*Article* **Nysfungin Production Improvement by UV Mutagenesis in** *Streptomyces noursei* **D-3-14**

**Ming Song <sup>1</sup> , Wubing He <sup>2</sup> , Sulan Cai <sup>1</sup> , Fuju Wang <sup>3</sup> , Weizhuo Xu 2,\* and Wei Xu 2,\***


**Abstract:** *Streptomyces noursei* D-3-14 was taken as a starting strain and treated with UV (15 W, 30 cm) mutagenesis for 40 s for three consecutive rounds. High yielding strains were screened using chemical and biological potency determination, and the components of the fermentation products were detected using HPLC. Finally, the mutant strain *Streptomyces noursei* 72-22-1 with a chemical potency of 8912 (U/mL) and a biological potency of 5557 (U/mL) was obtained after the genetic stability evaluation. After optimization of the fermentation conditions, the chemical potency and biological potency of *Streptomyces noursei* 72-22-1 reached 14,082 U/mL and 10579 U/mL, respectively, which is 1.58 and 1.91 times those before optimization. HPLC analysis indicated that the mutant strain 72-22-1 displayed a higher content of polyfungin B. When equimolar nystatin A1, A3, and polyfungin B were tested for their fungicidal activities towards *Saccharomyces cerevisiae* ATCC 2061, polyfungin B exhibited a better efficacy than nystatin A1 and A3.

**Keywords:** *Streptomyces noursei*; nysfungin; nystatin A1; nystatin A3; polyfungin B; UV mutagenesis

## **1. Introduction**

Nystatin is a kind of polyene macrolide antibiotic [1–3] that was first isolated by Hazen and Brown from soil in Fauquier County, Virginia in the 1950s [4]. At that time, it was named Fungicidin and demonstrated fungistatic and fungicidal activities, without antibacterial action. In 1954, this Fungicidin was first commercialized by Bristol Myers Squibb and renamed nystatin, with its major ingredient being nystatin A1. Later, Chinese researchers also isolated and identified additional ingredients of actinomycetes derived from Fungicidin in Guangdong province soil [5,6]. In 1981, Thomas et al. compared pharmaceutical grade samples of Fungicidin from China, Hungary, Italy, US, and Russia, and identified that nystatin A1 represented the majority of the ingredients in the US, Italy, and Hungarian samples, with a 70% concentration [7], which coincides with the British Pharmacopoeia in 1980 [8]. Meanwhile, he also reported that only 12% nystatin A1 was founded in the Chinese samples, with other components nystatin A3 and polyfungin B ranging from 20 to 50% [9]. Since then, nystatin has usually been assigned as western nystatin, which has a high A1 content as the major ingredient. While nysfungin is normally used to name the Chinese-derived nystatin, besides the A1, which may also have A3 and polyfungin B.

Nystatin is effective against *Candida*, *Cryptococcus*, *Aspergillus*, *Histoplasma*, and *Blastomyces* [10,11]. Nystatin is one of the most commonly used topical antifungal drugs, with a high efficacy, low cost, and fewer side effects, due to not being absorbed from the gastrointestinal tract [12,13].

**Citation:** Song, M.; He, W.; Cai, S.; Wang, F.; Xu, W.; Xu, W. Nysfungin Production Improvement by UV Mutagenesis in *Streptomyces noursei* D-3-14. *Catalysts* **2023**, *13*, 247. https://doi.org/10.3390/ catal13020247

Academic Editors: Zhilong Wang and Tao Pan

Received: 30 December 2022 Revised: 17 January 2023 Accepted: 18 January 2023 Published: 21 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

From their chemical structures, it could be seen that nystatin A3 has an additional digitoxose compared to nystatin A1 [9,14], while the polyfungin B has an absence of a C10 hydroxyl group compared with nystatin A3 [15]. All three components share the same macrolide polyene skeleton, as shown in Figure 1. of this study was to screen and characterize nystatin-producing *Streptomyces noursei* strains exposed to UV, to identify the strain that produced the highest activity of nystatin, thereby obtaining a high-activity and low-cost raw material for commercial nystatin production.

