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Article

Improved Fermentation Yield of Doramectin from Streptomyces avermitilis N72 by Strain Selection and Glucose Supplementation Strategies

by
Xiaojun Pan
1,2,3,4 and
Jun Cai
1,2,3,4,*
1
Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei University of Technology, Wuhan 430068, China
2
Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan 430068, China
3
Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei University of Technology, Wuhan 430068, China
4
College of Bioengineering and Food, Hubei University of Technology, Wuhan 430068, China
*
Author to whom correspondence should be addressed.
Fermentation 2023, 9(2), 121; https://doi.org/10.3390/fermentation9020121
Submission received: 15 December 2022 / Revised: 22 January 2023 / Accepted: 23 January 2023 / Published: 26 January 2023
(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)
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Abstract

:
Doramectin is a macrolide antiparasitic that is widely used in the treatment of mammalian parasitic diseases. Doramectin is usually produced by Streptomyces avermitilis fermentation using cyclohexanecarboxylic acid (CHC) as a precursor; however, the growth of S. avermitilis is usually inhibited by CHC, resulting in a low fermentation yield of doramectin. In this study, a high-yielding strain XY-62 was obtained using the S. avermitilis mutant strain S. avermitilis N72 as the starting strain, then combined with a CHC tolerance screening strategy using ultraviolet and nitrosoguanidine mutagenesis, and a 96 microtiter plate solid-state fermentation primary sieving and shake flask fermentation rescreening method. Compared with S. avermitilis N72, the doramectin fermentation yield increased by more than 1.3 times, and it was more adaptable to temperature, pH, and CHC concentration of the culture; additionally, the viability of the mycelial growth was enhanced. In addition, further studies on the high-yielding strain XY-62 revealed that the accumulation of doramectin could be further increased by glucose supplementation during the fermentation process, and the yield of doramectin reached 1068 μg/mL by scaling up the culture in 50 L fermenters; this has the potential for industrial production. Therefore, mutagenesis combined with CHC tolerance screening is an effective way to enhance the fermentation production of doramectin by S. avermitilis. Our strategy and findings can help to improve the production of doramectin in industrial strains of S. avermitilis.

1. Introduction

Doramectin is a macrolide disaccharide produced by the fermentation of S. avermitilis mutant strains using CHC as a precursor, and is a potent avermectin-like drug [1,2,3]. Doramectin is an anthelmintic active against internal and external parasites in animals, especially nematodes and mites. Doramectin does not easily cross the blood–brain barrier, and is safe for the treatment of parasitic diseases in mammals as it causes minimal damage to the central nervous system. It is used for the treatment of parasites in horses, cattle, sheep, pigs, and dogs [4,5,6,7]. It has a wider antiparasitic range and higher efficacy than avermectin [8,9], ivermectin [10,11] and other avermectin-based drugs [12,13]. It has also been shown to have an inhibitory effect on tumor cells [14].
The functions of the avermectin biosynthetic pathway and its biosynthetic genes have been well documented [15,16,17,18]. The biosynthetic pathway of doramectin is similar to that of avermectin, except that the starting unit for the synthesis of the doramectin macrolide backbone is cyclohexanol coenzyme A, rather than methylbutanoyl coenzyme A and isobutyryl coenzyme A [2,19,20,21].
Doramectin is a targeted biosynthetic metabolite of a mutant strain of S. avermitilis, and is not a natural product. Doramectin is produced using CHC-CoA as a starter unit, which can be achieved by adding CHC to the fermentation of S. avermitilis or by introducing a CHC-CoA biosynthetic gene cassette (PAC12) [2]. Yields of strains using the introduction of CHC-CoA are extremely low, and production still uses CHC in addition to fermentation. However, the addition of CHC had a significant inhibitory effect on the growth of the strain. In actual production, CHC is added in small amounts and several times during the stable period of mycelial growth or by microflow addition to reduce the inhibition of strain growth; however, flow addition requires additional equipment and increases the risk of bacterial contamination. The addition of CHC at 0.2~0.4 g/L in doramectin fermentation studies is likely to cause strain mortality [2,21]. To reduce the impact of CHC on strain survival, screening for CHC-tolerant strains is of interest. In addition, the production of doramectin still faces many technical problems. One of the difficulties in the production of doramectin is the very high requirements of the strain, which requires not only a high yield of doramectin (CHC-B1), but also a low level of CHC-B2 impurities; this makes the isolation and purification of the product more difficult [20]. In addition, the relatively long production cycle of doramectin, of which the fermentation and incubation time is 12~18 days, leads to increased production costs, thus limiting its application.
Currently, mutant selection and precursor addition strategies are effective methods to improve the yield of avermectin [22,23,24,25]. In industrial production, CHC is the starting precursor substance for the biosynthesis of doramectin by S. avermitilis, but CHC has an inhibitory effect on the growth of the bacterium, thus affecting the yield of doramectin [21,26]. Therefore, screening for strains with a high tolerance to CHC and reducing their growth inhibition is an effective way to increase the yield of doramectin; however, few applications of this strategy have been reported in the literature. Alternatively, high throughput screening of strains using liquid fermentation in microtiter plates is commonly used as an efficient method [27,28]. The primary screening of high-yielding strains of doramectin by surface culture in microtiter plates is a new endeavor.
In this study, the doramectin-producing strain S. avermitilis N72 was screened after ultraviolet and nitrosoguanidine mutagenesis for CHC tolerance, and high-yielding strains were selected by surface culture primary sieving in 96 microtiter plates and rescreening in shake flasks; subsequently, the stability of the passages was investigated. Preliminary studies on the growth in the fermentation characteristics of the high-yielding strains were then carried out, and combined with studies of their metabolic characteristics for glucose supplementation experiments and fermenter culture implementation in a 50 L fermenter.

