CN110066745B

CN110066745B - Marine micromonospora strain for fermenting high-yield Rakicidin H and application thereof

Info

Publication number: CN110066745B
Application number: CN201910175713.4A
Authority: CN
Inventors: 周剑; 方志锴; 陈丽; 赵薇; 江宏磊; 林风; 江红; 连云阳
Original assignee: Fujian Institute of Microbiology
Current assignee: Fujian Institute of Microbiology
Priority date: 2019-03-08
Filing date: 2019-03-08
Publication date: 2021-03-30
Anticipated expiration: 2039-03-08
Also published as: CN110066745A

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a marine micromonospora strain for producing Rakicidin H by fermentation and application thereof. The mutant strain marine micromonospora strain FIM02523 for producing the Rakicidin compound is subjected to mutation breeding, so that the mutant strain marine micromonospora strain FIM-R181103 for producing Rakicidin H with high yield is obtained, the strain can effectively improve the titer of Rakicidin H in fermentation liquor, in a fermentation experiment in a 20-1000L fermentation tank, the titer of Rakicidin H produced by fermenting the marine micromonospora strain FIM-R181103 is as high as about 430mg/L, the Rakicidin H extraction and purification work is greatly facilitated, and the industrial requirements can be met.

Description

Marine micromonospora strain for fermenting high-yield Rakicidin H and application thereof

Technical Field

The invention belongs to the technical field of microbial fermentation, and particularly relates to a marine micromonospora strain for fermenting high-yield Rakicidin H and application thereof.

Background

Rakicidins compounds are fermentation products of marine micromonospora, are antitumor active substances which are separated from metabolites of marine microorganisms and have clinical application values at present, and are important compounds.

A series of Rakicidins compounds with anti-tumor or bacteriostatic activity have been reported to be found in Micromonospora and Streptomyces, including: rakicidin A and B were found in Micromonospora sp.R385-2 by Kimberly D.Mcbrien et al, 1995; in 2000, Rakicidin C was found by Hu Jin-Feng et al in Streptomyces sp.GT61042; rakicidin D was discovered in 2010 by Yasuhiro Igarashi et al from Streptomyces sp. MWW064; in 2014, Rakicidin E was found in Micromonospora sp.TP-A0860 by Naoya Oku et al; 2016. the inventors' topic group isolated a novel compound of Rakicidins- -Rakicidin B1; in 2017, Rakicidin F was found in Streptomyces sp.GKU220 by Shigeru Kitani et al.

In addition, the inventor subjects discovered and separated new structural compounds Rakicidin G (described in Chinese patent CN108530379A) and Rakicidin H (described in Chinese patent CN108586380A) for the first time in 2017. Research further determines that the Rakicidin H compound has better in-vitro inhibition effect on human colon cancer cells HCT-8 and human pancreatic cancer cells PANC-1. Therefore, the Rakicidin H compound has excellent clinical application prospect.

However, few studies and reports on fermentation production processes of Rakicidin H compounds exist in the existing research. Although Kimberly D.Mcbrien et al report that processes beneficial to fermentation and separation and purification of Rakicidins compounds are only limited to the shaking flask process stage, suitable industrial production is not developed, the whole fermentation period is long, the main component in the fermentation liquor is mostly Rakicidin A, and the content of Rakicidin B is low, so that the method is not suitable for fermentation production of Rakicidin H compounds. Although the Chinese patent CN108586380A discloses a fermentation preparation method of Rakicidin H compounds, in the method, wild strain FIM-02523 is adopted to carry out fermentation production of Rakicidin compounds, and a small amount of Rakicidin H samples can be obtained through separation and purification for structure identification, the yield and the purification yield of the compound Rakicidin H are extremely low, and industrial production is difficult to realize.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide a marine micromonospora strain for fermenting high-yield Rakicidin H so as to solve the problem of low Rakicidin H fermentation yield in the prior art;

the second technical problem solved by the invention is to provide a method for producing Rakicidin H compound by using the strain through fermentation.

In order to solve the technical problems, the marine Micromonospora strain is classified and named as Micromonospora sp.FIM-R181103, is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, has the preservation number of CGMCC NO.17014, the preservation date of 2018, 12 and 19 days and the preservation address of No. 3 Hospital No.1 of North Chen of the sunward area in Beijing.

The micromonospora marinensis strain is obtained by mutating micromonospora marinensis FIM02523 serving as a starting strain.

The invention also discloses application of the marine micromonospora strain in fermentation production of Rakicidins compounds.

The Rakicidins compound is Rakicidin H.