Nystatin is effective against *Candida*, *Cryptococcus*, *Aspergillus*, *Histoplasma*, and *Blastomyces* [10,11]. Nystatin is one of the most commonly used topical antifungal drugs, with a high efficacy, low cost, and fewer side effects, due to not being absorbed from the gas-

From their chemical structures, it could be seen that nystatin A3 has an additional digitoxose compared to nystatin A1 [9,14], while the polyfungin B has an absence of a C10 hydroxyl group compared with nystatin A3 [15]. All three components share the same

In the FDA approved nystatin drugs, the major ingredient is the single nystatin A1, with the CAS No. 34786-70-4. While in the Chinese pharmacopoeia [16], nystatin is still considered a multiple-ingredient drug, and was named Nysfungin, so as to differentiate

Due to the actinomyces metabolic diversity, in this research, we used an environmentally isolated strain of *Streptomyces noursei* D-3-14 to perform ultraviolet (UV) mutagenesis, optimized the culture conditions, and finally obtained a higher production of Nysfungin. Mutation breeding is a powerful technique, in which microbial strains exposed to mutagen treatment are screened, to identify positive mutants with specific characteristics. This method has the advantages of simplicity, rapidity, and high efficiency [17]. UV is possibly one of the physical mutagens that causes genetic variation and enables the selection of traits as needed. UV has a strong genotoxic effect, producing DNA damage that leads to an altered DNA structure [18]. UV irradiation induces covalent crosslinks between neighboring pyrimidines. If left unrepaired, error-prone replication of this damaged DNA leads to an increased rate of mutagenesis and genome instability [19]. The aim

**Figure 1.** Chemical structures of nystatin A1, A3, and polyfungin B. **Figure 1.** Chemical structures of nystatin A1, A3, and polyfungin B.

*Catalysts* **2023**, *13*, x FOR PEER REVIEW 2 of 12

macrolide polyene skeleton, as shown in Figure 1.

it from the single major A1 nystatin.

trointestinal tract [12,13].

**2. Results and Discussion** *2.1. Determination of the Chemical and Biological Potency of the Starting Strain Streptomyces noursei D-3-14* In the FDA approved nystatin drugs, the major ingredient is the single nystatin A1, with the CAS No. 34786-70-4. While in the Chinese pharmacopoeia [16], nystatin is still considered a multiple-ingredient drug, and was named Nysfungin, so as to differentiate it from the single major A1 nystatin.

According to the experimental process in Section 3.3, the chemical potency of the starting strain *Streptomyces noursei* D-3-14 was detected as 3464 U/mL, as shown in Table 1, and the biological potency was detected as 2703 U/mL, as shown in Table 2. **Table 1.** Chemical potency detection of *Streptomyces noursei* D-3-14. **Expt. No OD<sup>319</sup> Chemical Potency (U/mL) Average Chemical Potency (U/mL)** Due to the actinomyces metabolic diversity, in this research, we used an environmentally isolated strain of *Streptomyces noursei* D-3-14 to perform ultraviolet (UV) mutagenesis, optimized the culture conditions, and finally obtained a higher production of Nysfungin. Mutation breeding is a powerful technique, in which microbial strains exposed to mutagen treatment are screened, to identify positive mutants with specific characteristics. This method has the advantages of simplicity, rapidity, and high efficiency [17]. UV is possibly one of the physical mutagens that causes genetic variation and enables the selection of traits as needed. UV has a strong genotoxic effect, producing DNA damage that leads to an altered DNA structure [18]. UV irradiation induces covalent crosslinks between neighboring pyrimidines. If left unrepaired, error-prone replication of this damaged DNA leads to an increased rate of mutagenesis and genome instability [19]. The aim of this study was to screen and characterize nystatin-producing *Streptomyces noursei* strains exposed to UV, to identify the strain that produced the highest activity of nystatin, thereby obtaining a high-activity and low-cost raw material for commercial nystatin production.

#### **2. Results and Discussion**

#### *2.1. Determination of the Chemical and Biological Potency of the Starting Strain Streptomyces noursei D-3-14*

According to the experimental process in Section 3.3, the chemical potency of the starting strain *Streptomyces noursei* D-3-14 was detected as 3464 U/mL, as shown in Table 1, and the biological potency was detected as 2703 U/mL, as shown in Table 2.