2. Materials and Methods

2.1. Microbial Strains, Culture Media and Culture Conditions

Strain: S. avermitilis N72 is a mutant strain of S. avermitilis ATCC 31267 with doramectin production capacity (300 μg/mL), selected and conserved by our laboratory and used as the starting strain in this study (labeled N72 in the diagram).
Medium: Solid medium G consisted of the following (g/L): soybean meal, 4; mannitol, 4; agar, 20; pH 7.0~7.2. Seed medium S consisted of (g/L): glucose, 5; maltodextrin, 20; soybean cake flour, 10; cottonseed cake flour, 10; pH 7.0~7.2. Fermentation medium F consisted of (g/L): soluble starch, 90; bean cake flour, 15; cottonseed flour, 15; yeast extract, 5; sodium chloride, 1; dipotassium hydrogen phosphate, 2.5; calcium carbonate, 7; magnesium sulfate, 5; CHC, 0.8; pH 7.0~7.2; for surface culture agar 20 g/L added to F. All media were autoclaved at 121 °C for 25 min.
Culture conditions: S. avermitilis was cultured on solid medium G for 7 days, and a spore suspension of 106~107 spores/mL was prepared in saline. The spore suspension was inoculated (1 mL) into seed medium S (30 mL in a 250 mL flask) and incubated for 48 h at 200 rpm in a shaking flask. Then, the inoculum (8%, v/v) was inoculated into fermentation medium F (40 mL in a 250 mL flask) in shaking flasks at 220 rpm for 12 days. The incubation temperature was 30 °C. Surface culture was carried out in 96 microtiter plates, and fermenter culture implementation was carried out in 10 L–50 L fermenter systems (EastBio, Zhenjiang, China).

2.2. Assessment of Strain CHC Tolerance and Mutagenic Lethality

2.2.1. CHC Tolerance Assessment

S. avermitilis N72 spore suspensions were inoculated on solid medium G plates containing different concentrations of CHC (0, 0.4, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 g/L) for 7 days (30 °C). Plates without CHC were used as a blank control, and the number of growing colonies was recorded to calculate the survival rate. The calculation formula is given as Equation 1 below.
S u r v i v a l   r a t e = 100 % × a b          
where a is the CFU of the CHC supplemented plate, and b is the CFU of the control plate.

