclic compound amphotericin B (AmB) is an important antifungal antibiotic for the clinical treatment of invasive fungal infections. To rationally guide the improvement of AmB production in the main producing strain Streptomyces nodosus, comparative metabolomics analysis was performed to investigate the intracellular metabolic changes in wild-type S. nodosus ZJB20140315 with low-yield AmB production and mutant S. nodosus ZJB2016050 with high-yield AmB production, the latter of which reached industrial criteria on a pilot scale. RESULTS: To investigate the relationship of intracellular metabolites, 7758 metabolites were identified in mutant S. nodosus and wildtype S. nodosus via LC-MS. Through analysis of metabolism, the level of 26 key metabolites that involved in carbon metabolism, fatty acids metabolism, amino acids metabolism, purine metabolism, folate biosynthesis and one carbon pool by folate were much higher in mutant S. nodosus. The enrichment of relevant metabolic pathways by gene overexpression strategy confirmed that one carbon pool by folate was the key metabolic pathway. Meanwhile, a recombinant strain with gene metH (methionine synthase) overexpressed showed 5.03 g/L AmB production within 120 h fermentation, which is 26.4% higher than that of the mutant strain. CONCLUSIONS: These results demonstrated that comparative metabolomics analysis was an effective approach for the improvement of AmB production and could be applied for other industrially or clinically important compounds as well.

Enhanced AmB Production in Streptomyces nodosus by Fermentation Regulation and Rational Combined Feeding Strategy.

Amphotericin B is a clinically important polyene macrolide antibiotic with a broad-spectrum antifungal activity. In this work, the addition of key precursors and differential metabolites, combined with staged fermentation process control strategies, was carried out to improve AmB production. Rationally designed addition strategies were proposed as follows: 4 mg/L isopropanol, 1 mM alanine, 1 g/L pyruvate, and 0.025 g/L nicotinamide were supplemented at 24 h. The AmB titer was ultimately enhanced to 6.63 g/L, with 28.5% increase in shake flasks fermentation. To further promote the biosynthesis of AmB, different glucose feeding strategies were investigated and the highest AmB titer (15.78 g/L) was obtained by constant speed fed-batch fermentation in a 5-L fermentor. Subsequently, compared with the batch fermentation (9.89 g/L), a novel combined feeding strategy was ultimately developed to improve the production of AmB by 85.9%, reaching 18.39 g/L that is the highest titer of AmB ever reported so far, in which the optimized components were fed at 24 h and the staged fermentation regulation strategies were used simultaneously. Moreover, the ratio of co-metabolite AmA decreased by 32.3%, from 3.1 to 2.1%. Through the detection of extracellular organic acids, the changes in α-ketoglutaric acid, pyruvate, and citric acid concentrations were identified as the most flexible metabolite nodes to further clarify the potential mechanism under different fermentation regulation strategies. These results demonstrated that the strategies above may provide new guidance for the industrial-scale production of AmB.

Amphotericin B biosynthesis in Streptomyces nodosus: quantitative analysis of metabolism via LC-MS/MS based metabolomics for rational design.

BACKGROUND: Amphotericin B (AmB) is widely used against fungal infection and produced mainly by Streptomyces nodosus. Various intracellular metabolites of S. nodosus were identified during AmB fermentation, and the key compounds that related to the cell growth and biosynthesis of AmB were analyzed by principal component analysis (PCA) and partial least squares (PLS). RESULTS: Rational design that based on the results of metabolomics was employed to improve the AmB productivity of Streptomyces nodosus, including the overexpression of genes involved in oxygen-taking, precursor-acquiring and product-exporting. The AmB yield of modified strain S. nodosus VMR4A was 6.58 g/L, which was increased significantly in comparison with that of strain S. nodosus ZJB2016050 (5.16 g/L). This was the highest yield of AmB reported so far, and meanwhile, the amount of by-product amphotericin A (AmA) was decreased by 45%. Moreover, the fermentation time of strain S. nodosus VMR4A was shortened by 24 h compared with that of strain. The results indicated that strain S. nodosus VMR4A was an excellent candidate for the industrial production of AmB because of its high production yield, low by-product content and the fast cell growth. CONCLUSIONS: This study would lay the foundation for improving the AmB productivity through metabolomics analysis and overexpression of key enzymes.