Method for improving rhodococcus erythropolis terpenoid production based on hydrogen peroxide adaptive evolution

Abstract

The application discloses a method for improving the ergothioneine yield of Rhodococcus based on hydrogen peroxide adaptive evolution, and a strain N1 can produce ergothioneine at 14.2 mg / L by utilizing glucose in a flask. The antioxidant capacity of the strain N1 can be further improved after adaptive evolution of hydrogen peroxide, and the ethereal Rhodococcus N1 can tolerate 582 mM hydrogen peroxide, and the ergothioneine yield is increased to 46.7 mg / L, which is the highest in non-recombinant strains.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a method for improving the yield of ergothionein in Rhodococcus based on adaptive evolution of hydrogen peroxide. Background Technology

[0002] Ergothioneine is a sulfur-containing amino acid derivative, first isolated in 1909 from the fungus Claviceps purpurea (a fungus that parasitizes the rye plant). It has strong antioxidant capacity and is a natural antioxidant. It also has anti-aging and anti-radiation activities, and plays a role in maintaining DNA synthesis, normal cell growth and metabolism, and various physiological functions. It is considered an essential functional active substance for maintaining human health.

[0003] Ergothioneine is widely used in the cosmetics industry due to its excellent antioxidant properties. On July 13, 2017, the European Commission authorized the market launch of L-ergothioneine as a new food ingredient. That same winter, the European Food Safety Authority (EFSA) issued a document recognizing the safety of synthetic L-ergothioneine as a new food resource for use in the diets of infants, pregnant women, and breastfeeding women. Currently, the main production method for ergothioneine is still plant extraction, and its low production capacity has resulted in high prices, severely hindering its market development and promotion.

[0004] Currently, the main methods for synthesizing ergothioneine include extraction, chemical synthesis, and biosynthesis. Extraction primarily involves separating and extracting ergothioneine from plants such as ergot and cereals. However, due to the low ergothioneine content in plants, this method produces ergothioneine with many impurities, high cost, and low yield, making large-scale industrial production difficult. Chemical synthesis of ergothioneine is costly and technically challenging, and it also poses certain safety risks. Biosynthesis, on the other hand, involves fermenting ergothioneine-producing microorganisms. Compared to extraction and chemical synthesis, biosynthesis offers advantages such as lower cost, readily available raw materials, and higher safety.

[0005] Rhodococcus belongs to the phylum Actinobacteria and is a Gram-positive bacterial strain. Currently, there are few reports on the production of ergothioneine using Rhodococcus fermentation. In this study, Rhodococcus ethereatus N1 was identified by HPLC as having the ability to produce ergothioneine, with an initial yield of 14.2 mg / L. To further increase the yield of the antioxidant ergothioneine, hydrogen peroxide adaptive evolution was employed to rapidly enhance the antioxidant capacity of strain N1, thereby increasing the yield of the antioxidant ergothioneine. The yield of ergothioneine reached 46.7 mg / L, verified by shake-flask and fermentation HPLC. Summary of the Invention

[0006] This invention aims to provide a method for improving ergothionein production in Rhodococcus based on adaptive evolution of hydrogen peroxide.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: A method for improving ergothionein production in Rhodococcus bacteria based on adaptive evolution of hydrogen peroxide includes the following steps: (1) Plate culture: Streaking of Rhodococcus ethereans onto LB solid medium and culture at 28-32℃ for 45-50 h; (2) Seed culture: Inoculate the colonies on the plate medium into LB seed medium and culture at a temperature of 28-32℃ for 28-32 h. (3) Adaptive evolution culture: Inoculate the seed culture solution into the adaptive evolution medium at an inoculation amount of 1~10% v / v; after growing for 24h~96h, add the adaptive evolution medium containing a higher concentration of H2O2 and continue to culture. (4) Fermentation culture: Add the culture after step 3) to the fermentation medium for culture at a temperature of 28-32℃ for 96-168 h to produce ergothioneine.

