CN117568183A

CN117568183A - High yield of neotame B 0 Mutant strain of echinocandin rosea and application

Info

Publication number: CN117568183A
Application number: CN202311302527.5A
Authority: CN
Inventors: 李辉; 宁雅芳; 邓志强; 陈伟清; 胥龙奇; 许行健; 郭霞凌
Original assignee: Dabang Hunan Bio Pharmacy Co ltd
Current assignee: Dabang Hunan Bio Pharmacy Co ltd
Priority date: 2023-10-09
Filing date: 2023-10-09
Publication date: 2024-02-20

Abstract

The invention belongs to the field of microbial pharmacy, and particularly discloses a high-yield neotame B ₀ An echinocandin rosea mutant strain and uses thereof. The high yield of the mokang B ₀ The mutant strain of the acanthopanax roxburghii is acanthopanax roxburghii (Glarea lozoyensis) A80PL9, and is obtained by performing ARTP-EMS composite mutation, preliminary screening and secondary screening on a starting strain TG178, and is preserved in China Center for Type Culture Collection (CCTCC) at the preservation number of M202313 at the 7 th month of 202320. The strain has stable genetic performance, and is neotame B ₀ The fermentation titer reaches 6.73g/L, the fermentation period of the strain is improved by 47.3% compared with that of the original strain under the same culture condition, the fermentation period of the strain is shortened by 35% compared with that of the original strain, the utilization rate of equipment and raw materials is improved, and the mokandine B can be greatly reduced ₀ The production cost has obvious economic and social benefits.

Description

High yield of neotame B 0 Mutant strain of echinocandin rosea and application

Technical Field

The invention belongs to the field of microbial pharmacy, and in particular relates to a high-yield neotame B ₀ An echinocandin rosea mutant strain and uses thereof.

Background

New-Mokang B ₀ (Pneumocandin B ₀ ) Also known as pneumocandin B ₀ Pneumocandin B ₀ Is a novel cyclic lipopeptide antibiotic produced by fermentation of a filamentous fungus Roche acanthopanacis Glarea lozoyensis, and belongs to an echinocandin compound. New-Mokang B ₀ The semisynthetic derivative caspofungin is the first echinocandin drug approved for treating invasive fungal infection worldwide, plays a role in sterilization by inhibiting the synthesis of the main component 1, 3-beta-D glucan of fungal cell walls, and is currently recommended first-line drug against fungal infection by clinical guidelines of various countries and is widely marketed worldwide. New-Mokang B ₀ The synthesis of caspofungin, the only precursor for the current synthesis of caspofungin, relies on high quality neotame B ₀ Is produced by the method. But neotame B ₀ The NRPS-PKS hybrid enzyme system produced by Glarea lozoyensis is synthesized through a complex metabolic network, has a relatively complex structure, is extremely difficult to synthesize by a chemical method and has no industrial application value. Thus, neotame B ₀ Is an important bottleneck for preparing caspofungin, and the fermentation titer directly affects the caspofunginProduction cost and market prospect.

The optimization of the breeding and fermentation process of high-quality and high-yield strains is to improve the neotame B ₀ The main means of fermentation potency is accumulated by continuous research of students at home and abroad over twenty years, and the new forms of concand B ₀ The fermentation titer and the product quality of the caspofungin are obviously improved, and the popularization and the application of the caspofungin are promoted. In recent years, pneumocandin B ₀ Whole genome sequencing of the producer has been completed and strain molecular genetic engineering has been achieved, but pneumocandin B ₀ The complete biosynthetic pathway and regulatory mechanisms have not been fully elucidated and the key gene sites available for modification are limited. Traditional mutation breeding still obtains the neomo kang B ₀ The method can not replace effective means for producing high-yield strains, and can play advantages when the comprehensive fermentation performance of the strains is improved. Chinese patent No. CN200910196286.4 reports that G.lozoyensis ATCC20957 is used as an initial strain, protoplast is treated by mutagen NTG, and a B strain is selected ₀ The potency reaches 5.2g/L and A ₀ Mutant strains with reduced impurity levels. CN201410555356.1 reports a G.lozoyensis Q45 mutant strain obtained by screening by double mutagenesis of plasma and lithium chloride, B ₀ The potency can reach 6.0g/L. CN201710575306.3 reports B isolated from natural environment ₀ The producing strain is a starting strain, and a strain B is obtained by NTG mutagenesis and screening ₀ Mutant G.lozoyensis HS-2158 with a yield of up to 7.6g/L and with sorbitol as the main carbon source. The fermentation titers of the mutants obtained by screening are obviously improved, but the fermentation period is relatively longer, the seed culture period in the CN200910196286.4 mutant is 8 days in the matched fermentation process, the fermentation period is 14 days, and the total period is more than 500 hours; the seed culture period in the CN201410555356.1 matched fermentation process is 3 days, the fermentation period is 11 days, and the total period is 336 hours; the culture period of a seed tank in the fermentation process of the CN201710575306.3 mutant strain is 98 hours, the culture period of the fermentation tank is 280 hours, and the total period is approximately 400 hours. CN201310032896.7 reports a synthesis of B ₀ The total fermentation period is shortened to about 300h, but the fermentation titer is only about 3g/L.

At the same potency level of synthesis of neotame B ₀ Under the condition of (1), the longer the fermentation period is, the higher the energy consumption is, the lower the equipment utilization rate is, and the higher the production cost and the higher the bacterial contamination rate are. Thus screening the temokadam B with high fermentation potency and short fermentation period ₀ The production strain is an extremely important work, so that the neotame B is improved at the same time ₀ Few mutagenesis breeding reports aimed at yield and shortening fermentation period are made. Breeding high-yield neomo-kang B ₀ And the strain with short fermentation period has important significance for the production and application of caspofungin.

Disclosure of Invention

In view of the deficiencies of the prior art, the invention provides a new and improved method for preparing the same ₀ High yield of neotame B with high fermentation potency and short fermentation period ₀ An echinocandin rosea mutant strain and uses thereof.

