CN113583881A - Penicillium strain for producing brefeldin A by fermentation and application thereof - Google Patents

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to an eupenicillium strain for producing brefeldin A by fermentation, and further discloses application of the eupenicillium strain for producing the brefeldin A by fermentation. According to the invention, a strain of Penicillium FIM-E-UN88-26(Penicillium brefeldianum) capable of producing brefeldin A with high yield is obtained by screening through a normal pressure room temperature plasma mutagenesis technology, the positive Penicillium FIM-E-UN88-26 can ferment brefeldin A with high yield, in a fermentation experiment, the titer of the brefeldin A produced by fermenting the positive Penicillium FIM-E-UN88-26 is as high as 2152 mu g/mL, the yield of the brefeldin A is greatly improved, the fermentation period is shortened to 72h, and the method is more suitable for industrial fermentation production.

Description

Penicillium strain for producing brefeldin A by fermentation and application thereof

Technical Field

The invention belongs to the technical field of microbial fermentation, and particularly relates to an eupenicillium strain for producing brefeldin A by fermentation, and further discloses application of the eupenicillium strain for producing the brefeldin A by fermentation.

Background

Brefeldin A (BFA for short) is a metabolite produced by fungi, has strong biological activities of resisting fungi, viruses, tumors, nematodes and the like, has great potential medicinal value, and can be used as a cytokine secretion inhibitor for development and application. BFAs are reported to have major functions of changing golgi and trans-golgi network structures, disrupting mass transport between endosomes and lysosomes, thereby affecting protein secretion and synthesis, and thus, have been widely used as an important molecular tool for the study of mammalian signaling pathways. Currently, BFA has attracted the interest of synthetic chemists because of its diverse biological activity and unique chemical structure, but because it also has multiple chiral centers, the chemical synthesis process is more complicated and the yield is lower; since many fungi can produce BFA, microbial fermentation becomes an effective way to produce BFA and is receiving wide attention. At present, much work is done in the biological activity aspect of BFA abroad, but separated and screened BFA producing strains have low or unstable fermentation level or long fermentation period, and have no industrial value.

At present, the breeding technology aiming at the improvement of brefeldin A fermentation strains mainly adopts the traditional breeding method, namely, ultraviolet mutagenesis is combined with breeding means such as different mutagens, and the like, so that a certain effect is achieved. For example, in previous researches, the applicant carries out UV + NTG composite mutagenesis screening on n-penicillium E-0506 suitable for fermenting brefeldin A, and obtains a mutagenic strain E-UN88, wherein the genetic character of the mutagenic strain is relatively stable, the fermentation level is 10.36 times higher than that of an original strain, but the fermentation level is not ideal. For another example, chinese patent CN101445784B discloses a penicillium breve variety ZJB082702 obtained by mutagenesis using low energy ion implantation technology, which is capable of producing BFA up to 943.8mg/L by fermentation; and the Wangzaiarmy et al in 2012 further optimizes the fermentation conditions of the strain, and the final fermentation level reaches 1304.7 mg/L. For another example, Chinese patent CN104877910B discloses that the yield of BFA of Penicillium bracteatum F4a in plant endophytic fungi reaches 1.9g/L, but the fermentation period is as long as 13 d.

The normal pressure room temperature plasma breeding technology (ARTP) is a microorganism genome rapid mutation technology, the generated plasma is rich in various chemical active particles, and has multiple effects of generating genetic substance damage to bacterial strain cells, causing cell membrane permeability and protein structure change, etc., the cells start an SOS repair mechanism, and generate various mismatching sites in the repair process.

In view of the above, obtaining a strain which is suitable for industrial production and can efficiently produce brefeldin a is urgently expected by practitioners and has positive significance.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide an n-penicillium strain for producing brefeldin A by fermentation, so as to solve the problem that the fermentation strain of the brefeldin A in the prior art is not ideal in performance;

the second technical problem to be solved by the invention is to provide the application of the eupenicillium strain in fermentation production of brefeldin A.