[0008] The Rhodococcus aetherivorans is Rhodococcus aetherivorans N1, with accession number CCTCC NO: M 20221270.

[0009] The adaptive evolution culture medium mentioned in step 3) is: glucose 30g / L, urea 0.5g / L, peptone 10g / L, yeast powder 5g / L, trace elements 1ml / L, hydrogen peroxide 0~8mM. More preferably, the initial hydrogen peroxide concentration is 3.5mM.

[0010] The adaptive evolution medium formula containing a higher concentration of H2O2 in step 3) is as follows: glucose 30g / L, urea 0.5g / L, peptone 10g / L, yeast powder 5g / L, trace elements 1ml / L, and hydrogen peroxide 10~600mM.

[0011] Step 4) The fermentation medium consists of 30 g / L glucose, 0.5 g / L urea, 10 g / L peptone, 5 g / L yeast extract, 1 ml / L trace elements, and 0-8 mM hydrogen peroxide.

[0012] Step 3) During adaptive evolution, the wavelength OD is detected using an ultraviolet spectrophotometer. 600 To determine the strain's resistance to hydrogen peroxide, the concentration of hydrogen peroxide in the adaptive evolution medium was gradually increased.

[0013] Step 3) Adaptive evolution: The initial hydrogen peroxide concentration is 0-8 mM, more preferably 3.5 mM. After 1-5 days of cultivation, the concentration is gradually increased from 5-50 mM to 50-600 mM.

[0014] The trace elements are: FeCl2·4H2O 1.5 g / L, CoCl2·6H2O 0.19 g / L, MnCl2·4H2O 0.1 g / L, ZnCl2 0.07 g / L, NiCl2·6H2O 0.024 g / L, Na2MO4·2H2O 0.036 g / L, and CuCl2·2H2O 0.002 g / L.

[0015] The strains used in this invention to produce ergothioneine include, but are not limited to, *Rhodococcus aetherivorans*. In one embodiment of this invention, the *Rhodococcus aetherivorans* strain used is classified as *Rhodococcus aetherivorans*, strain number N1, and has been deposited at the China Center for Type Culture Collection (CCTCC) on August 11, 2022, with accession number CCTCC NO: M20221270. The deposit address is Wuhan, China. This strain is a strain reported in the prior art (CN116855401A) that degrades various lignin-derived aromatic compounds. The inventors of this application have discovered that it can produce ergothioneine and have improved ergothioneine yield through adaptive evolution using hydrogen peroxide.

[0016] The Rhodococcus ethereans described in this invention is fermented and cultured under aerobic conditions.

[0017] The initial hydrogen peroxide evolution mechanism of Rhodococcus ethereinus described in this invention is as follows: (1) Plate culture: Streaking of Rhodococcus ethereans onto LB solid medium and cultured at 30℃ for 45-50 h; (2) Seed culture: Inoculate the colonies on the solid culture medium into LB seed culture medium and culture at a temperature of 28-32℃ for 28-32 h; (3) Evolutionary culture: The seed culture medium was inoculated into the adaptive evolutionary culture medium at an inoculation rate of 5% v / v. The adaptive evolution medium consisted of: glucose 30 g / L, urea 0.5 g / L, peptone 10 g / L, yeast extract 5 g / L, and trace elements 1 ml / L (FeCl2·4H2O 1.5 g / L, CoCl2·6H2O 0.19 g / L, MnCl2·4H2O 0.1 g / L, ZnCl2 0.07 g / L, NiCl2·6H2O 0.024 g / L, Na2MO4·2H2O 0.036 g / L, CuCl2·2H2O 0.002 g / L), with hydrogen peroxide 3.5 mM. OD was measured. 600 For concentrations greater than 10, the hydrogen peroxide concentration in the evolution medium was increased stepwise in increments of 10 mM, and OD was measured after each concentration increase. 600 If the concentration is greater than 10, the hydrogen peroxide concentration can be further increased to allow for adaptive evolution to tolerate hydrogen peroxide concentrations of 100-600 mM.