In one aspect of the invention, a high yield of neotame B is provided ₀ The mutant strain of the echinocandin rosea is a strain of the echinocandin rosea (Glarea lozoyensis) A80PL9, which is stored in China center for type culture collection (CCTCC, address: eight-channel 299 of Wuhan university in Wuhan, hubei province) of the university in 7 months 17 of 2023, and the preservation number is CCTCC NO: M20231320. The strain is sorbitol preferred type neotame B preserved by Dabang (Hunan) biopharmaceutical Co., ltd ₀ The industrial production strain Roche acanthopanax (Glarea lozoyensis) TG178 is obtained by ARTP-EMS composite mutagenesis breeding of an original strain. Compared with the original strain, the A80PL9 mutant strain, i.e. the pneumocandin B ₀ The fermentation titer on the tank can reach 6.73g/L, the total fermentation period is shortened to 13 days, and the serial passage has good genetic stability, thus being an unattainable production of the neotame B ₀ High-yield high-quality strain.

In another aspect of the present invention, the aforementioned high yield of neotame B is provided ₀ Mutant strain of echinocandin rosea for fermentation production of neotame B ₀ Or increase the neotame B ₀ Is used in fermentation yield.

In another aspect of the present invention, the aforementioned high yield of neotame B is provided ₀ Culture of a mutant strain of echinocandia rosea or extraction of the cultureTaking out the materials.

In another aspect of the present invention, the aforementioned high yield of neotame B is provided ₀ A culture of a mutant strain of echinocandin or an extract of a culture thereof is isolated in the presence of neotame B ₀ Neomoready B as a precursor ₀ The use in the synthesis of derivatives of (a); preferably, the neotame B ₀ Is caspofungin.

In another aspect of the present invention, there is provided a microbial agent comprising the aforementioned high yield of neotame B ₀ Is a mutant strain of echinocandin rosea or high yield of neotame B ₀ Is a culture of a mutant strain of echinocandia or an extract of a culture thereof.

High yield of neotame B as described above ₀ The breeding method of the mutant strain of the echinocandin rosea comprises the steps of carrying out composite mutagenesis by utilizing normal pressure room temperature plasma (ARTP) and Ethyl Methylsulfonate (EMS), and finally breeding a strain of the pneumocandin B through primary screening, secondary screening and stability verification ₀ The stable mutant strain of the acanthopanax roseus A80PL9 with improved fermentation titer and shortened fermentation period, and specifically, the breeding method comprises the following steps:

s1, spore suspension preparation: will be neotame B ₀ Inoculating the original strain Glarea lozoyensis TG178 onto solid slant spore culture medium slant, culturing at 25deg.C for 10-12 days, after the slant culture spores are ripe, digging out bacterial blocks with inoculating shovel into a triangle flask containing 50ml of sterile water and containing glass beads, violently shaking and scattering, filtering with four layers of sterilizing mirror-cleaning paper to obtain spore suspension to be mutagenized, using fungus spore counter and using physiological saline to release until the concentration is 10 ⁷ ～10 ⁸ CFU/ml。

S2, ARTP-EMS complex mutagenesis: and uniformly coating 10 mu l of spore suspension on a sterile metal slide arranged in a culture dish, transferring the culture dish into an operation bin of an ARTP mutagenesis system sterilized in advance, placing the metal slide on a corresponding hole site on a rotary table of the operation bin, and rotating the metal slide to the position right below a plasma generator. The operating voltage 110W, the irradiation distance 2mm, the operating gas flow 10slpm (standard liters per minute) and the mutagenesis time 120S were set. The mutagenized metal slide is placed into an EP tube filled with 1ml of sterile water, and is subjected to vibration treatment, so that spore liquid on the metal slide can be resuspended, and then the metal slide is placed into a constant-temperature shaking table at 30 ℃ for incubation for 30min. After incubation, adding the mixture into a triangular flask filled with 9mL of phosphate buffer solution, adding 0.1mL of Ethyl Methanesulfonate (EMS) stock solution to make the final concentration of EMS be 1%, shaking for 90min at a constant temperature of 30 ℃, adding 5mL of 25% sodium thiosulfate solution to terminate mutagenesis, carrying out gradient dilution, and then coating the mixture on a solid flat spore culture medium, and culturing at 25 ℃ for 10-12 days until single bacterial colonies can be observed. Ten ARTP-EMS complex mutagenesis operations are carried out to construct a large mutation library.

S3, primary screening of a bacteriostasis area: the mutant strains after the composite mutagenesis were observed for growth of single colonies on the plate medium at different times and counted. Single colonies with obvious colony morphology and color change after mutagenesis are synchronously inoculated into a flat spore culture medium and a 24-deep pore plate (the loading amount of the pore plate is 5 ml) containing a fermentation culture medium, and colony numbers are made in a one-to-one correspondence. Placing the solid flat spore culture medium in a constant temperature of 24 ℃ for static culture for 10-12 days, continuously observing the growth condition of the bacterial colony, and measuring and recording the growth diameter of the bacterial colony on the flat spore culture medium. Shake culturing the 24 deep hole plate on a shaker at 24deg.C and 225rpm for 15 days, transferring 1ml fermentation broth from each hole to 24 hole deep hole plate containing 5 times volume of absolute ethanol, shake extracting in a horizontal shaker microplates machine to obtain intracellular product of neotame B ₀ Leaching into ethanol for standby. Pouring a PDA bottom layer solid culture medium into a culture dish, cooling the hot melted PDA culture medium to about 50 ℃ after solidification, adding a proper amount of candida albicans liquid, pouring an upper layer semi-solid culture medium, uniformly placing a plurality of oxford cups on the bottom layer solid culture medium before pouring, and pulling out after cooling. And (3) dropwise adding an appropriate amount of ethanol extract of the mutant strain to be detected into holes formed by oxford cups, wherein 1 oxford cup hole is dropwise added with ethanol extract of the original strain as a contrast, placing the double-layer flat plate into a 28 ℃ incubator for culturing for 24 hours, observing and measuring the diameter of a bacteriostasis ring by using a vernier caliper. Selecting mutant strains with the diameter of the inhibition zone increased by more than 30% compared with the original strain, carrying out shake flask fermentation re-screening, and carrying out slant subculture on single colonies with the diameter of the inhibition zone increased by more than 30% on the corresponding flat spore culture medium according to the marks.