**Table 1.** Chemical potency detection of *Streptomyces noursei* D-3-14.


**Table 2.** Biological potency detection of *Streptomyces noursei* D-3-14. **Diameter (mm) Diameter (mm) Inhibition Inhibition** 

**Table 2.** Biological potency detection of *Streptomyces noursei* D-3-14.

*Catalysts* **2023**, *13*, x FOR PEER REVIEW 3 of 12

2 0.249 3116 3464

1 0.295 3709

3 0.284 3567

**Inhibition** 

**Inhibition** 

#### *2.2. HPLC Analysis of the Starting Strain Streptomyces noursei D-3-14* was similar to the nysfungin reference, as seen in Figure 2.

According to the experimental process in Section 3.3, the HPLC analysis of D-3-14 was similar to the nysfungin reference, as seen in Figure 2.

According to the experimental process in Section 3.3, the HPLC analysis of D-3-14

**Average** 

**Figure 2.** HPLC analysis of the fermentation products of *Streptomyces noursei* D-3-14 and the refer-**Figure 2.** HPLC analysis of the fermentation products of *Streptomyces noursei* D-3-14 and the reference.

#### ence. *2.3. Determination of the UV Irradiation Duration of Mutagenesis*

*2.3. Determination of the UV Irradiation Duration of Mutagenesis* The lethal rate should increase with UV radiation duration. According to our experience, a 90% lethal rate is acceptable for further research. Figure 3 shows that the UV radi-The lethal rate should increase with UV radiation duration. According to our experience, a 90% lethal rate is acceptable for further research. Figure 3 shows that the UV radiation duration was set to 40 s. *Catalysts* **2023**, *13*, x FOR PEER REVIEW 4 of 12

**Figure 3.** Lethality curve for UV irradiation duration. **Figure 3.** Lethality curve for UV irradiation duration.

mutagenesis.

UV mutagenesis.

*2.4. Preliminary and Secondary Screening for Three Consecutive Rounds 2.4. Preliminary and Secondary Screening for Three Consecutive Rounds*

ical and biological potency of the starting strain, as seen in Table 3.

**Strain No. Chemical Potency (U/mL) Biological Potency (U/mL) Polyfugin B Content (%)**

chemical and biological potency of *Streptomyces noursei* 74-14-8 (Table 4).

**Strain No. Chemical Potency (U/mL) Biological Potency (U/mL) Polyfugin B Content (%)**

72-22-1 8912 5557 53.63 72-22-3 5328 3467 47.64 72-22-5 7214 5679 31.61 72-22-10 5912 3578 44.72 72-22-11 6879 4344 35.11 72-22-14 6020 4684 36.79 72-22-19 5829 3689 32.77 72-22-20 5741 4121 33.04 72-22-49 5311 3877 28.64

According to the experimental process in Section 3.4, three consecutive rounds of preliminary and secondary screening were performed following the UV mutagenesis. According to the experimental process in Section 3.4, three consecutive rounds of preliminary and secondary screening were performed following the UV mutagenesis.

**Table 3.** Biological potency and Polyfungin B contents of various strains after the first round of UV

In the second round of screening, 22 out of 72 preliminary screened mutants were selected. The resultant *Streptomyces noursei* 72-22-1 was identified as having a chemical and biological potency of 8912 U/mL and 5557 U/mL, exhibiting 1.54 and 1.20 times the

**Table 4.** Biological potency and Polyfungin B contents of various strains after the second round of

In the first round of preliminary screening, 74 mutants were obtained. Then 14 out of these 74 mutants were selected for the secondary screen. In this round, *Streptomyces nour-*

74-14-8 5783 4626 33.63 74-14-61 5177 3467 27.64 74-14-67 5101 3825 21.61

In the first round of preliminary screening, 74 mutants were obtained. Then 14 out of these 74 mutants were selected for the secondary screen. In this round, *Streptomyces noursei* 74-14-8 was identified as the resultant strain, with a chemical potency of 5783 U/mL and biological potency of 4626 U/mL, which exhibited about 1.67 and 1.71 times the chemical and biological potency of the starting strain, as seen in Table 3.