2.2.2. Mutagenic Lethality

S. avermitilis N72 spore suspensions were mutagenized at a distance of 20 cm from a 15 W UV lamp at various times (0, 30, 60, 90, 120, 150, 180 s). For nitrosoguanidine (NTG) mutagenesis, S. avermitilis N72 spore suspensions were mutagenized by adding NTG (Macklin, Shanghai, China) to a concentration of 600 μg/mL, and NTG was mutagenized for various times (0,10, 20, 30, 40, 50, 60 min). For combined UV and NTG mutagenesis, S. avermitilis N72 spore suspensions were first treated with UV for different times (30 s, 60 s) and then mutagenized with NTG for different times (0, 10, 20, 30, 40, 50, 60 min). After the spore mutagenesis treatment, the spores were inoculated into solid medium G plates (100 μL per dish) and incubated in the dark for 7 days (30 °C). Spore suspension plates without mutagenesis were used as blank controls, and the number of growing colonies (CFU) was recorded to calculate the lethality [29]. The calculation formula is given in Equation 2 below.
L e t h a l i t y       r a t e = 100 % × 1 − a b      
where a is the CFU of the plate seeded with the mutagenized spores, and b is the CFU of the control plate.

2.3. Screening and Genetic Stability of High-Yielding Strains

2.3.1. Testing of the Analytical Method for Doramectin Content

Doramectin standards (Sigma-Aldrich, USA) were diluted to a range of concentrations (25, 50, 100, 200, 400 μg/mL) and analyzed (detection wavelength was 245 nm) by HPLC (LC-20AD, Shimadzu, Japan), and the standard curve plotted. The doramectin standards were diluted to a range of concentrations (25, 50,100,150, 200, and 250 μg/mL) and analyzed (detection wavelength was 245 nm) by ELISA (Synergy2, Gene Company Limited, USA) and the standard curve plotted. Twenty single colonies of mutant strains were randomly selected and inoculated in 96 microplates for surface culture (30 °C, 12 d), and the samples were analyzed by HPLC and ELISA, respectively. Sixteen single colonies of mutant strains were randomly selected and incubated in seed flasks (30 °C, 48 h), then inoculated in 96 microplates and fermentation flasks for surface culture (30 °C, 12 d) and fermentation flask culture (30 °C, 12 d); the samples were analyzed by HPLC.

2.3.2. 96-Well Plate Surface Culture Primary Sieve

Three different mutagenesis treatments of S. avermitilis N72 spore suspensions (106~107 spores/mL) were inoculated (100 μL per dish) onto CHC-tolerant plates (30 °C, 7 days), and the growing single colonies were inoculated simultaneously into 96 microplate A (G medium, 30 °C, 7 days) and 96 microplate B incubated at (F solid medium, 30 °C, 12 days). Microplate A (for growth and screening) and microplate B (for fermentation and detection) were inoculated with the same mutant strain at the same serial number of wells. Samples from microplate B were processed and analyzed for doramectin content by ELISA for preliminary screening to identify positive mutant strains (over 20% increase in doramectin production compared to control), and the corresponding strains in microplate A were further studied.

2.3.3. Shake Flask Fermentation Rescreening

Positive mutant strains, after initial screening, were passed through slant expansion, inoculated onto shake flask medium S, and cultured (30 °C, 48 h), and transferred to shake flask medium F (30 °C, 12 days). The fermentation broth was tested by HPLC for doramectin yield, and the strain with a high yield was selected.

2.3.4. Assessing the Genetic Stability of High-Yielding Strains

The high-yielding strains obtained in this study were passaged six times on solid medium G. The strains from each passaging were expanded in S seed flasks under the same conditions and then inoculated into F fermentation shake flasks for 12 days to detect doramectin production.

2.4. Comparison of the Differences in Growth and Fermentation Characteristics between S. avermitilis N72 and the High-Yielding Strain XY-62

2.4.1. Growth Characteristics of the Strains in Seed Shake Flasks

For the examination of fermentation temperature, XY-16 and S. avermitilis N72 seed solutions were inoculated into shake flasks of medium S and incubated at different temperatures (20, 25, 30, 35 and 40 °C) for 48 h. For the initial pH examination, XY-16 and S. avermitilis N72 seed solutions were inoculated into medium S with different initial pH values (6.0, 6.5, 7.0, 7.5and 8.0) and incubated at 30 °C for 48 h. For the CHC tolerance study, XY-16 and S. avermitilis N72 seed solutions were inoculated into medium S with different concentrations of CHC (0.6, 0.8, 1.0, 1.2, 1.4 g/L) and incubated at 30 °C for 48 h. At the end of the incubation, the seed solution PMV was tested and growth was analyzed.