S4, shaking and re-screening: after the strains screened in the step S3 are subjected to subculture by a solid slant spore culture medium, inoculating the strains into a shake flask seed culture medium, culturing for 4 days at 24 ℃ and 250rpm, transferring the strains into a fermentation culture medium at 10% of inoculum size, culturing for 15 days at 24 ℃ in parallel at 250rpm, and sampling and detecting N Mo Kangding B at 24h intervals every day from the 7 th day ₀ Titers and hyphal morphology varied until the end of 15 days. Combining three screening indexes of fermentation titer, fermentation period and mycelium morphology to finally obtain a mutant strain with the number of A80PL9.

S5, genetic stability investigation: continuously passaging the A80PL9 mutant strain obtained by the screening in the step S4 on a solid slant spore culture medium for 8 times, carrying out shake flask fermentation for each generation, and sampling from the 7 th day to determine the neotame B ₀ Titers and components, the genetic stability of the high-yield strain obtained through primary screening and secondary screening is confirmed through the fermentation period and the fermentation titers.

Through the steps, the composite mutagenesis of ARTP combined with EMS is adopted, and 109 forward mutant strains are primarily screened from more than two thousand mutant single colonies through primary screening of a bacteriostasis zone. And (3) obtaining a high-yield high-quality mutant strain with the number of A80PL9 from 109 forward mutant strains by shaking and re-screening and combining fermentation titer, fermentation period and mycelium morphology screening indexes. The cost performance of the A80PL9 mutant strain in shake flask fermentation is improved by more than 50 percent, the fermentation period is shortened by more than 30 percent, and the strain has good genetic stability. The A80PL9 mutant strain has obvious colony color change, high growth speed and high spore producing capacity on solid plate spore culture medium and obvious hypha form change in liquid culture medium.

In another aspect of the present invention, there is provided the use of said mutant strain, i.e., echinocandin A80PL9 in enhancing pneumocandin B ₀ The application in fermentation production is realized by culturing the high-yield neotame B ₀ Is realized by using a mutant strain of echinocandin rosea; preferably, the preparation method comprises the following steps:

(1) Spore slant culture: inoculating the mutant strain A80PL9 to a slant culture medium, culturing for 10-12 days at 24 ℃, eluting mature spores obtained by culturing by using sterile water, and obtaining spore suspension;

(2) Shake flask seed culture: inoculating the spore suspension to a shake flask seed culture medium, and culturing for 72 hours at 24 ℃ under the condition that the rotation speed of a shaking table is 225rpm to obtain shake flask seeds;

(3) Seed pot culture: inoculating the shake flask seeds into a seed tank culture medium, and culturing for 72 hours at the temperature of 24 ℃ under the conditions of stirring rotation speed of 100-200 rpm, ventilation of 0.6-1.2 VVM and tank pressure of 0.05Mpa to obtain seed liquid;

(4) Culturing in a fermentation tank: inoculating the seed liquid to a fermentation tank culture medium, and culturing at 24-25deg.C, stirring speed of 100-250 rpm, tank pressure of 0.05-0.07 Mpa, ventilation of 0.6-1.5 VVM, dissolved oxygen not less than 20%, and pH of 5.0-6.0 for 10 days to obtain the product containing neotame B ₀ Is a fermentation broth of (a); preferably, the aqueous sorbitol solution with a concentration of 20% is added daily from day 7, the volume of added aqueous sorbitol solution being 5% of the volume of the initial fermentation broth.

The composition of each culture medium is as follows:

the composition of the slant spore culture medium and the flat spore culture medium is the same, and the slant spore culture medium and the flat spore culture medium comprise the following components in concentration: glucose 5.0%, yeast extract 1.0%, malt extract 0.5%, soybean peptone 0.5%, hydrolyzed casein 0.1%, ammonium sulfate 0.1%, agar 2.0%, and distilled water in balance; before sterilization, the pH is adjusted to 6.5, and the sterilization condition is 121 ℃ for 30min;

the shake flask seed medium included the following concentrations of each component: 4 to 6 percent of sorbitol, 0.5 to 1.5 percent of glucose, 1 to 3 percent of soybean cake powder, 0.5 to 1.5 percent of cottonseed cake powder, 0.5 to 1.5 percent of corn steep liquor dry powder, 0.1 to 0.3 percent of monopotassium phosphate, 0.01 percent of magnesium sulfate heptahydrate and the balance of distilled water; sterilizing for 30min at 121 ℃ with pH of 4.8-5.2;

the seed pot medium comprises the following components in concentration: 4 to 6 percent of sorbitol, 0.5 to 1.5 percent of glucose, 1 to 3 percent of soybean cake powder, 0.5 to 1.5 percent of cottonseed cake powder, 0.5 to 1.5 percent of corn steep liquor dry powder, 0.1 to 0.3 percent of monopotassium phosphate, 0.01 percent of magnesium sulfate heptahydrate, 0.05 percent of SAG471 organic silicon defoamer and the balance of distilled water; sterilizing for 30min at 121 ℃ with pH of 4.8-5.2;

the shake flask fermentation medium comprises the following components in concentration: 8 to 12 percent of sorbitol, 0.5 to 1.5 percent of glucose, 0.5 to 1.5 percent of soybean oil, 1 to 3 percent of cottonseed meal, 1 to 3 percent of corn gluten meal, 0.5 to 1.5 percent of soybean meal, 0.5 to 1.5 percent of proline, 0.1 to 0.3 percent of threonine, 0.05 to 0.2 percent of ammonium sulfate, 0.2 to 0.6 percent of dipotassium hydrogen phosphate, 0.04 to 0.06 percent of ferrous phosphate, 0.02 to 0.04 percent of manganese sulfate and the balance of distilled water;

the fermenter medium comprises the following components in the following concentrations: 8 to 12 percent of sorbitol, 0.5 to 1.5 percent of glucose, 0.5 to 1.5 percent of soybean oil, 1 to 3 percent of cottonseed cake powder, 1 to 3 percent of corn gluten meal, 0.5 to 1.5 percent of soybean cake powder, 0.5 to 1.5 percent of proline, 0.1 to 0.3 percent of threonine, 0.05 to 0.2 percent of ammonium sulfate, 0.2 to 0.6 percent of dipotassium hydrogen phosphate, 0.04 to 0.06 percent of ferrous phosphate, 0.02 to 0.04 percent of manganese sulfate, 0.05 percent of SAG471 organosilicon defoamer and the balance of distilled water; sterilizing at 121 deg.c for 30min at pH 4.5-5.0.