[0018] For example, the adaptive evolution medium was: glucose, 30 g / L, urea, 0.5 g / L, peptone, 10 g / L, yeast extract, 5 g / L, and trace elements 1 ml / L (FeCl2·4H2O 1.5 g / L, CoCl2·6H2O 0.19 g / L, MnCl2·4H2O 0.1 g / L, ZnCl2 0.07 g / L, NiCl2·6H2O 0.024 g / L, Na2MO4·2H2O 0.036 g / L, CuCl2·2H2O 0.002 g / L), with an initial hydrogen peroxide concentration of 3.5 mM. The OD600 was measured.

[0019] Furthermore, the fermentation broth of the adaptive evolution medium was inoculated into an adaptive evolution medium containing a higher concentration of hydrogen peroxide. This adaptive evolution medium contained the following components: glucose, 30 g / L; urea, 0.5 g / L; peptone, 10 g / L; yeast extract, 5 g / L; trace elements, 1 ml / L (FeCl2·4H2O 1.5 g / L, CoCl2·6H2O 0.19 g / L, MnCl2·4H2O 0.1 g / L, ZnCl2 0.07 g / L, NiCl2·6H2O 0.024 g / L, Na2MO4·2H2O 0.036 g / L, CuCl2·2H2O 0.002 g / L), and hydrogen peroxide, 10–100 mM. The OD600 was then measured.

[0020] The ergothionein detection method of this invention is as follows: Standard sample preparation: Prepare ergothioneine standards at concentrations of 0.8 mg / L, 1.6 mg / L, 3.2 mg / L, 16 mg / L, and 32 mg / L.

[0021] Sample preparation method: Take 1 ml of fermentation broth, 12000 rpm, 4 min, resuspend in 85% methanol solution, 40℃, shake for 30 min, 12000 rpm, 3 min, take the supernatant and pass it through a membrane for testing.

[0022] High performance liquid chromatography (HPLC) detection method: Column: C18; Mobile phase A: ultrapure water, Mobile phase B: acetonitrile, Elution ratio A:B=3:7; Column temperature: 30℃; Detection wavelength: 254nm; Injection volume: 5uL; Detection time: 20min per sample. Beneficial effects

[0023] The original Rhodococcus ethereatus N1 could produce ergothioneine at 14.2 mg / L through fermentation. Through multiple rounds of hydrogen peroxide-adaptive evolution, Rhodococcus ethereatus N1 eventually became tolerant to 582 mM hydrogen peroxide, achieving an ergothioneine yield of 46.7 mg / L. This yield represents the highest ergothioneine production using Rhodococcus ethereatus. Attached Figure Description

[0024] Figure 1 Rhodococcus strain N1 utilizes glucose for growth and ergothioneine production (left OD value, right yield). Figure 2 Initial hydrogen peroxide pressure screening of Rhodococcus strain N1; Figure 3 Rhodococcus strain N1 was cultured for 51 days and underwent adaptive evolution through 3.5mM~100mM hydrogen peroxide. Figure 4 Rhodococcus strain N1 underwent adaptive evolution from day 51 to day 105 using 100mM~134mM hydrogen peroxide. Figure 5 Growth of Rhodococcus strain N1 after 150 days of adaptive evolution; Figure 6 Diluted and plated with hydrogen peroxide-adapted evolutionary Rhodococcus strain N1 on plates; Figure 7 The Rhodococcus strain N1, which underwent hydrogen peroxide adaptive evolution, was inoculated into fermentation medium for fermentation and testing. Figure 8 Ergothioneine standard, HPLC chromatogram of sample. Implementation

[0025] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims. Example 1

[0026] The Rhodococcus aetherivorans N1 strain utilizes glucose to produce 14.2 mg / L of ergothioneine. Figure 1 A single colony of strain N1 was picked from the solid culture medium and inoculated into 5 mL LB liquid culture tubes. The culture was carried out at 30 °C and 180 r·min⁻¹ for 24 h. Then, it was inoculated into 100 mL LB medium at an inoculation rate of 2% v / v and incubated at 30 °C and 180 r·min⁻¹ for 24 h. Finally, it was inoculated into fermentation medium at 5% v / v and fermented for 144 h.