**Table 3.** Biological potency and Polyfungin B contents of various strains after the first round of UV mutagenesis.


In the second round of screening, 22 out of 72 preliminary screened mutants were selected. The resultant *Streptomyces noursei* 72-22-1 was identified as having a chemical and biological potency of 8912 U/mL and 5557 U/mL, exhibiting 1.54 and 1.20 times the chemical and biological potency of *Streptomyces noursei* 74-14-8 (Table 4).

**Table 4.** Biological potency and Polyfungin B contents of various strains after the second round of UV mutagenesis.


In the third round of screening, 24 out of 112 preliminary screened mutants were selected. The resultant *Streptomyces noursei* 112-24-63 was identified as having a chemical and biological potency of 11097 U/mL and 10751 U/mL, which was 1.25 and 1.93 times the chemical and biological potency of *Streptomyces noursei* 72-22-1 (Table 5).

**Table 5.** Biological potency and Polyfungin B contents of various strains after the third round of UV mutagenesis.


#### *2.5. Genetic Stability Experiment*

According to the experimental process in 3.5, genetic stability experiments were performed on *Streptomyces noursei* 72-22-1 and 112-24-63. The results can be seen in Figure 4.

**Figure 4.** Genetic stability experiment results for *Streptomyces noursei* 72-22-1 and 112-24-63. **Figure 4.** Genetic stability experiment results for *Streptomyces noursei* 72-22-1 and 112-24-63.

#### *2.6. Single-Factor Evaluation for the Nysfungin Fermentation 2.6. Single-Factor Evaluation for the Nysfungin Fermentation*

Single factor glucose concentration, peanut meal concentration, starting pH, and inoculation volume ration were evaluated to generate the results in Figure 5. The results showed that when the content of glucose in the fermentation medium was 5.5%, the yield of nystatin was higher. A carbon source is one of the essential nutrients in a microbial culture medium, providing energy for the growth, reproduction, and metabolic activities of microorganisms. Our previous work found that during streptomycin fermentation, the glucose concentration in the fermentation broth must be controlled to be lower than a certain level in the later stage of fermentation. If the glucose concentration is higher than 10 mg/mL, the synthesis of mannosidase will be inhibited and the production of streptomycin will be significantly reduced. When the culture medium contains two or more carbon sources, microorganisms generally use glucose first and then the other carbon sources. These results indicate that secondary metabolites, such as antibiotics, are regulated by carbon metabolites, such as glucose. Single factor glucose concentration, peanut meal concentration, starting pH, and inoculation volume ration were evaluated to generate the results in Figure 5. The results showed that when the content of glucose in the fermentation medium was 5.5%, the yield of nystatin was higher. A carbon source is one of the essential nutrients in a microbial culture medium, providing energy for the growth, reproduction, and metabolic activities of microorganisms. Our previous work found that during streptomycin fermentation, the glucose concentration in the fermentation broth must be controlled to be lower than a certain level in the later stage of fermentation. If the glucose concentration is higher than 10 mg/mL, the synthesis of mannosidase will be inhibited and the production of streptomycin will be significantly reduced. When the culture medium contains two or more carbon sources, microorganisms generally use glucose first and then the other carbon sources. These results indicate that secondary metabolites, such as antibiotics, are regulated by carbon metabolites, such as glucose.

The nitrogen source not only plays a nutritional role in the fermentation of microorganisms, but also contains inducers, precursors, and other substances required for the synthesis of secondary metabolites. When there are multiple nitrogen sources in the fermentation medium, microorganisms always use the simple nitrogen sources first, and then decompose complex nitrogen sources. Moreover, when the concentration of these

**Figure 5.** Evaluation results for single factors of glucose concentration, peanut meal concentration, starting pH, and inoculation volume ratio for fermentation.

**Figure 5.** Evaluation results for single factors of glucose concentration, peanut meal concentration, starting pH, and inoculation volume ratio for fermentation. *2.7. HPLC Analysis for Streptomyces noursei 72-22-1* According to the experimental process in 3.3, the fermentation product in the HPLC analysis of 72-22-1 was similar to the nysfungin reference, as seen in Figure 6. The nitrogen source not only plays a nutritional role in the fermentation of microorganisms, but also contains inducers, precursors, and other substances required for the synthesis of secondary metabolites. When there are multiple nitrogen sources in the fermentation medium, microorganisms always use the simple nitrogen sources first, and then decompose complex nitrogen sources. Moreover, when the concentration of these simple nitrogen sources (such as ammonium ions, amino acids) is high, they synthesize few secondary metabolites. The results showed that when the content of peanut meal in the fermentation medium was 2.5%, the yield of nystatin was higher.