In order to solve the technical problems, the Penicillium notatum strain is classified and named as Penicillium brefeldianum FIM-E-UN88-26, is preserved in the Guangdong province microorganism strain preservation center, is preserved in the experimental building 5 of the institute of Hospital, Dazhou 100, Mineli, Zuixian, Guangzhou, Guangdong province, has the preservation number of GDMCC No.61807 and the preservation date of 2021, 7 and 14 days.

The invention also discloses application of the eupenicillium strain in fermentation production of brefeldin A.

The invention also discloses a method for producing brefeldin A by fermentation, which comprises the step of inoculating the eupenicillium strain into a suitable fermentation culture medium for fermentation culture.

Specifically, the fermentation medium comprises the following components in mass percentThe components of the amount: 2-4 wt% of soluble starch, 1-3 wt% of soybean flour, 1-3 wt% of glucose, 0.1-0.5 wt% of peptone, 0.2-1.0 wt% of yeast extract and KH₂PO₄ 0.05-0.8wt％、MgSO₄.7H₂0.01-0.12 wt% of O, 0.15-0.15 wt% of Tween 800.02, 0.05-0.1 wt% of calcium carbonate and 6.0-7.2 of pH value.

Preferably, the fermentation medium comprises the following components in percentage by mass: 3 wt% of soluble starch, 2 wt% of soybean meal, 2 wt% of glucose, 0.2 wt% of peptone, 0.5 wt% of yeast extract and KH₂PO₄0.2wt％、MgSO₄.7H₂0.04 wt% of O, 0.1 wt% of Tween 800.05, 0.1 wt% of calcium carbonate and pH 6.5.

Specifically, the conditions of the fermentation culture step comprise: the rotation speed is controlled to be 100 and 250rpm, and fermentation culture is carried out for 68-120h at the temperature of 24-30 ℃.

Specifically, the method for producing brefeldin A by fermentation further comprises the step of inoculating the eupenicillium strain into a seed culture medium for seed liquid culture;

the seed culture medium comprises the following components in percentage by mass: 2-4 wt% of soluble starch, 1-3 wt% of soybean flour, 1-3 wt% of glucose, 0.1-0.5 wt% of peptone, 0.2-1.0 wt% of yeast extract and KH₂PO₄ 0.1-0.8wt％、MgSO₄.7H₂0.01-0.12 wt% of O, 0.15-0.78 wt% of Tween 800.02, and pH 6.2-7.0.

Preferably, the seed culture medium comprises the following components in percentage by mass: 3 wt% of soluble starch, 2 wt% of soybean meal, 2 wt% of glucose, 0.2 wt% of peptone, 0.5 wt% of yeast extract and KH₂PO₄ 0.2wt％、MgSO₄.7H₂O0.04 wt%, Tween 800.05 wt%, pH 6.5.

Specifically, the conditions of the seed liquid culture step include: the seed liquid culture is carried out for 32-48h at 24-30 ℃ by controlling the rotation speed of 100 and 250 rpm.

Specifically, the method for producing brefeldin A by fermentation further comprises the step of inoculating the penicillium eupenicillium strain into a slant culture medium for activation;

the slant culture medium comprises the following components in percentage by mass: 15-25 wt% of potato, 1-3 wt% of glucose and 2.0 wt% of agar, and the pH value is natural.

Preferably, the slant culture medium is a PDA culture medium, and comprises the following components in percentage by mass: 20 wt% of potato, 2 wt% of glucose and 2.0 wt% of agar, and the pH is natural.

Specifically, the conditions of the slant culture medium activation step include: culturing at 24-30 deg.C for 8-12 days.