[0027] The fermentation medium was as follows: glucose, 30 g / L; urea, 0.5 g / L; corn steep liquor powder, 2.5 g / L; and trace elements, 1 ml / L (FeCl₂·4H₂O 1.5 g / L, CoCl₂·6H₂O 0.19 g / L, MnCl₂·4H₂O 0.1 g / L, ZnCl₂ 0.07 g / L, NiCl₂·6H₂O 0.024 g / L, Na₂MO₄·2H₂O 0.036 g / L, CuCl₂·2H₂O 0.002 g / L). Figure 1 As shown: Cell growth OD 600 The concentration can reach 38.7, and the accumulated ergothioneine can reach 14.2 mg / L. Example 2

[0028] Hydrogen peroxide adaptive evolution was performed on Rhodococcus aetherivorans N1 strain, with 0-8 mM hydrogen peroxide as the initial selection pressure. Figure 2 A single colony of strain N1 was picked from solid culture medium and inoculated into 5 mL LB liquid culture tubes. The culture was incubated at 30 °C and 180 r·min⁻¹ for 24 h. Then, a 2% v / v inoculation was carried out into 100 mL LB medium and incubated at 30 °C and 180 r·min⁻¹ for 24 h. Finally, a 5% v / v inoculation was carried out into adaptive evolution medium for fermentation for 72 h. OD₂ was then measured. 600 .

[0029] The above-mentioned adaptive evolution culture medium consisted of: glucose 30 g / L, urea 0.5 g / L, peptone 10 g / L, yeast extract 5 g / L, trace elements 1 ml / L (FeCl2·4H2O 1.5 g / L, CoCl2·6H2O 0.19 g / L, MnCl2·4H2O 0.1 g / L, ZnCl2 0.07 g / L, NiCl2·6H2O 0.024 g / L, Na2MO4·2H2O 0.036 g / L, CuCl2·2H2O 0.002 g / L), and hydrogen peroxide 0–8 mM. Figure 2 As shown, 3.5 mM hydrogen peroxide was used as the initial concentration. Example 3

[0030] Rhodococcus aetherivorans N1 strain grows under higher concentrations of hydrogen peroxide stress ( Figure 3 ). A single colony of strain N1 was picked from solid culture medium and inoculated into 5 mL LB liquid culture tubes. The culture was incubated at 30 °C and 180 r·min⁻¹ for 24 h. Then, at a 2% v / v inoculation rate, it was transferred to 100 mL LB medium and incubated at 30 °C and 180 r·min⁻¹ for 24 h. Finally, at a 5% v / v inoculation rate, it was transferred to an adaptive evolution medium (glucose, 30 g / L, urea, 0.5 g / L, peptone, 10 g / L, yeast extract, 5 g / L, trace elements 1 mL / L (FeCl₂·4H₂O 1.5 g / L, CoCl₂·6H₂O 0.19 g / L, MnCl₂·4H₂O 0.1 g / L, ZnCl₂ 0.07 g / L, NiCl₂·6H₂O 0.024 g / L, Na₂MO₄·2H₂O 0.036 g / L, CuCl₂·2H₂O) Fermented in 0.002 g / L hydrogen peroxide (3.5 mM) for 120 h, 5 ml of the fermentation broth was inoculated at 5% v / v into an adaptive evolution medium containing a higher concentration of hydrogen peroxide, and the OD was measured. 600， The concentration of hydrogen peroxide in the evolution medium was gradually increased in increments of 10 mM, and OD was measured after each concentration increase. 600 They continued to increase the hydrogen peroxide concentration, thereby adaptively evolving to be able to tolerate a concentration of 100 mM hydrogen peroxide.

