How Is Amphotericin B API Powder Manufactured?

Amphotericin B 1397-89-3 Chemical Structure
Amphotericin B Chemical Structure

Amphotericin B API is a commonly seen antibiotic used for a wide range of infections caused by bacteria.

It is a polyene antifungal antibiotic that inhibits fungal growth by affecting cell membrane permeability.

As one the largest Amphotericin B manufacturers in China, we share its general production process as below:

The general production process

Industrially people choose Streptomyces nodosus as strains and carry out aeration deep fermentation in a liquid medium containing carbohydrates and organic nitrogen sources.

When considerable potency units are reached, amphotericin is extracted from the fermentation broth.

The amphotericin produced by fermentation contains two components, A and B.

The A component is less toxic and has a weak antifungal effect, so it doesn’t exert much economic value.

Our target molecule, the B component, has a strong antibiotic effect and is named amphotericin B.

The final product is obtained by isolating component A and purifying B.

The solid medium suitable for the growth of the bacterium is screened out as the MS medium.

The optimal carbon and nitrogen sources were glucose and beef paste.

One of the optimal glucose concentrations was 45 g/L, the optimal beef paste concentration was 40 g/L, and the optimal carbon-to-nitrogen ratio was 1:1.

The yield by the process above could reach 2710 mg/L.

Next, the fermentation conditions of flask-shake were optimized:

2% inoculum of fermentation broth, 48 h incubation time of seed solution, initial pH of fermentation broth at 7.0, fermentation temperature at 28°C, 40 mL of liquid volume in shake flask, and 0.2 g/L of Mg~(2+) addition.

The effect of shear force on the fermentation was investigated, and it was found that adding a certain amount of glass beads to the fermentation broth could significantly promote the production of Amphotericin B.

The yield could reach 4563.2 mg/L when glass beads were added at 60 beads per bottle, which was 341.1% higher than that of the control group.

The microscopic observation of the fermentation broth showed that the mycelium was significantly improved, and the mycelium was loosened, which was more favorable for the strain to take in nutrients and oxygen.

Both the pointed baffle bottle and the flat baffle bottle could significantly promote Amphotericin B production.

When studying the fermentation process of Streptomyces nodosus ZJB15076, it was found that after a short adaptation period, Amphotericin B synthesis started rapidly around the second day after inoculation, stopped growing around the sixth day, and reached the highest yield around the seventh day.

After that, the biomass and product concentration of the fermentation continued to decrease, and the pH of the fermentation broth increased significantly during the whole fermentation process due to the accumulation of products and the cleavage of the bacterium at the later stage of fermentation.

Finally, the effects of stirring paddle type, stirring speed, and aeration on the fermentation yield of Amphotericin B in a 5L fermenter were investigated.

The experimental results showed that the six straight-bladed disc turbine stirrer was more suitable for Amphotericin B fermentation production than the other two types of stirrers, and the optimal stirring speed was determined to be 400 rpm and the optimal aeration rate was 1.5 vvm.

The effect of staged temperature-controlled incubation on Amphotericin B synthesis was investigated.

However, it was found that the fermentation yield was not significantly promoted by the staged temperature-controlled incubation, and only increased by 5.9% compared with the control group.

Fermentation Process Control

The mechanism of metabolic regulation in the biosynthesis of amphotericin B is complex.

Modern genetic engineering and metabolic engineering techniques have been widely used to enhance the production performance of strains to improve the yield, but less research has been conducted on the control of the fermentation process.

To further optimize the fermentation process of amphotericin B and reduce the production cost, it is important to develop novel fermentation control strategies.

We screened a strain of Streptomyces nodosus with high amphotericin B production and investigated the effects of key precursors and differential metabolites as an additive on amphotericin B biosynthesis based on the existing metabolome analysis.

The fermentation process was optimized by combining the phase-control strategy and the supplementation method to increase amphotericin B production and reduce amphotericin A’s content as a by-product.

The essential precursors and differential metabolites were screened based on the analysis of significant metabolic differences during the shake flask fermentation process, and a rational design of single-factor optimization and orthogonal co-addition strategy was proposed.

The final Amphotericin B yield at the shake level was 6.47 g/L, an increase of 25.4%.

It was shown that different additives could significantly affect biosynthesis yields by influencing important intermediate metabolic processes.

To further promote biosynthesis, the optimum initial glucose concentration, and stirring speed in the 5 L fermenter were optimized, and the optimum initial glucose concentration and speed were determined to be 70 g/L and 500 rpm, respectively.

Because of the partial growth coupling type of amphotericin B fermentation, a phased control strategy was used to investigate the effects of phased pH, temperature, and dissolved oxygen control strategies on Am B production.

We found that the phased control strategy of pH 7.0, temperature 30℃/26℃, and DO 20% was optimal.

Under these three parameters, the production finally increased from 9.89 g/L to 12.66 g/L, 11.79 g/L, and 11.28 g/L, respectively.

The effect of different regulatory strategies on the metabolic process of the bacterium during the synthesis was also elucidated by extracellular organic acid analysis, which further indicated that the fermentation regulatory strategy combined with metabolic control played a decisive role in the whole process of biosynthesis.

Based on the study of shake flask additive, we conducted an amplification experiment of additive supplementation in a 5 L fermenter and demonstrated the effectiveness of adding the above four compounds simultaneously in a 5 L fermenter,

We speculate that the increase in yield may be due to the decrease in the catalytic activity of the enoyl reductase structural domain in Amph C PKS module 5, which led to the inhibition of Am A synthesis;

The 5 L fermentor was optimized for 6 types of replenishment, including pulse replenishment, p H-Stat, and DO-Stat replenishment, constant rate replenishment, variable-rate replenishment, and constant residual glucose concentration replenishment, and the optimal replenishment strategy was determined to be constant rate replenishment at a flow rate of 1.5 g/(L-h).

The maximum yield reached 15.79 g/L, which was 59.7% higher than that of the split fermentation, but the by-product Am A content also increased from 3.1% to 7.1%;

Considering that the composition of the supplementation medium was relatively single, the optimal supplementation medium was optimized and determined to be 200 g/L glucose, 5 g/L peptones, and 0.3 g/L ammonium sulfate.

Based on the previous experimental study, the best-combined control strategy was carried out to improve the yield and reduce the by-product content, i.e., the fermentation was supplemented with 4 mg/L isopropanol, 89 mg/L alanine, 1 g/L pyruvate, and 25 mg/L nicotinamide at 24 h of fermentation.

The yield finally reached 18.39 g/L, which was 85.9% higher than that of the split fermentation, and the maximum bacterial volume (PMV) reached 45.0%.

The by-product content was 2.1%, which was 32.8% lower than that of the split fermentation;

Through the detection of extracellular organic acids, changes in the concentrations of α-ketoglutarate, pyruvate, and citric acid were identified as the most critical metabolite nodes, thus further elucidating the possible metabolic mechanisms under this fermentation regulation strategy.

The essence of this strategy is to monitor the metabolic flow of α-ketoglutarate and other substances to analyze the fermentation process, and to regulate the growth and metabolism of the bacterium so that the metabolism of target substances can be achieved to the maximum extent possible so that the Amphotericin B potency increases.

Finally, we conducted a scale-up study on a 50 L fermenter, and the above different fermentation control strategies have provided new guidance for production on an industrial scale.

The manufacturing process above is just a general idea of the production, and the actual production process is far more complicated than we said above.

Either way, in case you have different or better ideas on the production, you are welcome to comment or contact us directly.

References

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