The Penicillium FIM-E-UN88-26(Penicillium brefeldianum) with high brefeldin A yield is obtained by screening through a normal-pressure room-temperature plasma mutagenesis technology, and can ferment the brefeldin A with high yield. In a fermentation experiment, the titer of the brefeldin A produced by fermenting the penicillium notatum FIM-E-UN88-26 is as high as 2152 mu g/mL, the yield of the brefeldin A is greatly improved, the fermentation period is shortened to 72h, and the method is more suitable for industrial fermentation production; the screened strain FIM-E-UN88-26 has good stability, the titer of the brefeldin A produced by the strain is basically stable after five generations of continuous passage, the strain is maintained at the same higher level, and the strain can be used as a production strain for further research and development.

Drawings

In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,

FIG. 1 is a graph of ARTP efflux time versus lethality in mutagenesis experiments;

FIG. 2A phylogenetic tree of the strain FIM-E-UN88-26 according to the invention.

Detailed Description

In the following examples of the present invention, the culture medium includes:

the separation plate culture medium and the slant culture medium comprise the following components in percentage by mass: adding potato 20%, glucose 2%, agar 2.0%, and distilled water in balance, adjusting pH to natural, and sterilizing with high pressure steam at 121 deg.C for 20 min;

the seed culture medium comprises the following components in percentage by mass: 3% of soluble starch, 2% of soybean meal, 2% of glucose and proteinPeptone 0.2%, yeast extract 0.5%, KH₂PO₄ 0.2％、MgSO₄.7H₂O0.04%, Tween 800.05%, pH6.5, and sterilizing with high pressure steam at 121 deg.C for 30 min;

the fermentation medium comprises the following components in percentage by mass: 3% of soluble starch, 2% of soybean meal, 2% of glucose, 0.2% of peptone, 0.5% of yeast extract and KH₂PO₄ 0.2％、MgSO₄.7H₂O0.04%, Tween 800.05%, calcium carbonate 0.1%, pH6.5, and autoclaving at 121 deg.C for 30 min.

In the following embodiments of the present invention, the detection of brefeldin a content is performed by a high performance liquid chromatography method: centrifuging 2mL of fermentation liquor, extracting for 2 times by using methanol with the same volume, combining supernate and extract, filtering by using an organic filter membrane to obtain primary fermentation extract, and taking 10 mu L of primary fermentation extract for HPLC detection. Chromatographic conditions are as follows: c18(4.6 mm. times.250 mm,5 μm) column, measuring wavelength 230nm, in methanol: water 57: 43(V/V) as a mobile phase, a flow rate of 1.0mL/min, a column temperature: at 30 ℃. And (3) taking a brefeldin A standard substance as a reference substance, calculating the titer according to the peak area of the sample/the peak area of the standard liquid, the concentration of the standard liquid and the dilution factor, and taking the average value of the fermentation titer for 3 times.

EXAMPLE 1 acquisition of the mutagenized Strain FIM-E-UN88-26

Taking an eupenicillium E-UN88 strain with relatively stable genetic character as an initial strain, transferring the strain to a slant culture medium, and culturing in a constant temperature incubator for 8-12d at 26 ℃; washing off spores on the slant culture medium with normal saline, scattering glass beads, filtering with warp cloth, and making into 10⁶Spore suspension per mL.

10 μ L of the spore suspension prepared above was pipetted by a pipette onto a circular iron plate with a diameter of 1cm, placed in a normal pressure room temperature plasma mutagenesis system with a working gas of helium, a power supply of 110W, and a working gas flow of 10L/min, and a treatment distance of 2mm, and treated for 10s, 15s, 30s, 40s, 50s, 60s, and 70s, respectively, and the treated spore suspension was subjected to gradient dilution and plating to prepare a mortality curve (as shown in fig. 1). As can be seen from FIG. 1, there is a clear dose-effect relationship between the mutagenic treatment dose and the lethality of the strain E-UN88, which gradually increases with the duration of the treatment.

Selecting irradiation time with three different lethal doses of 15s, 40s and 60s according to the lethality curve, and carrying out plasma mutagenesis on the spore suspension of the strain E-UN 88; and mixing the spore suspensions with different processing time in a test tube filled with normal saline, and uniformly mixing to obtain the mutagenized spore suspension for later use.

