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.

Improvement of amphotericin B production by a newly isolated Streptomyces nodosus mutant.

Amphotericin B is an effective antifungal antibiotic. However, its production in the wild-type strain of Streptomyces nodosus is relatively low. In this study, a strain of ZJB 20130827, capable of producing amphotericin B, was isolated and identified as S. nodosus based on its physiological and biochemical characteristics as well as its 16S rRNA gene sequence analysis. With N-methyl-N-nitroso-N'-nitroguanidine (NTG) and ultraviolet (UV) treatment, this wild-type strain was mutated for improving the yield of amphotericin B. After NTG and UV treatment, the best mutant N3 was obtained for optimization of fermentation conditions. The production of amphotericin B with N3 was 5,260 mg/L, while the wild-type strain ZJB 20130827 was about 580 mg/L, an increase of 906.9%. A genetically stable mutant strain with high yield of amphotericin B was obtained using NTG and UV. The mutant obtained in this work could potentially be utilized in industrial production of amphotericin B.

Large-scale synthesis of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate by a stereoselective carbonyl reductase with high substrate concentration and product yield.

To biosynthesize the (3R,5S)-CDHH in an industrial scale, a newly synthesized stereoselective short chain carbonyl reductase (SCR) was successfully cloned and expressed in Escherichia coli. The fermentation of recombinant E. coli harboring SCR was carried out in 500 L and 5000 L fermenters, with biomass and specific activity of 9.7 g DCW/L, 15749.95 U/g DCW, and 10.97 g DCW/L, 19210.12 U/g DCW, respectively. The recombinant SCR was successfully applied for efficient production of (3R,5S)-CDHH. The scale-up synthesis of (3R,5S)-CDHH was performed in 5000 L bioreactor with 400 g/L of (S)-CHOH at 30°C, resulting in a space-time yield of 13.7 mM/h/g DCW, which was the highest ever reported. After isolation and purification, the yield and d.e. of (3R,5S)-CDHH reached 97.5% and 99.5%, respectively. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:612-620, 2017.