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

Abstract

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.

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

“…Precursor supplement and key metabolite addition significantly influence the secondary metabolite production (Mao et al, 2009 ; Xia et al, 2013 ). As shown in Table 1 , seven precursors and 15 key differential metabolites were determined as additives and tested in shake flask fermentation based on the biosynthesis of AmB and the metabolite analysis (Zhang et al, 2020 ). When 10 mg/L sodium propionate was supplemented, the highest AmB titer (6.93 g/L) was achieved, with 34% increase.…”

“…When different amino acids were ingested into the medium at 24 h, 4 mM serine addition showed the highest titer increase (34.2%), followed by alanine (28.4%), and glycine (10%). Based on our previous metabolite analysis, 5,6,7,8-tetrahydrofolate (THF), 5,10-methyltetrahydrofolate, and 5-formiminotetrahydrofolate were selected as several metabolites contributing the most into the groups' discrimination (Zhang et al, 2020 ). The amphotericin yields were increased by serine and glycine.…”

“…To improve the production of AmB, seven isometric short-chain fatty acids, lower alcohols, and salt substance with a final concentration (0–12 mg/L) were added as precursors in the medium at 0 h. After 108 h, the AmB titer was detected to investigate which was the optimal additive and the addition concentration of these precursors. Subsequently, addition time was optimized between 0 and 72 h. Furthermore, 15 key differential metabolites based on metabolomics analysis were screened as additives into the fermentation medium at 24 h (Zhang et al, 2020 ). The optimum addition volumes of these compounds from 0.01 to 2 g/L were investigated.…”

“…AmA and AmB were then detected at 304 and 405 nm using a UV detector (Hitachi, Ltd., Japan), respectively. In addition, a commercial standard of AmB was obtained from Sigma-Aldrich (CAS: 1397-89-3) (Zhang et al, 2020 ).…”

“…New strategies in our lab were developed to optimize the biosynthesis of AmB based on in-depth analysis of key metabolites and pathways. Through overexpression genes involved in oxygen taking, precursor acquiring, and product exporting, the titer of AmB increased to 6.58 g/L, which is the highest titer of AmB in a shake flask as far as we know (Zhang et al, 2020 ). However, there has been no research to determine the key differential metabolites as additives to enhance AmB production.…”