[0031] The adaptive evolution medium for higher concentrations of hydrogen peroxide mentioned above is as follows: glucose, 30 g / L; urea, 0.5 g / L; peptone, 10 g / L; yeast extract, 5 g / L; trace elements 1 ml / L (FeCl2·4H2O 1.5 g / L, CoCl2·6H2O 0.19 g / L, MnCl2·4H2O 0.1 g / L, ZnCl2 0.07 g / L, NiCl2·6H2O 0.024 g / L, Na2MO4·2H2O 0.036 g / L, CuCl2·2H2O 0.002 g / L), hydrogen peroxide 3.5~100 mM. Figure 3 As shown, after 51 days of adaptive evolution, strain N1 can ferment and grow in an adaptive evolution medium containing 100 mM hydrogen peroxide, with an OD of 600 It can reach 23.55. Example 4

[0032] Example 3: Rhodococcus aetherivorans N1 strain tolerant to 100mM hydrogen peroxide was cultured at a higher concentration. Figure 4 In Example 3, the culture medium was mixed with adaptive evolution medium and inoculated into adaptive evolution medium containing 100 mM to 134 mM hydrogen peroxide. The medium was incubated at 30 °C and 180 r·min⁻¹ for 120 h. On day 51, the hydrogen peroxide concentration in the evolution medium was gradually increased in increments of 8 mM. OD was measured after each concentration increase. 600 Continue to increase the hydrogen peroxide concentration. The above-mentioned adaptive evolution medium consisted of: glucose, 30 g / L; urea, 0.5 g / L; peptone, 10 g / L; yeast extract, 5 g / L; trace elements 1 ml / L (FeCl2·4H2O 1.5 g / L, CoCl2·6H2O 0.19 g / L, MnCl2·4H2O 0.1 g / L, ZnCl2 0.07 g / L, NiCl2·6H2O 0.024 g / L, Na2MO4·2H2O 0.036 g / L, CuCl2·2H2O 0.002 g / L), and hydrogen peroxide 100 mM~134 mM. Figure 4 As shown, after 105 days of cultivation, strain N1 can ferment and grow in an adaptive evolution medium containing 134 mM hydrogen peroxide, with an OD600 of up to 20.45. Example 5

[0033] The adaptively evolved Rhodococcus aetherivorans N1 strain grew under higher concentrations of hydrogen peroxide stress ( Figure 55 ml of fermentation broth was taken from an adaptive evolution medium containing 134 mM hydrogen peroxide and inoculated at 5% v / v into an adaptive evolution medium containing a higher concentration of hydrogen peroxide. OD was then measured. 600 The concentration of hydrogen peroxide in the evolution medium was gradually increased in increments of 10 mM, and OD was measured after each increase in concentration. 600 Continue to increase the hydrogen peroxide concentration. The above-mentioned adaptive evolution culture medium consisted of: glucose, 30 g / L; urea, 0.5 g / L; peptone, 10 g / L; yeast extract, 5 g / L; trace elements 1 ml / L (FeCl2·4H2O 1.5 g / L, CoCl2·6H2O 0.19 g / L, MnCl2·4H2O 0.1 g / L, ZnCl2 0.07 g / L, NiCl2·6H2O 0.024 g / L, Na2MO4·2H2O 0.036 g / L, CuCl2·2H2O 0.002 g / L), and hydrogen peroxide 134 mM~582 mM. Figure 5 As shown, after 150 days of cultivation, the adaptive evolutionary Rhodococcus strain can grow in an adaptive evolutionary medium containing 582 mM hydrogen peroxide, with an OD600 of 25.62. Example 6

[0034] The adaptively evolved Rhodococcus aetherivorans N1 strain was fermented in an adaptive evolution medium containing 582 mM hydrogen peroxide for 120 h. 1 ml of the fermentation broth was then plated. Figure 6 ) like Figure 6 As shown, there were multiple single colonies on the plate. Six single colonies were randomly selected and inoculated into 5 mL LB liquid culture tubes. The culture was incubated at 30 °C and 180 r·min⁻¹ for 24 h. Then, at an inoculation rate of 2% v / v, the culture was transferred to 100 mL LB medium and incubated at 30 °C and 180 r·min⁻¹ for 24 h. Finally, the culture was inoculated into fermentation medium at 5% v / v and fermented for 144 h. Figure 7 ).