The invention also discloses a method for producing Rakicidin H by fermentation, which comprises the step of inoculating the marine micromonospora strain into a suitable fermentation culture medium for fermentation culture.

The fermentation medium comprises the following components in percentage by mass: 3.0-5.0% of soluble starch, 0.8-1.5% of cane sugar, 2.0-4.0% of cold-rolled soybean cake powder, 0.2-0.8% of ammonium sulfate, 0.1-0.5% of alanine and MgSO₄·7H₂O 0.03-0.06％，FeSO₄·7H₂O 0.004-0.008％，CuSO₄·5H₂O 0.004- 0.008％，CoCl₂·6H₂O 0.0004-0.0008％，CaCO₃0.4-0.8 percent of the total weight of the mixture, and adjusting the pH value to 7.0-7.5.

Preferably, the fermentation medium comprises the following components in percentage by mass: 5.0 percent of soluble starch, 0.8 percent of cane sugar, 3.0 percent of cold-rolled soybean cake powder, 0.5 percent of ammonium sulfate and 0.3 percent of alanine，MgSO₄·7H₂O 0.04％，FeSO₄·7H₂O 0.005％，CuSO₄·5H₂O 0.005％，CoCl₂·6H₂O 0.0005％， CaCO₃0.5 percent of water, adjusting the pH value to 7.5, and sterilizing for 30min at 121 ℃.

The conditions of the fermentation culture step are as follows: inoculating the strain with an inoculum size of 5.0-15.0%, controlling the rotation speed of 200-500rpm, ventilating at 0.5-1.8vvm, controlling the DO value in the fermentation process to be more than 20%, and performing fermentation culture at 28-36 ℃.

Preferably, the strain is inoculated into a fermentation tank at the inoculation amount of 10.0 percent, fermentation culture is carried out at the temperature of 30 ℃, the rotation speed is controlled to be 200rpm in the early stage of fermentation, the aeration is carried out at the speed of 0.8vvm, and the rotation speed is gradually increased to be 500rpm along with the growth of hypha; according to the change of DO parameters of the fermentation tank, the ventilation is gradually increased to 1.8vvm, and the DO value is controlled to be more than 20% all the time in the fermentation process.

Preferably, the fermentation process further comprises the step of supplementing amino acids, organic acid salts and/or organic alcohols to the fermentation liquor for feeding.

Specifically, the amino acid comprises alanine and/or 2-aminopentanoic acid;

the organic acid comprises butyric acid, caproic acid, caprylic acid and/or capric acid;

the organic acid salt comprises ammonium salt, sodium salt and/or potassium salt of butyric acid, caproic acid, caprylic acid or capric acid;

the organic alcohol comprises n-butanol and/or n-hexanol.

Most preferably, during the fermentation, a 2-aminopentanoic acid solution of 1.0% of the total volume of the fermentation broth is added by feeding between 15 and 72 hours after the fermentation has been carried out.

The method also comprises the step of inoculating the strain into a seed culture medium for seed liquid culture, wherein the seed culture medium comprises the following components in percentage by mass: 1.0-3.0% of soluble starch, 0.5-1.5% of glucose, 1.0-3.0% of yeast powder, MgSO4 & 7H₂O 0.04-0.08％，KH₂PO₄ 0.02-0.06％， CaCO₃0.1-0.3%, preparing with tap water, adjusting pH to 7.0-7.5, sterilizing at 121 deg.C for 30miAnd n, cooling, inoculating a certain amount of spore suspension, and culturing in seed liquid at 28-35 ℃.

Preferably, the seed culture medium comprises the following components in percentage by mass: 2.0 percent of soluble starch, 1.0 percent of glucose, 2.0 percent of yeast powder, MgSO4 & 7H₂O 0.05％，KH₂PO₄ 0.05％，CaCO₃0.2 percent of tap water, adjusting the pH value to 7.0-7.5, sterilizing for 30min at 121 ℃, cooling, inoculating a certain amount of spore suspension, and culturing the seed liquid at 32 ℃.

The seed liquid culture step comprises the steps of seed liquid culture in a shake flask and seed tank culture.

The method also comprises the step of inoculating the strain to a solid slant culture medium of the aspartokinase for activation culture, wherein the solid slant culture medium comprises the following components in percentage by mass: 1-3% of soluble starch, 0.04-0.06% of L-aspartyl and KNO₃ 0.08-0.12％，K₂HPO₄·3H₂O 0.04-0.06％， NaCl 0.04-0.06％，MgSO₄·7H₂O 0.04-0.06％，CaCO₃0.08-0.12% of agar and 1-2% of agar, and the pH value is adjusted to 7.5.