The beneficial effects of the invention are as follows:

ARTP is a novel physical mutagenesis technology, which utilizes atmospheric pressure glow discharge to generate various active particles, acts on DNA to trigger gene mutation, and has the advantages of simple operation, high mutation rate and good mutagenesis effect. EMS is used as a high-efficiency chemical mutagen, can induce and generate high-density series allelic point mutation, and has the advantages of high efficiency, small side effect and the like. The invention carries out composite mutagenesis by adopting a method combining the two mutagenesis modes, and compared with a single mutagenesis method, the invention can not cause the strain to generate resistance, and the obtained mutagenesis strain has more stable performance. The invention utilizes ARTP-EMS composite mutagenesis technology to treat the pneumocandin B ₀ The industrial production strain Glarea lozoyensis TG178 is subjected to mutagenesis, and a strain of neotame B is screened from a huge amount of mutant stock through primary screening of a bacteriostasis area and shaking re-screening ₀ The inventors designated Glarea lozoyensis A PL9 as a mutant strain having a significantly improved yield over the original strain. The A80PL9 mutant of the invention has a new temokadam B compared with the original strain TG178 ₀ The total period of seed culture and fermentation culture is shortened to 13 days while the yield is greatly improved, which is 35 percent shorter than the original strain, thereby greatly improving the single-pot productivity and production efficiency and reducing the comprehensive energy consumptionHas very ideal industrial application value.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute an undue limitation on the application, in which:

FIG. 1 is a schematic diagram of the overall operation flow of the present invention;

FIG. 2 is a graph showing the mortality of strains at various ARTP mutagenesis times;

FIG. 3 is a graph showing the mortality of strains at different EMS mutagenesis times;

FIG. 4A is a colony morphology of the starting strain TG178 (plate spore culture);

FIG. 4B is a colony morphology (slab spore culture) of mutant strain A80PL 9;

FIG. 5A is a diagram of the mycelium morphology of the starting strain TG178 (shake flask seed culture);

FIG. 5B is a hypha morphology map of mutant A80PL9 (shake flask seed culture);

FIG. 6 is a graph showing comparison of fermentation titers of the starting strain TG178 and the mutant strain A80PL9.

Detailed Description

The present invention will be described in further detail with reference to the following examples, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent, and the description thereof is merely illustrative of the present invention and not intended to be limiting. The starting strain used in the invention is sorbitol preference type neotame B preserved by Dabang (Hunan) biopharmaceutical Co., ltd ₀ Industrial producer strain Glarea lozoyensis TG178. The materials, reagents, instruments and methods used in the examples of the present invention, without any particular description, are conventional in the art and are commercially available.

The culture medium according to the embodiment of the invention comprises the following components:

slant spore culture medium: glucose 5.0%, yeast extract 1.0%, malt extract 0.5%, soybean peptone 0.5%, hydrolyzed casein 0.1%, ammonium sulfate 0.1%, agar 2.0%, and distilled water in balance; the pH was adjusted to 6.5 before sterilization, and the sterilization conditions were 121℃for 30min. The prepared slant culture medium is packaged in 250ml eggplant bottles, each eggplant bottle is packaged in 50ml, and a solid slant spore culture medium is prepared by swinging the slant after wet heat sterilization, or a flat spore culture medium is prepared by pouring the sterilized solid slant culture medium into a sterile culture dish.

Shake flask seed medium: 5.0% of sorbitol, 1.0% of glucose, 2.0% of soybean cake powder, 1.0% of cotton seed cake powder, 1.0% of corn steep liquor dry powder, 0.2% of potassium dihydrogen phosphate, 0.01% of magnesium sulfate heptahydrate and the balance of distilled water, wherein the pH is adjusted to 5.0 before sterilization, and the sterilization condition is 121 ℃ for 30min. Subpackaging the prepared shake flask seed culture medium and 500ml triangular flasks, subpackaging 60ml each triangular flask, and performing wet heat sterilization for later use.

Shake flask fermentation medium: 10% of sorbitol, 1.0% of glucose, 1.0% of soybean oil, 2.0% of cottonseed meal, 2.0% of corn gluten meal, 1.0% of soybean meal, 1.0% of proline, 0.2% of threonine, 0.1% of ammonium sulfate, 0.4% of dipotassium hydrogen phosphate, 0.05% of ferrous phosphate, 0.03% of manganese sulfate and the balance of distilled water, wherein the pH is natural, the packaging amount of 5ml of the soybean meal is packaged according to 24 deep-hole plates, the packaging amount of 80ml of the soybean meal is packaged according to 500ml triangular flasks, and the soybean meal is sterilized at 121 ℃ for 30min for standby.