The experimental results showed that when the initial pH of the fermentation medium was 7.0, and the inoculation volume was 12%, the yield of nystatin fermentation was higher.

According to the above single-factor evaluation results, a four-factor and three-level L<sup>9</sup> (3<sup>4</sup> ) orthogonal experiment was designed, as seen in Table S1. Orthogonal experimental results and analysis (Table S2) showed that a glucose concentration of 5.5%, peanut melt concentration of 2.5%, initial pH of 7.5, and inoculation volume ratio of 14% were the optimal conditions. The final optimized chemical and biological potency results for *Streptomyces noursei* 72-22-1 were 14,082 U/mL and 10579 U/mL, which are 1.58 and 1.91 times those before optimization. **Figure 5.** Evaluation results for single factors of glucose concentration, peanut meal concentration, starting pH, and inoculation volume ratio for fermentation. *2.7. HPLC Analysis for Streptomyces noursei 72-22-1* According to the experimental process in 3.3, the fermentation product in the HPLC

#### **Figure 6.** HPLC analysis of the fermentation products of *Streptomyces noursei* 72-22-1. *2.7. HPLC Analysis for Streptomyces noursei 72-22-1* analysis of 72-22-1 was similar to the nysfungin reference, as seen in Figure 6.

It can be seen that in the mutant *Streptomyces noursei* 72-22-1, nystatin A1, A3, and polyfungin B exhibited retention times of 9.085 min, 12.288 min, and 16.599 min. The ratio According to the experimental process in 3.3, the fermentation product in the HPLC analysis of 72-22-1 was similar to the nysfungin reference, as seen in Figure 6.

using semi-preparative HPLC, and their fungicidal activity was evaluated according to the experimental process in 3.3. **Figure 6.** HPLC analysis of the fermentation products of *Streptomyces noursei* 72-22-1. **Figure 6.** HPLC analysis of the fermentation products of *Streptomyces noursei* 72-22-1.

of polyfungin B was about 53%, which is much higher than the reference.

The results are summarized in Table 6. It can be seen that polyfungin B displayed a better fungicidal activity than nystatin A1 and A3, which is a novel result in nysfungin activity research. It can be seen that in the mutant *Streptomyces noursei* 72-22-1, nystatin A1, A3, and polyfungin B exhibited retention times of 9.085 min, 12.288 min, and 16.599 min. The ratio It can be seen that in the mutant *Streptomyces noursei* 72-22-1, nystatin A1, A3, and polyfungin B exhibited retention times of 9.085 min, 12.288 min, and 16.599 min. The ratio of polyfungin B was about 53%, which is much higher than the reference.

**Table 6.** Results of nystatin A1, A3, and polyfungin B inhibiting *Saccharomyces cerevisiae* ATCC 2061.

*2.8. Fungicidal Activity of Nystatin A1, A3, and Polyfungin B towards Saccharomyces cerevisiae*

using semi-preparative HPLC, and their fungicidal activity was evaluated according to

**Table 6.** Results of nystatin A1, A3, and polyfungin B inhibiting *Saccharomyces cerevisiae* ATCC 2061.

**Inhibition Zone Diameter of Nystatin A3 (mm)**

**Inhibition Zone Diameter of Nystatin A3 (mm)**

The results are summarized in Table 6. It can be seen that polyfungin B displayed a better fungicidal activity than nystatin A1 and A3, which is a novel result in nysfungin

**Inhibition Zone Diameter of Polyfugin B (mm)**

**Inhibition Zone Diameter of Polyfugin B (mm)**

1 20.68 16.44 21.62 2 19.26 16.84 21.18

1 20.68 16.44 21.62 2 19.26 16.84 21.18

**Expt. No. Inhibition Zone Diameter of** 

activity research.

**Expt. No. Inhibition Zone Diameter of** 

*ATCC 2061*

**Nystatin A1 (mm)**

the experimental process in 3.3.

**Nystatin A1 (mm)**
