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

Bo Zhang,Yu-Han Zhang,Kai Huang,Yu-Guo Zheng,Kai Chen,Zhi-Qiang Liu,Sheng-Xian Jiang,Yu Chen

doi:10.3389/fbioe.2020.00597

Bo Zhang, Yu-Han Zhang + Show 6 more

Open Access

https://doi.org/10.3389/fbioe.2020.00597

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Abstract

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.

Highlights

Amphotericin B (AmB), a polyene macrolide antibiotic predominantly produced by Streptomyces nodosus (Caffrey et al, 2001), is a broad-spectrum antifungal drug mainly used in the clinical treatment of deep fungal invasive infections (Vasquez et al, 2018)
When the ER5 domain in module AmphC plays roles, which is responsible for the reduction of the C28–C29 unsaturated bond during the biosynthesis of macrolides, cometabolite amphotericin A (AmA, C28–C29 saturated bond), with low antifungal activity, is produced simultaneously (Borgos et al, 2006), the result of which is that AmB and AmA are relatively difficult and time-consuming to separate
The weakened affinity between the NADPH cofactor and the ER5 domain likely explains why isopropanol is a better additive for reducing the AmA production, which apparently arises from the failure of enoyl reductase to function during the antibiotic biosynthesis (Borgos et al, 2006)

Summary

Introduction

Amphotericin B (AmB), a polyene macrolide antibiotic predominantly produced by Streptomyces nodosus (Caffrey et al, 2001), is a broad-spectrum antifungal drug mainly used in the clinical treatment of deep fungal invasive infections (Vasquez et al, 2018). Enhanced Amphotericin B Production responsible for AmB biosynthesis have been cloned and sequenced (Caffrey et al, 2001; Sweeney et al, 2015). When the ER5 domain in module AmphC plays roles, which is responsible for the reduction of the C28–C29 unsaturated bond during the biosynthesis of macrolides, cometabolite amphotericin A (AmA, C28–C29 saturated bond), with low antifungal activity, is produced simultaneously (Borgos et al, 2006), the result of which is that AmB and AmA are relatively difficult and time-consuming to separate. The clinical usage of AmB is limited because of its side effects, such as acute nephrotoxicity (Larabi et al, 2003), it is still irreplaceably applied as a useful antibiotic for more than 50 years for treating systemic fungal infections (Deray, 2002)

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