EXAMPLE 1ARTP-EMS Complex mutagenesis

1.1 preparation of bacterial inclined plane culture medium and spore suspension

Taking neotame B ₀ Inoculating the original strain Glarea lozoyensis TG178 to a slant spore culture medium, culturing at constant temperature of 24 ℃ for 10-12 days, after the slant spores are mature, digging out even and full fungus blocks by an inoculating shovel into a triangle flask containing 50ml of sterile water and provided with glass beads, scattering by vigorous shaking, filtering by four layers of sterilizing mirror wiping paper to prepare spore suspension to be mutagenized, counting by a fungus spore counter and releasing to the concentration of 10 by normal saline ⁷ ～10 ⁸ CFU/ml。

1.2 Single factor mutagenesis condition screening by atmospheric pressure room temperature plasma (ARTP)

The Glarea lozoyensis TG spore suspension prepared in step 1.1 was subjected to a physical mutagenesis pretreatment experiment using an art p-IIS atmospheric pressure room temperature plasma mutagenesis instrument (available from the company of the biotechnology of notharbour, inc.) and the mortality was calculated. The ARTP mutagenesis step before the composite mutagenesis and the mortality calculation method are specifically as follows:

mu.l of spore suspension was spread evenly on sterile metal slides placed in petri dishes, allowing the spore suspension to cover all slides. Transferring the culture dish to an operation bin of an ARTP mutagenesis system sterilized in advance, and placing a metal slide on a corresponding hole site on a rotary table of the operation bin and rotating the metal slide to the position right below a plasma generator. High purity helium is used as working gas of the plasma, and the mutagenesis treatment is carried out under the conditions of 110W working voltage, 2mm irradiation distance and 10slpm working gas flow (standard liter per minute). Different treatment groups are arranged, three parallel samples are arranged in each treatment group, and the treatment time of each treatment group is respectively 0s (control), 10s, 20s, 40s, 60s, 80s, 100s, 120s, 140s, 160s, 180s and 200s. After mutagenesis, the metal slide was placed in an EP tube containing 1ml of sterile water and subjected to shaking treatment with a vortex shaker to allow the spore liquid on the metal slide to be resuspended. Gradient diluted to 10 with physiological saline ^-3 、10 ^-4 、10 ^-5 200ul of the culture medium is uniformly coated on the surface of a flat-plate culture medium (the flat-plate culture medium consists of the same slant culture medium), the flat-plate culture medium is placed in a 24 ℃ incubator for culturing for 10 days, the colony forming unit number on each flat plate is counted, and the mortality rate of each mutagenesis treatment time is calculated, wherein the mortality rate (%) = (number of colonies subjected to mutagenesis treatment-number of colonies subjected to mutagenesis treatment)/number of colonies subjected to mutagenesis treatment is multiplied by 100%. And drawing a mortality curve, wherein the mortality reaches 87% when the treatment time is 120s, and the probability of obtaining the optimal mutant is maximum when the positive mutation rate is highest when the mutation mortality is 90-95% according to the modern microorganism breeding theory and experience, as shown in figure 2. In order to ensure the survival amount of the bacterial cells with more complex mutagenesis, the ARTP treatment time is not too long, and 120s is selected as the optimal mutagenesis time.

1.3 Ethyl Methylsulfonate (EMS) Single factor mutagenesis condition screening

The Glarea lozoyensis TG spore suspension prepared in step 1.1 was subjected to a pre-experiment with Ethyl Methanesulfonate (EMS) for chemical mutagenesis treatment and the mortality was calculated. The EMS mutagenesis step before the composite mutagenesis and the mortality calculation method are specifically as follows:

transferring 4.5ml spore suspension into a sterile test tube, adding 0.5ml EMS ethanol solution with concentration of 100mg/ml to make final concentration of EMS 10mg/ml, shaking gently, placing on a shaking table test tube rack, and respectively treating at 30deg.C and 220rpm for 0min, 30min, 60min, 90min and 120min, wherein three parallel samples are obtained for each treatment group. After the treatment, 0.5ml of spore suspension was added to a test tube containing 10ml of sterile 5% sodium thiosulfate solution and shake-cultured for 30min, EMS was inactivated, and mutagenesis was terminated. Respectively taking spore suspensions with different mutagenesis time, properly diluting, uniformly coating 200ul of spore suspensions on the surface of a flat culture medium (the flat culture medium consists of the same slant culture medium), and placing the flat culture medium in a 24 ℃ incubator for culturing for 10 days. The number of colony forming units on each plate was counted, the mortality rate was calculated for each mutagenesis treatment time, and a mortality rate curve was drawn, as shown in fig. 3, and the mortality rate reached 91% at a treatment time of 90min. 90min was chosen as the optimal EMS mutagenesis time.

1.4ARTP-EMS Complex mutagenesis

And (2) uniformly coating 10 mu l of the Glarea lozoyensis TG spore suspension prepared in the step (1.1) on a sterile metal slide, transferring to an operation bin of an ARTP mutagenesis system for advanced sterilization, placing the metal slide on a corresponding hole site on a rotary table of the operation bin, and rotating the metal slide to the position right below a plasma generator. High-purity helium is used as working gas of plasma, and working voltage 110W, irradiation distance 2mm, working gas flow 10slpm and mutagenesis time 120s are set. The mutagenized metal slide is placed into an EP tube filled with 1ml of sterile water, and is subjected to vibration treatment, so that spore liquid on the metal slide can be resuspended, and then the metal slide is placed into a constant-temperature shaking table at 30 ℃ for incubation for 30min. After incubation, all the incubation products were added to a sterile test tube containing 3.5ml of phosphate buffer, and then 0.5ml of EMS solution with a concentration of 100mg/ml was added, and the mixture was placed on a test tube rack and subjected to shaking at a constant temperature of 30℃for 90 minutes. After the treatment, 0.5ml of spore suspension was added to a tube containing 10ml of sterile 5% sodium thiosulfate solution, and the mixture was shaken for 30min, EMS was inactivated, and mutagenesis was terminated. After proper dilution, 200ul of the mixture is evenly spread on the surface of a plate culture medium, and the plate culture medium is placed in a 24 ℃ incubator for 10 days, and the colony forming unit number on each plate is counted. And (3) repeatedly taking the spore suspension prepared in the step (1.1), and carrying out ten ARTP-EMS composite mutagenesis operations to construct a large mutation library.