Performing gradient dilution on the obtained mutagenic spore suspension, and controlling the dilution degree to be 10 respectively^-1、10^-2、10^-3、10^-4、10^-5、10^-6 Choose 10^-4、10^-5、10^-6The spore suspension of three dilutions is coated on the separation plate culture medium respectively, and cultured for 8-12 days at 28 ℃ in the dark.

Transferring the single colony grown on each separation plate to a slant culture medium at 28 deg.C for 10 days, inoculating 0.5 cm/0.5 cm thallus Porphyrae to the seed culture medium, and culturing at 26 deg.C and 220r/min for 36-48 hr to obtain seed solution; inoculating the seed liquid into a fermentation culture medium with an inoculation amount of 5%, and culturing at 26 deg.C and 230r/min for 72-120h to obtain a fermentation liquid.

Taking a proper amount of the obtained fermentation liquor, adding methanol with the same volume for extraction for 2 times, combining the supernatant and the extract, filtering the mixture through an organic filter membrane to obtain a primary fermentation extract, measuring the yield of brefeldin A by using a high performance liquid chromatography, and determining that the product in the fermentation liquor has a correct structure.

By this method, the strain producing the largest amount of brefeldin A was selected, and for convenience of description, the selected strain was named as strain FIM-E-UN88-26, and strain FIM-E-UN88-26 was preserved in glycerol.

EXAMPLE 2 identification of the Strain FIM-E-UN88-26

Physiological and biochemical characteristic identification

Streaking the obtained strain FIM-E-UN88-26 on a separation culture medium plate, inserting a cover glass, culturing at 28 deg.C for 7-20d, and observing morphological characteristics and hyphae of single colony with optical microscope, transmission and scanning electron microscope.

The main morphological and physiological and biochemical characteristics of the obtained strain FIM-E-UN88-26 are as follows: the bacterial colony on the flat plate is round and white, the surface is villous, no soluble pigment is produced, the bacterial colony appears radioactive wrinkles after maturation, the center is slightly raised, the back surface is earthy yellow, observation under a mirror shows that conidiophores have transverse septa, the top does not have an expanded top sac, conidiophores at the top end form a string shape, and the conidiophores are approximately spherical. The strain FIM-E-UN88-26 is a high oxygen consumption bacterium, the optimal growth temperature is 24-30 ℃, and the optimal growth pH is 5.5-7.5; when the rotating speed of a shaking table is 210-280r/min and the culture lasts for 3-5d, the yield of the brefeldin A is highest, and dissolved oxygen has a remarkable effect on the production of the brefeldin A in the fermentation process.

Molecular biological identification

Sequencing the ITS sequence of the strain FIM-E-UN88-26, comparing the ITS sequence of the tested strain with the existing sequence in a GenBank database, and carrying out homology analysis; selecting a corresponding ITS gene sequence of a model strain on an LPSN (http:// www.bacterio.cict.fr) website, comparing the systematic evolution analysis by CLUSTAL-X software, carrying out the systematic evolution analysis on a generated comparison file by a MEGA software adjacency method, wherein the topological analysis is the result of 1000 repeated sampling; the ITS sequence analysis shows that the sequence homology of the strain FIM-E-UN88-26 and Penicillium brefeldianum (Penicillium brefeldianum) is 99.12%.

By combining the above morphological, physiological and biochemical characteristics and molecular biological identification, the strain FIM-E-UN88-26 is finally determined to be Penicillium (Penicillium brefeldianum) genus, which is classified and named as Penicillium (Penicillium brefeldianum) FIM-E-UN88-26, and is preserved in the Guangdong province collection center of microbial strains, and the address is No. 5 th of Hakkaido 100 college 59 in the Vietnam of Guangdong province, the preservation number is GDMCC No.61807, and the preservation date is 14 months 2021.

Example 3 genetic stability verification of Strain FIM-E-UN88-26

The screened high-yield brefeldin A strain FIM-E-UN88-26 was continuously cultured and passaged (F1, F2, F3, F4, F5 and F6), and fermentation titer was measured after 500mL shake flask fermentation, and the primary strain (F0) with good growth was used as a control, and the results are shown in Table 1.