2.4.2. Metabolic Characteristics of the Strains in Fermentation Shake Flasks

XY-16 and S. avermitilis N72 seed solutions were inoculated with fermentation medium F in shake flasks (30 °C) and assayed for total sugars, reducing sugars, PMV, and doramectin production at different time points of the culture (0, 48, 96, 144, 192, 240, 288 h). The fermentation metabolic characteristics of the strains were compared at different growth periods.

2.5. Supplementary Fermentation of High-Yielding Strain XY-62 with a 50 L Fermenter Scale-Up Culture

2.5.1. Shake Flask Fermentation with Glucose Supplementation

XY-62 inoculated fermentation shake flasks were supplemented with glucose (0, 0.5%, 1%, 1.5%, 2%, 2.5%, w/v) at different fermentation time points (0, 96, 144, 192, 240 h). A total of 12 days of fermentation incubation at 30 °C and assayed for doramectin production. The effect of glucose on doramectin yield was analyzed.

2.5.2. Scaled-Up Culture in 50 L Fermenters

XY-62 was expanded in seed bottles and inoculated into 10 L fermenters (seed medium S, filling volume 6 L, inoculum 5%) for 48 h. The seed solution was transferred to 50 L fermenters (fermentation medium F, filling volume 30 L, inoculum 8%) for incubation. The tank temperature was 30 °C, tank pressure 0.05 MPa, stirring speed 60~180 r/min, air flow rate 0.8~1.2 vvm, and the fermentation-dissolved oxygen value remained above 35% by controlling the aeration flow rate and stirring speed. The fermentation was supplemented with 1.5% glucose at 192 h. The culture cycle was 12 days, and samples were taken every 24 h. The fermentation broth was tested for pH, total sugars, reducing sugars, amino nitrogen, bacterial concentration, and doramectin production. The metabolic characteristics of the strains were analyzed at different growth periods.

2.6. Analysis Methods

Microscopy was used for mycelial observation and spore counting. Biomass was estimated as the mycelial volume (PMV) of 10 mL of culture medium by centrifugation at 5000 rpm for 5 min [30]. The pH was determined by an acidity meter. Reducing sugars and total sugars in the fermentation broth were determined by titration with Felling’s reagent [31]. The amino nitrogen content of the fermentation broth was determined by titration with formalin solution [32]. Surface culture samples were analyzed in 96-well microplates using ELISA. Then, 800 μL of methanol was added to each sample for 2 h. The supernatant was collected by centrifugation (8000 rpm, 10 min), and an aliquot (200 μL) of the supernatant was transferred to a 96 microplate and measured at 245 nm. Shake flask and fermenter samples were measured by HPLC; 1 mL of fermentation broth was mixed with 4 mL of methanol, and the cells were extracted by sonication for 2 h. The supernatant was then collected by centrifugation, and filtered through 0.22 μm; 10 μL was separated on a Waters C18 column. The elution was carried out at 35 °C with methanol/water (90:10, v/v) as the mobile phase at a flow rate of 1 mL/min for 25 min. The UV detection wavelength was 245 nm. The doramectin content in the samples was calculated from the doramectin standard curve.

2.7. Statistical Analysis

Experiments were performed in triplicate. The error lines in the graphs indicate the standard deviation of the three replicates of the corresponding experiments. All analyses were performed using SPSS software (version 26, IBM Inc., Armonk, NY, USA), and plots were processed using Origin software (version 2021b, OriginLab Corporation, Northampton, MA, USA).

3. Results

3.1. Assessment of Strain CHC Tolerance and Mutagenic Conditions

3.1.1. Effect of CHC Concentration on the Viability of S. avermitilis N72 Cells

The growth inhibition of CHC was significant, with cell mortality increasing with increasing CHC concentrations. Less than 50% and less than 4% survivals were detected when the concentration of CHC was increased to 1.2 and 1.8 g/L, respectively (Figure 1A). In the CHC resistance screening assay, a CHC concentration of 1.4~1.6 g/L was chosen as reasonable.