[0035] The fermentation medium was as follows: glucose, 30 g / L; urea, 0.5 g / L; corn steep liquor powder, 2.5 g / L; and trace elements, 1 ml / L (FeCl₂·4H₂O 1.5 g / L, CoCl₂·6H₂O 0.19 g / L, MnCl₂·4H₂O 0.1 g / L, ZnCl₂ 0.07 g / L, NiCl₂·6H₂O 0.024 g / L, Na₂MO₄·2H₂O 0.036 g / L, CuCl₂·2H₂O 0.002 g / L). Figure 7As shown, compared with the newly evolved strains that have not undergone adaptation, the adaptive evolved strains have increased the yield of ergothionein accumulated in the fermentation medium, reaching a maximum of 46.7 mg / L.

Claims

1. A method for improving ergothioneine production in Rhodococcus based on hydrogen peroxide adaptive evolution, characterized in that, Includes the following steps: (1) Plate culture: Streaking of Rhodococcus ethereans onto LB solid medium and cultured at 28-32℃ for 45-50 h; (2) Seed culture: The colonies on the plate medium were inoculated into LB seed medium and cultured at a temperature of 28-32℃ for 28-32 h. (3) Adaptive evolution culture: Inoculate the seed culture solution into the adaptive evolution medium at an inoculation amount of 1~10% v / v; after growing for 24h~96h, add the adaptive evolution medium containing a higher concentration of H2O2 and continue to culture. (4) Fermentation culture: The culture after step 3) is added to the fermentation medium for culture at a temperature of 28-32℃ for 96-168 h to produce ergothionein; The Rhodococcus aetherivorans is Rhodococcus aetherivorans N1, with accession number: CCTCCNO: M 20221270; The adaptive evolution culture medium mentioned in step 3) is: glucose 30g / L, urea 0.5g / L, peptone 10g / L, yeast extract 5g / L, trace elements 1ml / L, hydrogen peroxide 3.5mM; The adaptive evolution medium formula containing a higher concentration of H2O2 in step 3) is as follows: glucose 30 g / L, urea 0.5 g / L, peptone 10 g / L, yeast extract 5 g / L, trace elements 1 ml / L, and hydrogen peroxide 10~600 mM; The initial hydrogen peroxide concentration for adaptive evolution was 3.5 mM, and after 1 to 5 days of cultivation, the concentration was gradually increased from 5 to 50 mM to 100 to 600 mM. Step 4) The fermentation medium consists of 30 g / L glucose, 0.5 g / L urea, 10 g / L peptone, 5 g / L yeast extract, 1 ml / L trace elements, and 0-8 mM hydrogen peroxide.

2. The method for improving ergothioneine production in Rhodococcus bacillus based on adaptive evolution of hydrogen peroxide according to claim 1, characterized in that, Step 3) During adaptive evolution, the wavelength OD is detected using an ultraviolet spectrophotometer. 600 To determine the strain's resistance to hydrogen peroxide, the concentration of hydrogen peroxide in the adaptive evolution medium was gradually increased.

3. The method for improving ergothioneine production by Rhodococcus bacillus based on hydrogen peroxide adaptive evolution according to claim 1, characterized in that, The trace elements are: FeCl2·4H2O 1.5 g / L, CoCl2·6H2O 0.19 g / L, MnCl2·4H2O 0.1 g / L, ZnCl2 0.07 g / L, NiCl2·6H2O 0.024 g / L, Na2MO4·2H2O 0.036 g / L, and CuCl2·2H2O 0.002 g / L.

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