EXAMPLE 2 Primary screening, rescreening and stability investigation of mutant strains

2.1 diameter Primary screening of antibacterial zone

And (3) observing and counting the growth condition of single colonies on the plate culture medium at different times for the mutant strain subjected to the composite mutagenesis, synchronously inoculating the single colonies with obvious colony morphology and color change after the mutagenesis into the plate spore culture medium (the plate culture medium consists of the same inclined plane culture medium) and a 24-deep pore plate (the pore plate filling amount is 5 ml) containing the fermentation culture medium, and carrying out one-to-one correspondence and making colony numbers. The flat spore culture medium is placed at the constant temperature of 24 ℃ for static culture for 10-12 days, the growth condition of the bacterial colony is continuously observed, and the growth diameter of the bacterial colony on the flat spore culture medium is measured and recorded. Shake culturing the 24 deep hole plate on a shaker at 24deg.C and 225rpm for 15 days, transferring 1ml fermentation broth from each hole after culturing, transferring into 24 hole deep hole plate filled with anhydrous ethanol with volume of about 5 times of fungus mass, shake extracting in a horizontal shaker microplates machine, and collecting intracellular product of neotame B ₀ Leaching into ethanol for standby.

Taking a sterile culture dish with the diameter of 90mm and the height of 16mm, injecting 20ml of hot melted PDA culture medium, solidifying to obtain a bottom solid culture medium, and uniformly placing 14 oxford cups (round small tubes with the inner diameter of 6nm, the outer diameter of 8nm and the height of 10 nm) on the bottom solid culture medium at equal intervals. And in addition, after a proper amount of PDA culture medium is melted by heat, cooling to about 50 ℃ and starting water bath heat preservation, sucking a proper amount of overnight cultured candida albicans bacterial liquid by using a sterile suction tube, adding the liquid into the heat-preserving PDA culture medium, and slightly shaking the liquid to be uniform to be used as a bacterial layer culture medium. Sucking 5ml of fungus layer culture medium with a sterile large-mouth straw, rapidly adding into a culture dish paved with a bottom layer culture medium, and pulling out the oxford cup for later use after solidification. And (3) dropwise adding 100ul of ethanol extract of the mutant strain to be detected into holes formed by oxford cups, wherein 1 oxford cup hole is dropwise added with ethanol extract of the original strain as a control, placing the double-layer flat plate into a 28 ℃ incubator for culturing for 24 hours, observing and measuring the diameter of a bacteriostasis ring by using a vernier caliper. Selecting mutant strains with the diameter of the inhibition zone increased by more than 30% compared with the original strain, carrying out shake flask fermentation re-screening, and carrying out slant subculture on single colonies with the diameter of the inhibition zone increased by more than 30% on the corresponding flat spore culture medium according to the marks. The total of 2240 single colonies are initially screened, and the forward mutation rate is 16.7% by counting the forward mutant 376 strains, wherein the diameter of the inhibition zone is increased by more than 30% by counting the forward mutant 109 strains.

2.2 shaking flask fermentation double Screen

After the 109 mutant strains screened in the step 2.1 are subjected to slant subculture, the 109 mutant strains are inoculated into a shake flask seed culture medium, and are cultured at 24 ℃ and 250rpm for 4 days, then are transferred into a shake flask fermentation culture medium with 10% of inoculum size, and are cultured for 15 days at 24 ℃ in parallel at 250rpm with the initial strain serving as a control (the seed culture period of the initial strain is 5 days). Sampling detection New Mo Kangding B every 24h from day 7 ₀ Titers and mycelium morphology, until the end of 15 days. Hypha morphology was observed by microscopy, neotame B ₀ The potency is detected by high performance liquid chromatography, specifically, 1ml fermentation liquor is taken daily to be centrifugated, ethanol with the volume of 5 times is added to dilute, the mixture is centrifugated to take supernatant after shaking extraction for 30min, and liquid phase detection is carried out after filtration by a 0.22 mu m filter membrane. The liquid chromatography conditions were: chromatographic column Hypersil ODS2 (250×4.6mm,5 μm), mobile phase acetonitrile/water= (40:60, V/V), sample injection amount 20 μl each time, flow rate 1.0mL/min, detection wavelength 210nm, column temperature 40 ℃, peak time about 9.64min. On the basis of fermentation titer screening, fermentation period screening and mycelium morphology screening, a mutant strain with the number of A80PL9 is obtained from 109 forward mutant strains, the shake flask fermentation titer reaches 4.3g/L peak value after 10 days, the shake flask titer peak value is improved by 53.5% compared with that of the original strain after 15 days, and the total fermentation period is shortened to 13 days.

The mutant strain A80PL9 spore suspension and the original strain TG178 spore suspension are respectively diluted in a gradient way and then uniformly coated on a flat spore culture medium, and the flat spore culture medium is placed in a 24 ℃ incubator for culturing for 10 days, and the colony morphology is observed and the colony diameter is measured. The colony morphology of the original strain TG178 and the mutant strain A80PL9 is different from that of the original strain A80PL9, and compared with the original strain, the colony morphology and the color of the mutant strain A80PL9 are obviously changed, and the colony morphology of the mutant strain A80PL9 is round, has protrusions, surface wrinkles, irregular edges, black brown color and white villus at the periphery.

The mutant strain A80PL9 black brown single colony and the original strain pure white single colony are respectively selected and cultured in shake flask seed culture medium, 3 are respectively in parallel, and the culture is carried out at 24 ℃ for 72 hours until hyphae grow out. The mycelium morphology of the original strain TG178 and the mycelium of the mutant strain A80PL9 are observed by an optical microscope on a mycelium glass, the mycelium morphology of the mutant strain A80PL9 is obviously changed compared with that of the original strain, and the single uniform reticular mycelium of the original strain is rarely differentiated into two mycelium morphologies of thick and fine meshes, so that the mycelium is clearly distinguished under the microscope.

2.3 genetic stability investigation and seed preservation

The obtained neotame B is subjected to the repeated screening in the step 2.2 ₀ High-yielding mutant A80PL9 was subcultured to investigate phase stability, serially passaged 8 times on a slant, shake flask fermented for each passage and assayed for neotame B ₀ The titers and components are arranged in three parallel for each generation, the results are shown in the following table, the average titer of fermentation of the 1 st generation is 4.58g/L, the average titer of fermentation of the 8 th generation is 4.37g/L, the lowest titer is 4.25g/L, the highest titer is 4.63g/L, and the fluctuation range of the titers is not more than 10%, so that the mutant strain A80PL9 has good genetic stability.