3.1.2. Effect of UV Light and NTG on the Viability of S. avermitilis N72 Cells

Cell mortality increased with increasing UV irradiation time. Less than 40% survival was detected when the spores were irradiated for 90 s, while cell mortality reached 96% after exposure to UV for 180 s (Figure 1B). A 600 μg/mL concentration of NTG affected the growth of the strain, and the mortality rate increased with increasing NTG mutagenesis time. The survival rate detected after 20 min of NTG mutagenesis was less than 50%, while the cell mortality rate detected after 50 min of NTG mutagenesis was 98% (Figure 1C). The combination of UV irradiation and NTG treatment increased the lethality of S. avermitilis. Cell mortality could reach 80%~90% after UV irradiation for 30~60 s and 600 μg/mL NTG mutagenesis for 20 min, while cell mortality could reach 100% after UV irradiation for 30 s and 600 μg/mL NTG mutagenesis for 30 min (Figure 1D). Some investigators have suggested that mutagenesis with a lethality of 80% to 90% is more effective (Wang et al., 2011 [21]; Cao et al., 2018 [27]). Considering the operability of the experiment, the UV mutagenesis time was therefore chosen to be 120~150 s, and the NTG mutagenesis time was chosen to be 30~40 min. The combination of two mutagens was chosen, to be followed by UV mutagenesis for 30~60 s and NTG mutagenesis for 20~30 min.

3.2. Selection and Breeding of High-Yielding Strains and Their Genetic Stability

3.2.1. Analysis of Doramectin Content and Feasibility of Fermentation Methods

The HPLC method for doramectin was reliable, with good linearity between concentration and peak area for doramectin standards over a fairly wide range (R2 = 0.9987) (Figure 2A). The analytical efficiency can be improved by using the microtiter-plate based ELISA method over a wide range (R2 = 0.9979); a good linear relationship exists between the concentration of doramectin standards and OD245 (Figure 2B). Extracts from 96 microtiter plates were selected for analysis by HPLC and showed a good correlation between OD245 and concentrations measured by HPLC (R2 = 0.9856) (Figure 2C). In addition, there was a good correlation between single-colony solid fermentation and shake flask fermentation (R2 = 0.9597) (Figure 2D). Therefore, the ELISA assay methods and HPLC-estimated concentration of doramectin are linearly correlated.

3.2.2. 96-Well Plate Primary Sieve for Surface Culture

The fermentation yield of doramectin was measured in 96-well plates by selecting 450 UV-mutagenized and CHC-tolerant single colonies. Fifteen strains displayed improved doramectin yields ranging from 120 to 180 μg/mL in surface culture. This corresponded to a 20~80% increase in doramectin yield compared to S. avermitilis N72 (Figure 3A). The fermentation yield of doramectin was measured also in 443 NTG-mutagenized 30 min CHC-tolerant single colonies, and they were fermented in 96-well plates. Ten strains with higher yields ranging from 120 to 190 μg/mL in surface culture were identified, and displayed a 20~90% increase in doramectin yield compared to S. avermitilis N72 (Figure 3B). The fermentation yield of doramectin was also measured by selecting 446 UV-mutagenized 30 s and NTG-mutagenized 20 min tolerant single colonies in 96-well plates. Twelve strains with higher yields had solid fermentation yields of 120~230 μg/mL compared to S. avermitilis N72. This corresponded to a 20~130% higher doramectin yield (Figure 3C).

3.2.3. Shake Flask Fermentation Rescreening and Genetic Stability of High-Yielding Strains

Thirty-seven positive mutant strains from the initial screening were seeded on slants and fermented in a shake flask (15 strains were UV mutagenized, 10 strains were NTG mutagenized and 12 strains were compound mutagenized) for 12 days, with S. avermitilis N72 fermentation as a control. The yield of strain XY-62 doramectin reached 700 μg/mL, representing a 1.3-fold increase compared to S. avermitilis N72 (Figure 3D). The morphologies of XY-62 and S. avermitilis N72 differed slightly in solid medium G, with XY-62 colonies being more distinctly raised, and there was no significant difference in growth patterns between the two in liquid medium S (Figure 3E). The ability of XY-62 to produce doramectin is relatively genetically stable, as indicated by the fact that there was no significant change in doramectin production when the high-yielding strain XY-62 was passed through six consecutive generations (Figure 3F).