TABLE 1 Effect of passages on the production of brefeldin A by the strain FIM-E-UN88-26

Strain algebra	F0	F1	F2	F3	F4	F5	F6
								Relative potency (%)	100	100.2	100.4	99.3	98.8	97.0	93.3

As can be seen from the above table 1, the five-generation fermentation level of the strain FIM-E-UN88-26 screened by the invention has no obvious influence, the titer of the brefeldin A produced by the strain FIM-E-UN88-26 is basically stable and is maintained at the same higher level, thereby showing that the strain FIM-E-UN88-26 has better genetic stability.

Example 4 fermentation of Brefeldin A by the Strain E-UN88

Activation of the Strain E-UN 88: the strain E-UN88 preserved by glycerol is transferred to a slant culture medium and cultured in a constant temperature incubator for 8-12 days at the culture temperature of 28 ℃.

E-UN88 seed liquid preparation: the single colony obtained by activating the strain E-UN88 was inoculated into a seed medium (50 mL in a 500mL triangular flask) and cultured at 26 ℃ for 36 hours at 250r/min to obtain a seed solution.

Fermentation culture: inoculating the prepared seed solution into a fermentation culture medium (100 mL of the fermentation culture medium is filled in a 500mL triangular flask) with the inoculation amount of 5% (v/v), fermenting and culturing for 96h under the conditions of 26 ℃ and 250r/min, and detecting the obtained fermentation liquid.

The detection result shows that the yield of brefeldin A in three batches of shake flask fermentation is 751 mu g/mL, 733 mu g/mL and 710 mu g/mL respectively; further testing that the purity of brefeldin A in the fermentation liquor reaches 65.0%, 66.1% and 67.8% respectively.

EXAMPLE 5 fermentation of brefeldin A by the mutagenized Strain FIM-E-UN88-26

Activation of the strain FIM-E-UN 88-26: the strain FIM-E-UN88-26 preserved by glycerol is transferred to a slant culture medium and cultured in a constant temperature incubator for 8-12d, and the culture temperature is 28 ℃.

Preparing FIM-E-UN88-26 seed liquid: inoculating 0.5cm x 0.5cm thallus Porphyrae obtained by activating the above strain FIM-E-UN88-26 into seed culture medium (50 mL seed culture medium in 500mL triangular flask), and culturing at 26 deg.C and 220r/min for 36 hr to obtain seed solution.

Fermentation culture: inoculating the prepared seed solution into a fermentation culture medium (100 mL of the fermentation culture medium is filled in a 500mL triangular flask) with the inoculation amount of 5% (v/v), fermenting and culturing for 96h under the conditions of 26 ℃ and 230r/min, and detecting the obtained fermentation liquid.

The detection result shows that the yield of brefeldin A obtained by three batches of shake flask fermentation is 1980 mug/mL, 2060 mug/mL and 2031 mug/mL respectively; further testing that the purity of brefeldin A in the fermentation liquor reaches 85.0%, 86.3% and 87.2% respectively. Therefore, the screened strain can efficiently ferment the brefeldin A, and the purity of the brefeldin A in the fermentation liquid is better.

EXAMPLE 6 fermentative production of brefeldin A by the mutagenized Strain FIM-E-UN88-26

And (3) seed culture in a shaking flask: inoculating the strain FIM-E-UN88-26 lawn into seed culture medium (200 mL seed culture medium in 1000mL triangular flask), and culturing at 26 deg.C and 200r/min for 36h to obtain shake flask seed solution.

Seed tank seed culture: the shake flask seed solution was inoculated in a seed medium (100L of a 60L seed medium contained in a jar) at 0.5% by volume at a culture temperature of 26 ℃, a jar pressure of 0.05MPa, an air flow rate of 1: culturing for 32h under the conditions of 1vvm and the stirring speed of 100-.

