Process Optimization Of Amphotericin B Produced By Streptomyces Nodosus

Amphotericin B(Amphotericin B,Am B)is a clinically important polyene macrolide antibiotic with broad-spectrum antifungal activity and it is widely used to treat systemic fungal infections.The metabolic regulation mechanism in the process of Am B biosynthesis is relatively complex.Modern genetic engineering and metabolic engineering technologies have been widely used to improve the production performance of strains to meet the growth of market demand for Am B.However,there are less researches for fermentation.In order to further improve the fermentation level of Am B and reduce production costs,the development of new fermentation regulation strategies is extremely important.In this paper,in order to achieve the goal of increasing the yield of Am B and reducing the content of by-product Am A,an Am B high-yield S.nodosus screened in our lab was used to study the effects of the key precursors and differential metabolites as additives on the biosynthesis of Am B based on the analysis of existing metabolomics.Moreover,the fermentation process optimization combined with the staged control strategies and feeding regulation strategies was successfully built.Based on the analysis of significant metabolic differences in shake-flask fermentation,some key precursors and differential metabolites were screened.Rationally designed single-factor optimization and orthogonal mixed addition strategy were proposed.It was found that the united addition of 4 mg/L isopropanol,89 mg/L alanine,1 g/L pyruvate,and 25 mg/L nicotinamide at 24 h was the optimal supplementation strategy for exogenous additives.Finally,the Am B titer was improved to 6.47 g/L with 25.4% increase at shake-flask level.The results showed that different supplements affected important intermediate metabolic processes,thus significantly affected the biosynthesis process of Am B.In order to further promote the biosynthesis of Am B,the optimal initial glucose concentration and stirring speed in the 5-L fermentor were optimized.Ultimately,the optimal initial glucose concentration and stirring speed were determined to be 70 g/L and 500 rpm,respectively.In view of the fact that Am B fermentation is a partial growth-coupling type,different staged regulation strategies were proposed.Furthermore,the effects of staged p H,temperature,and dissolved oxygenfermentation control strategies on the production of Am B were performed.The optimal control strategies were the staged p H 7.0,temperature 30 ?/26 ? and DO20% control strategy,separately.The yield of Am B was finally increased from 9.89g/L in batch fermentation to 12.66 g/L,11.79 g/L and 11.28 g/L under these three strategies.In addition,extracellular organic acid analysis showed the effects of different regulatory strategies on the cell metabolic process,further illustrating that the fermentation control strategies combined with metabolic control played a decisive role on the entire Am B biosynthesis.Based on the research of additives in shake flasks,a scale-up experiment of additives in a 5-L fermentor was conducted to determine the effectiveness of adding the above four compounds to 5-L fermentor.The yield of Am B can be improved to12.56 g/L with a 27.0% increase,and the content of by-product Am A was 2.3% which has significantly decreased by 24.8%.It was presumed that the reduction of catalytic activity of the enoyl reductase domain in Amph C PKS module 5 might lead to the inhibition of Am A synthesis.Furthermore,different feeding control strategies in 5-L fermentor were optimized,including 6 kinds of feeding methods: pulse feeding,p H-Stat and DO-Stat fed-batch,constant speed fed-batch,variable speed fed-batch and constant residual glucose concentration fed-batch,and finally it was determined that constant-speed feeding at the flow rate of 1.5 g/(L·h)was the optimal feeding strategy.At this time,the maximum Am B yield reached 15.79 g/L,which was 59.7%higher than batch fermentation.And the by-product Am A content increased from3.1% to 7.1%.Considering that the composition of the feeding medium is relatively single,it was optimized to determine the optimal feeding medium as 200 g/L glucose,5 g/L peptone and 0.3 g/L ammonium sulfate.Based on previous studies,in order to most effectively improve the yield of Am B and reduce the content of by-product Am A,the fed-batch fermentation under the optimal combined control strategy was carried out,that is,the staged p H,temperature,and DO control strategy were used,and 4 mg/L isopropanol,89 mg/L alanine,1 g/L pyruvate and 25 mg/L nicotinamide were unitedly added at 24 h.With the addition of four additives,the optimal feeding medium was fed under constant speed fed-batch strategy at a flow rate of 1.5 g/(L·h).Finally,Am B titer accumulated to 18.39 g/L,which was 85.9% higher than batch fermentation,the maximum biomass(PMV)reached 45.0%,and the by-product Am A content was 2.1%,which was significantly reduced by 32.8% compared with batch fermentation.Through extracellular organic acids detection,changes of ?-ketoglutarate,pyruvate and citric acid concentration were identified as the most critical metabolite nodes,which further clarified the possible metabolic mechanism under this fermentation regulation strategy.Its essence is showned.Under the characteristics analysis of fermentation process,the metabolic flow of substances such as ?-ketoglutarate is taken as a control point to regulate cell growth and metabolism,so that the substance metabolism is maximized to themetabolic pathway which is conducive to product synthesis to make the Am B titer increase.Finally,the scale-up study was carried out in 50-L fermentor.The above-mentioned different fermentation regulation strategies provided new guidance for the industrial production of Am B.