3.3. Comparison of the Differences in Growth and Fermentation Characteristics between the High-Yield Strains XY-62 and S. avermitilis N72

3.3.1. Growth Characteristics of the Strain

S. avermitilis N72 growth on seed medium S (Figure 4A) was characterized by a rapid increase in PMV from 0 to 48 h. After a peak in PMV between 48 and 72 h, it gradually decreased, and the growth of the bacterium was seen to slow down; the bacterium gradually declined as the incubation time increased. In comparison, the PMV of XY-62 was significantly higher than that of S. avermitilis N72 when cultured on seed medium S. The effect of different temperatures on the growth of mutant strains XY-62 and S. avermitilis N72 was similar (Figure 4B). This was characterized by incubation at 20~40 °C, with a gradual increase in PMV with temperature reaching a maximum at 30 °C, and a gradual decrease with increasing temperature. Comparing XY-62 and S. avermitilis N72, the growth activity of the mutant strain XY-62 was significantly higher than that of S. avermitilis N72 at 30~40 °C. XY-62 has better adaptability to culture temperature. The effect of pH on the growth of mutant strains XY-62 and S. avermitilis N72 was similar (Figure 4C). pH values of 6.5~7.5 showed relatively stable growth. Beyond this range, the growth of the strains was reduced, but the growth of XY-62 was less inhibited. Therefore, the mutant strain XY-62 was more stable in its adaptation to environmental pH. The growth of mutant strains XY-62 and S. avermitilis N72 was affected by different concentrations of CHC (Figure 4D). At CHC concentrations above 0.8 g/L, S. avermitilis N72 growth was reduced, while XY-62 was able to grow at 1.2 g/L CHC and above, and S. avermitilis N72 was barely able to grow. This showed that XY-62 was more tolerant of CHC.

3.3.2. Metabolic Characteristics of the Strains

The growth metabolism curves of XY-62 and S. avermitilis N72 in fermentation medium F were similar (Figure 4E). From 0 to 144 h, PMV increased rapidly, and the total and reducing sugar contents decreased, implying rapid growth of the organism and accelerated sugar utilization. Production of doramectin started at 96 h, followed by a rapid increase in yield, which slowed down at 240~288 h. Compared to S. avermitilis N72, the biomass and sugar consumption of XY-62 were higher, the viability of the organism was stronger and the production capacity for doramectin was greater.

3.4. Supplementary Fermentation of High-Yielding Strain XY-62 and Scale-Up Culture in 50 L Fermenters

Based on the metabolic characteristics of XY-62 fermentation, sugar consumption was enhanced, and the amount of reducing sugars was low in the late stage, so glucose was supplemented. The supplementation of glucose promoted the accumulation of doramectin (Figure 5A), especially at the 192nd hour of fermentation when supplemented with 1.5% glucose. The doramectin yield reached 810 μg/mL, which was 14.8% and 10.2% higher than that of the control group without glucose supplementation and with 1.5% glucose added to the base medium, respectively.
The metabolic characteristics of the growth of the bacterium in the 50 L fermenter magnified culture (Figure 5B,C) were roughly divided into three stages: the early stage (0~48 h), the middle stage (48~192 h) and the late stage (192~288 h). In the early stage, PMV increased, the growth of the bacterium was faster, total sugar and reducing sugar consumption was rapid, amino nitrogen decreased, pH decreased, DO decreased, and doramectin was not produced. In the middle stage, PMV reached a maximum, total sugar and reduced sugar consumption accelerated, the pH increased to 7.0, and the bacterium started to produce a large amount of doramectin. At the later stage, the PMV decreased, the growth of the bacteria gradually slowed down, the consumption of total sugars and reducing sugars decreased, the pH increased, the DO increased, and the increase in doramectin slowed down. The yield of doramectin reached 1068 μg/mL, representing an increase of 30% compared with shake flask fermentation.