Fermentation culture in a fermentation tank: the prepared seed solution was inoculated into a fermentation medium (1 ton of a tank-packed 700L fermentation medium) at an inoculation amount of 5% (v/v), and the mixture was cultured at a culture temperature of 26 ℃, a tank pressure of 0.05MPa, and an air flow rate of 1: 0.8-1.5vvm, the stirring speed is 100-.

The detection result shows that the yield of the brefeldin A produced by three batches of fermentation is 2139 mu g/mL, 2152 mu g/mL and 2098 mu g/mL respectively; further determining that the purity of the brefeldin A in the fermentation liquor is not lower than 86.0%, 86.6% and 86.1%, and further proving that the screened strain can efficiently ferment the brefeldin A and the purity of the brefeldin A in the fermentation liquor is better.

In conclusion, the highest yield of brefeldin A of the screened strain FIM-E-UN88-26 can reach 2152 mu g/mL, the fermentation period of a 1-ton tank is only 72h, the purity of the brefeldin A in the fermentation liquid is not lower than 85%, the impurity content is effectively controlled, and the downstream purification of the brefeldin A is facilitated.

It can be seen that the screened mutant strain Penicillium sp FIM-E-UN88-26(Penicillium brefeldianum) can be used as a production strain for further research and development.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims (9)

1. An Penicillium notatum FIM-E-UN88-26, which has been deposited with the Guangdong province collection of microorganisms with the accession number GDMCC No.61807 and the date of deposit at 2021/7/14.

2. Use of the eupenicillium strain of claim 1 for the fermentative production of brefeldin a.

3. A method for producing brefeldin A by fermentation, comprising the step of inoculating the eupenicillium strain of claim 1 in a suitable fermentation medium for fermentation culture.

4. The method for producing brefeldin A by fermentation according to claim 3, wherein the fermentation medium comprises the following components in mass content: 2-4 wt% of soluble starch, 1-3 wt% of soybean flour, 1-3 wt% of glucose, 0.1-0.5 wt% of peptone, 0.2-1.0 wt% of yeast extract and KH₂PO₄0.05-0.8wt％、MgSO₄.7H₂0.01-0.12 wt% of O, 0.15-0.15 wt% of Tween 800.02, 0.05-0.1 wt% of calcium carbonate and 6.0-7.2 of pH value.

5. The method for the fermentative production of brefeldin a according to claim 3 or 4, wherein the conditions of the fermentation culture step comprise: the rotation speed is controlled to be 100 and 250rpm, and fermentation culture is carried out for 68-120h at the temperature of 24-30 ℃.

6. The method for producing brefeldin A by fermentation according to any one of claims 3-5, further comprising the step of inoculating the eupenicillium strain of claim 1 in a seed culture medium for seed culture;

the seed culture medium comprises the following components in percentage by mass: 2-4 wt% of soluble starch, 1-3 wt% of soybean flour, 1-3 wt% of glucose, 0.1-0.5 wt% of peptone, 0.2-1.0 wt% of yeast extract and KH₂PO₄0.1-0.8wt％、MgSO₄.7H₂0.01-0.12 wt% of O, 0.15-0.78 wt% of Tween 800.02, and pH 6.2-7.0.

7. The method for fermentative production of brefeldin a according to claim 6, wherein the conditions of the seed culture step comprise: the seed liquid culture is carried out for 32-48h at 24-30 ℃ by controlling the rotation speed of 100 and 250 rpm.

8. The method for producing brefeldin A by fermentation according to any one of claims 3 to 7, further comprising the step of inoculating the eupenicillium strain of claim 1 in a slant culture medium for activation;

the slant culture medium comprises the following components in percentage by mass: 15-25 wt% of potato, 1-3 wt% of glucose and 2.0 wt% of agar, and the pH value is natural.

9. The method for fermentative production of brefeldin a according to claim 8, wherein the conditions of the slant medium activation step comprise: culturing at 24-30 deg.C for 8-12 days.

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