Table 1 mutant a80PL9 strain passaging times and average shake flask titers

Passage times (generation)	G1	G2	G3	G4	G5	G6	G7	G8
									Average potency (g/L)	4.58	4.33	4.63	4.47	4.25	4.49	4.51	4.37

According to the results of example 1 and example 2, screening a strain with stable genetic performance and New Mo Kangding B by adopting ARTP-EMS complex mutagenesis in the application by taking acanthopanax roseus TG178 as an initial strain ₀ The Roche acanthopanax senticosus Glarea lozoyensis A PL9 with high fermentation titer and short fermentation period is preserved in China Center for Type Culture Collection (CCTCC) for 7 months and 17 days in 2023, wherein the address is the China center for type culture collection of the university of Wuhan, china, post code 430072) with the preservation number of CCTCC NO of M20231320, and is hereinafter called Roche acanthopanax senticosus A80PL9.

EXAMPLE 3 echinocandin rosea A80PL9 fortified pneumocandin B ₀ Fermentation production

In this example, the screening of the obtained acanthopanax loxoprofen A80PL9 was used as an industrial strain to strengthen the pneumocandin B ₀ Comprising the steps of:

s1, spore suspension preparation: picking single colony of a flat plate of the acanthus roseus A80PL9 or mature parent inclined spore to an eggplant bottle inclined plane culture medium by using an inoculating shovel, culturing the spores at the temperature of 24 ℃ for 10-12 days, eluting the spores by using 20ml of sterile normal saline after the culturing and the maturing, preparing spore eluent, and filtering by using four layers of sterilizing mirror wiping paper to obtain spore suspension.

S2, shake flask seed culture: inoculating the spore suspension prepared in the step 1 into a shake flask seed culture medium for shake flask seed culture, inoculating the inoculum size (spore suspension) according to 0.05% of the volume of the shake flask seed culture medium, culturing for 72h under the conditions of the culture temperature of 24 ℃ and the humidity of 50%, the rotation speed of a shaking table of 225rpm and the amplitude of 5cm, and combining seed bottles to obtain a shake flask seed culture solution.

S3, culturing in a seed tank: adding 0.7 cubic seed culture medium into 1 cubic seed tank, mixing the seed tank culture medium with shake flask seed culture medium, adding 0.05% SAG471 organosilicon defoamer, sterilizing by steam wet heat sterilization, maintaining pressure at 121deg.C for 30min, cooling to 24deg.C, and inoculating 700ml shake flask seed liquid according to the ratio of 0.1% of seed culture medium to start seed tank culture. The culture temperature is 24 ℃, the stirring rotation speed is 100-200 rpm, the aeration rate is 0.6-1.2 VVM, the tank pressure is 0.05Mpa, the dissolved oxygen is 30-70%, and the culture period is 72 hours. Wherein the stirring rotation speed is controlled to be 100rpm for 0-24 h, 125rpm for 24-48 h and 150rpm for 48h and seed moving; the ventilation rate is controlled to be 0.6VVM in 0-36 h, 0.9VVM in 36-48 h, and 1.2VVM in 48 h. Before the culturing is finished and the transplanting is finished, the growth condition of hypha is observed by sampling microscopic examination, the hypha meets the transplanting standard, and the hypha has no mixed bacteria and has a thallus concentration of 40%.

S4, culturing in a fermentation tank: adding 0.65 cube fermentation culture medium into 10 cube fermentation tank, mixing the culture medium formula of the fermentation tank with shake flask fermentation culture medium, adding 0.05% SAG471 organic silicon defoamer, sterilizing by steam wet heat sterilization, maintaining pressure at 121deg.C for 30min, cooling to 24deg.C, transferring culture solution in seed tank into fermentation tank according to 10% inoculation amount, and fermenting.

The fermentation culture conditions and the feed supplement in the fermentation process are controlled as follows:

1) And (3) temperature control: the whole process is controlled at 24-25 ℃;

2) pH control: the pH is natural in 0-72 hours, the pH is automatically controlled to be 6.0 by adding 20% of phosphoric acid in a flowing way in 72-144 hours, and the pH is automatically controlled to be 5.5 by adding 25% of ammonia water in a flowing way in 144 hours to a tank;

3) Tank pressure control: controlling the tank pressure to be 0.05Mpa in 0-96 hours, and controlling the tank pressure to be 0.07Mpa in 96 hours to be discharged;

4) Stirring control: controlling the stirring rotation speed to 120rpm in 0-48 hours, controlling the stirring rotation speed to 200rpm in 48-72 hours, and controlling the stirring rotation speed to 250rpm in 72 hours to tank discharging;

5) And (3) air quantity control: controlling the air quantity to be 0.6VVM in 0-24 hours, controlling the air quantity to be 1.0VVM in 24-48 hours, and controlling the air quantity to be 1.5VVM in 48 hours and tank discharging;

6) Dissolved oxygen control: controlling the dissolved oxygen to be more than or equal to 60% in 0-48 hours, controlling the dissolved oxygen to be more than or equal to 40% in 48-72 hours, and controlling the dissolved oxygen to be more than or equal to 20% after 72-72 hours fermentation by controlling the air quantity, the stirring rotating speed and the tank pressure;

7) Sorbitol feed supplement control: and (3) starting fermentation at 168h, adding 400-500L of sorbitol aqueous solution with concentration of 20% every day (the volume of each material adding is about 5% of the volume of the initial fermentation liquid), and controlling the sorbitol content in the fermentation liquid to be 3-4%.

The fermentation process starts from the period of transplanting, and samples are taken at intervals of 12 hours to detect the neotame B in the fermentation liquid ₀ Titer, fermentation broth pH, thallus concentration PMV and sorbitol residue, and microscopic observation of mycelium morphology changes. The mycelium was aged and tended to autolyze after fermentation for about 240 hours (10 days), the pH was raised significantly, the potency was no longer increased and slightly decreased, the fermentation was terminated, the fermentation results were counted as follows, and the fermentation metabolism curve is shown in FIG. 5.