4. Discussion

We report a method for screening high-yielding strains of doramectin. Mutagenesis is a cost-effective method for the creation of high-yielding strains, Song et al. studied a genetic mutant Streptomyces avermitilis S-233 with high-avermectins B1a by comobtained treatment with carbon heavy ion irradiation and sodium nitrite, and Xu et al. obtained a strain Streptomyces viridochromogenes F-23 with high-avilamycin production by combined mutagenesis with UV and ARTP [23,33]. The growth of the strains was inhibited by CHC, which is consistent with literature reports [21]. In addition, mutagenesis is a viable method for selecting strains with increased adaptability to the environment [34]. The significant increase in doramectin levels in the CHC-tolerant mutant strain in this study may be due to the enhanced stress of the strain to the precursor substance CHC. Mutagenic selection can further enhance the adaptability of the strain in CHC-containing media, resulting in a strain more suitable for industrial production.
Microorganisms have an enhanced capacity to consume nutrients and supplemental fermentation is an effective method. Supplementation experiments have shown that glucose has a positive effect on doramectin yield enhancement. From the biosynthetic pathway of doramectin [2,17], it is known that glucose is not only used as a carbon source to promote microbial growth but also as a raw material for the direct synthesis of the doramectin disaccharide side chain. In addition, the degradation of glucose to form malonyl-CoA and methyl malonyl-CoA can provide more precursors for doramectin biosynthesis and facilitate the formation of macrolides. Glucose supplementation promoted the accumulation of doramectin, especially in the middle and late stages of supplementation.
The growth and metabolism of S. avermitilis are aerobic, and compared to shake flask culture, fermenter culture can better regulate the fermentation process, especially the regulation of dissolved oxygen and replenishment operation. The dissolved oxygen level has a great influence on the production of secondary metabolites during the fermentation of S. avermitilis [33,35,36]. The 30% increase in fermentation yield in doramectin fermenters compared to shake flask fermentation may be caused by the better-dissolved oxygen conditions provided by the fermenters. In addition, the doramectin yield of strain XY-62 was relatively high compared to those reported in the reviewed literature for the same fermentation time [2,19,20,21].
In conclusion, our approach has been successful in enhancing the ability of S. avermitilis N72 to produce doramectin. This is mainly based on the selection and breeding of the CHC-resistant strain XY-62 to improve its survival in CHC-containing media and on the enhanced viability of the strain and enhanced sugar metabolism, especially glucose metabolism, which promotes the accumulation of doramectin. Based on this study, the fermentation process can be further optimized, and the mechanism of high doramectin production by mutant strain XY-62 can be investigated to better enhance the fermentation potential of high-yielding strain XY-62.

5. Conclusions

In this study, S. avermitilis N72 was treated for mutagenesis and CHC tolerance, and a high-yielding strain XY-62 was obtained using an efficient screening strategy of solid fermentation in 96-well microplates. Comparing the growth and metabolic characteristics of the strains before and after screening indicated that XY-62 was more suitable for industrial production. The strategy of adding 1.5% glucose in the middle and late stages of fermentation could further promote the accumulation of doramectin. It was also validated in a 50 L fermenter. In conclusion, the screening of CHC-tolerant strains and the glucose supplementation strategy are effective methods to significantly enhance the production of doramectin by S. avermitilis fermentation.

Author Contributions

X.P.: conceived and designed the study, performed the research, and formal analysis, and wrote the original draft. J.C.: conceived and designed the study, resources, supervision, writing—review, and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declared no conflict of interest.