TABLE 2 results of fermentation verification of mutant Roche acutangulis A80PL9

EXAMPLE 4 fermentation production of Neomodine B by the starting Strain, roche acanthopanax senticosus TG178 ₀

Neomoready B in example 3 ₀ Reference example 3 was conducted by replacing the production strain of echinocandin rosea a80PL9 with the starting strain of echinocandin rosea TG178, spore suspension preparation, shake flask seed culture, seed tank culture, fermenter fermentation, and the like. Unlike example 3The culture period of the seed tank in this example was 120 hours (5 days), the fermentation period of the fermenter was 360 hours (15 days), and the results of the fermentation were counted as follows.

TABLE 3 fermentation verification results of the starting strain, roche acanthopanacis TG178

According to the experimental results of example 3 and example 4, the mutant strain, i.e., rockwell A80PL9, compared with the starting strain, i.e., rockwell A TG178, whether from Neumokadam B ₀ The fermentation titer is greatly broken through, the fermentation titer is improved from 4.57g/L to 6.73g/L, the fermentation titer is improved by 47.3% compared with the original strain, the total fermentation period is shortened from 20 days to 13 days, and the fermentation titer is shortened by 35% compared with the original strain. The comparison of the fermentation titer curves of the mutant strain Roche acanthopanax rotundus A80PL9 of example 3 and the starting strain Roche acanthopanax rotundus TG178 of example 4 is shown in FIG. 6.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

Claims

1. High-yield neotame B ₀ The mutant strain of the acanthopanax roxburghii is characterized in that the strain is acanthopanax roxburghii (Glarea lozoyensis) A80PL9, and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20231320 in 7 months of 2023.

2. The high yield of neotame B of claim 1 ₀ Mutant strain of echinocandin rosea for fermentation production of neotame B ₀ Or increase the neotame B ₀ Is used in fermentation yield.

3. The high yield of neotame B of claim 1 ₀ Mutant strain of echinocandin roseaOr an extract of a culture thereof.

4. The high yield of neotame B of claim 3 ₀ A culture of a mutant strain of echinocandin or an extract of a culture thereof is isolated in the presence of neotame B ₀ Neomoready B as a precursor ₀ Is used for synthesizing the derivative.

5. The use according to claim 4, wherein the neotame B ₀ Is caspofungin.

6. A microbial agent comprising the highly productive neotame B as defined in claim 1 ₀ Is a mutant strain of echinocandin rosea, or a highly productive neotame B as claimed in claim 3 ₀ Is a culture of a mutant strain of echinocandia or an extract of a culture thereof.

7. Neumokoding B ₀ Is characterized by culturing the highly productive neotame B of claim 1 ₀ Is realized by using the mutant strain of the echinocandin rosea.

8. The preparation method according to claim 7, characterized in that the preparation method comprises:

(1) Spore slant culture: high yield of neotame B as claimed in claim 1 ₀ The mutant strain of the acanthus roseus is used as a production strain, the production strain is inoculated to a slant spore culture medium, the culture is carried out for 10 to 12 days at the temperature of 24 ℃, and the mature spores obtained by the culture are eluted by sterile water to obtain spore suspension;

(4) Culturing in a fermentation tank: inoculating the seed liquid to a fermentation tank culture medium, and culturing at 24-25deg.C, stirring speed of 100-250 rpm, tank pressure of 0.05-0.07 Mpa, ventilation of 0.6-1.5 VVM, dissolved oxygen not less than 20%, and pH of 5.0-6.0 for 10 days to obtain the product containing neotame B ₀ Is a fermentation broth of (a).

9. The method according to claim 8, wherein in the step (4), the aqueous sorbitol solution having a concentration of 20% is added daily from the time of the culture to the time of the 7 th day, and the volume of the added aqueous sorbitol solution is 5% of the volume of the initial fermentation liquid.

10. The method of claim 8, wherein the slant spore media comprises the following concentrations of each component: glucose 5.0%, yeast extract 1.0%, malt extract 0.5%, soybean peptone 0.5%, hydrolyzed casein 0.1%, ammonium sulfate 0.1%, agar 2.0%, and distilled water in balance; before sterilization, the pH is adjusted to 6.5, and the sterilization condition is 121 ℃ for 30min;

and/or, the shake flask seed medium comprises the following components in concentration: 4 to 6 percent of sorbitol, 0.5 to 1.5 percent of glucose, 1 to 3 percent of soybean cake powder, 0.5 to 1.5 percent of cottonseed cake powder, 0.5 to 1.5 percent of corn steep liquor dry powder, 0.1 to 0.3 percent of monopotassium phosphate, 0.01 percent of magnesium sulfate heptahydrate and the balance of distilled water; sterilizing for 30min at 121 ℃ with pH of 4.8-5.2;

and/or, the seed tank culture medium comprises the following components in concentration: 4 to 6 percent of sorbitol, 0.5 to 1.5 percent of glucose, 1 to 3 percent of soybean cake powder, 0.5 to 1.5 percent of cottonseed cake powder, 0.5 to 1.5 percent of corn steep liquor dry powder, 0.1 to 0.3 percent of monopotassium phosphate, 0.01 percent of magnesium sulfate heptahydrate, 0.05 percent of SAG471 organic silicon defoamer and the balance of distilled water; sterilizing for 30min at 121 ℃ with pH of 4.8-5.2;

and/or, the fermenter medium comprises the following components in the following concentrations: 8 to 12 percent of sorbitol, 0.5 to 1.5 percent of glucose, 0.5 to 1.5 percent of soybean oil, 1 to 3 percent of cottonseed cake powder, 1 to 3 percent of corn gluten meal, 0.5 to 1.5 percent of soybean cake powder, 0.5 to 1.5 percent of proline, 0.1 to 0.3 percent of threonine, 0.05 to 0.2 percent of ammonium sulfate, 0.2 to 0.6 percent of dipotassium hydrogen phosphate, 0.04 to 0.06 percent of ferrous phosphate, 0.02 to 0.04 percent of manganese sulfate, 0.05 percent of SAG471 organosilicon defoamer and the balance of distilled water; sterilizing at 121 deg.c for 30min at pH 4.5-5.0.