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Figure 1. Assessment of CHC tolerance and the effect of mutagenic conditions on the viability of S. avermitilis N72. (A) The survival rate of S. avermitilis N72 at different concentrations of CHC, (B) lethality of S. avermitilis N72 by UV, (C) lethality of S. avermitilis N72 by NTG, (D) lethality of S. avermitilis N72 by the combined effect of UV and NTG.
Figure 1. Assessment of CHC tolerance and the effect of mutagenic conditions on the viability of S. avermitilis N72. (A) The survival rate of S. avermitilis N72 at different concentrations of CHC, (B) lethality of S. avermitilis N72 by UV, (C) lethality of S. avermitilis N72 by NTG, (D) lethality of S. avermitilis N72 by the combined effect of UV and NTG.
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Figure 2. Analysis of the correlation between doramectin ELISA assay and HPLC quantification. (A) doramectin HPLC standard curve, (B) doramectin ELISA standard curve, (C) correlation of HPLC assay with ELISA method, (D) correlation of single-colony microplate surface culture with shake flask fermentation.
Figure 2. Analysis of the correlation between doramectin ELISA assay and HPLC quantification. (A) doramectin HPLC standard curve, (B) doramectin ELISA standard curve, (C) correlation of HPLC assay with ELISA method, (D) correlation of single-colony microplate surface culture with shake flask fermentation.
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Figure 3. Stability of mutagenic selection and high-yielding strains of S. avermitilis N72. (A) single colonies of S. avermitilis N72 after UV mutagenesis and CHC-tolerance screening in a 96-microplate surface culture primary sieve, (B) single colonies of S. avermitilis N72 after NTG mutagenesis and CHC-tolerance screening in a 96-microplate surface culture primary sieve, (C) single colonies of S. avermitilis N72 after UV and NTG mutagenesis and CHC-tolerance screening in 96-microplate solid-state fermentation primary sieve, (D) shake flask rescreening of positive mutant strains (* represents p < 0.05), (E) growth pattern of S. avermitilis N72 and high-yielding strain XY-62 in solid medium G and seed medium S, (F) enetic stability of S. avermitilis N72 and the high-yielding strain XY-62.
Figure 3. Stability of mutagenic selection and high-yielding strains of S. avermitilis N72. (A) single colonies of S. avermitilis N72 after UV mutagenesis and CHC-tolerance screening in a 96-microplate surface culture primary sieve, (B) single colonies of S. avermitilis N72 after NTG mutagenesis and CHC-tolerance screening in a 96-microplate surface culture primary sieve, (C) single colonies of S. avermitilis N72 after UV and NTG mutagenesis and CHC-tolerance screening in 96-microplate solid-state fermentation primary sieve, (D) shake flask rescreening of positive mutant strains (* represents p < 0.05), (E) growth pattern of S. avermitilis N72 and high-yielding strain XY-62 in solid medium G and seed medium S, (F) enetic stability of S. avermitilis N72 and the high-yielding strain XY-62.
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Figure 4. Differences in growth and metabolism between S. avermitilis N72 and XY-62 shake flasks. (A) effect of different incubation times on biomass, (B) effect of different incubation temperatures on biomass, (C) effect of different pH values on biomass, (D) effect of different CHC concentrations on biomass, (E) fermentation shake flask growth and metabolism curves.
Figure 4. Differences in growth and metabolism between S. avermitilis N72 and XY-62 shake flasks. (A) effect of different incubation times on biomass, (B) effect of different incubation temperatures on biomass, (C) effect of different pH values on biomass, (D) effect of different CHC concentrations on biomass, (E) fermentation shake flask growth and metabolism curves.
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Figure 5. Fermentation of high-yielding strain XY-62 with increased glucose amount and culture in a 50 L fermenter. (A) effect of glucose supplementation on the yield of doramectin synthesis by XY-62 (* represents p < 0.05), (B) mycelial morphology of XY-62 at different growth periods in the 50 L fermenter, (C) growth and metabolic curves of XY-62 in 50 L fermenter.
Figure 5. Fermentation of high-yielding strain XY-62 with increased glucose amount and culture in a 50 L fermenter. (A) effect of glucose supplementation on the yield of doramectin synthesis by XY-62 (* represents p < 0.05), (B) mycelial morphology of XY-62 at different growth periods in the 50 L fermenter, (C) growth and metabolic curves of XY-62 in 50 L fermenter.
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Pan, X.; Cai, J. Improved Fermentation Yield of Doramectin from Streptomyces avermitilis N72 by Strain Selection and Glucose Supplementation Strategies. Fermentation 2023, 9, 121. https://doi.org/10.3390/fermentation9020121

AMA Style

Pan X, Cai J. Improved Fermentation Yield of Doramectin from Streptomyces avermitilis N72 by Strain Selection and Glucose Supplementation Strategies. Fermentation. 2023; 9(2):121. https://doi.org/10.3390/fermentation9020121

Chicago/Turabian Style

Pan, Xiaojun, and Jun Cai. 2023. "Improved Fermentation Yield of Doramectin from Streptomyces avermitilis N72 by Strain Selection and Glucose Supplementation Strategies" Fermentation 9, no. 2: 121. https://doi.org/10.3390/fermentation9020121

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