Preferably, the solid slant culture medium comprises the following components by mass: 2 percent of soluble starch, 0.05 percent of L-aspartyl and KNO₃ 0.1％，K₂HPO₄·3H₂O 0.05％，NaCl 0.05％， MgSO₄·7H₂O 0.05％，CaCO₃0.1 percent of agar and 1.5 percent of agar, and the pH value is adjusted to 7.5.

The mutant strain marine micromonospora strain FIM02523 for producing the Rakicidin compound is subjected to mutation breeding, so that the mutant strain marine micromonospora strain FIM-R181103 for producing Rakicidin H with high yield is obtained, the strain can effectively improve the titer of Rakicidin H in fermentation liquor, in a fermentation experiment in a 20-1000L fermentation tank, the titer of Rakicidin H produced by fermenting the marine micromonospora strain FIM-R181103 is as high as about 430mg/L, and is improved by about 6 times compared with the titer of a starting strain, so that the extraction and purification work of Rakicidin H is greatly facilitated, and the industrial requirements can be met.

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 an electron microscope scanning image of the mutant strain FIM-R181103 of the present invention;

FIG. 2 is a graph of the on-tank fermentation curve of the mutant strain FIM-R181103 of the present invention.

Detailed Description

EXAMPLE 1 mutation Breeding of the Strain

The mutation breeding method of micromonospora marinum FIM-R181103 comprises the following steps:

(1) preparing spore suspension: adding a proper amount of sterile normal saline into a fresh inclined surface of a mature cultured starting strain FIM02523 (preservation number is CGMCC NO.12132), lightly scraping by using an inoculation shovel, pouring into a sterile shake flask with glass beads, vibrating and scattering, and filtering hyphae to leave a spore suspension for later use;

(2) nitrosoguanidine (NTG) mutagenesis: weighing 200mg of NGT in a 100ml triangular flask, adding 2ml of acetone, and then adding 18ml of Tris-aminomethane maleic acid buffer solution to completely dissolve and uniformly mix the mixture to obtain 20ml of NTG solution with the concentration of 10 mg/ml; mixing NTG mother liquor with the prepared bacterial suspension to make NTG final concentration respectively 1mg/ml, 2mg/ml, 3mg/ml, and shake culturing at 32 deg.C for 60 min; centrifuging the mutagenized spore liquid immediately to remove NTG, repeatedly centrifuging and washing the spores for 3 times by using sterile physiological saline to prepare bacterial suspension with proper concentration; diluting the bacterial suspension to a proper multiple, and respectively taking 0.1mL of the bacterial suspension to coat the bacterial suspension on a Gao-shi aspartic acid flat plate; placing the uniformly coated flat plate in a constant-temperature incubator at 32 ℃ for 12 days, and using the well-cultured flat plate for calculating the lethality and screening strains;

(3) ARTP (atmospheric room temperature plasma) mutagenesis: setting the working power of an ARTP mutation breeding instrument to be 120W, setting the gas source to be argon, setting the gas flow to be 10L/min, setting the irradiation distance between a plasma emission source and a sample to be 2mm, and setting the irradiation time to be 0, 60, 120, 180, 240 and 360S; uniformly coating 10ul of prepared spore suspension on a sterile metal slide, and putting the sterile metal slide on a sample carrying platform in an ARTP instrument; performing plasma irradiation according to a program, after the sample is treated, placing a slide into an EP tube filled with 1ml of normal saline by using sterile forceps, oscillating the EP tube on an oscillator for 1min, and eluting microorganisms attached to the slide into liquid to form new bacterial suspension; properly diluting the new bacterial suspension, coating a flat plate with 0.1ml of diluent, putting the flat plate into a constant-temperature incubator at 32 ℃ for 12 days, and using the well-cultured flat plate for calculating the lethality and screening strains;

(4) NTG-ARTP compound mutation and Rakicidin H high-yield mutant strain screening: carrying out NTG and ARTP mutagenesis according to the above method to obtain preferred mutagenesis conditions, setting NTG mutagenesis conditions as NTG final concentration of 1mg/ml in bacterial suspension and chemical mutagenesis treatment time of 60min according to the standard that the lethality reaches 65%; the ARTP mutagenesis condition is 120W of the working power of an ARTP instrument, the gas source is argon, the gas flow is 10L/min, the irradiation distance between a plasma emission source and a sample is 2mm, and the irradiation time is 90S; directly using bacterial suspension obtained by NTG mutagenesis for ARTP mutagenesis; coating and separating the bacterial suspension obtained after the compound mutagenesis on a Gao's 1 culture medium, culturing for 10-15 days in a constant-temperature incubator at 28 ℃, and using the cultured plate for calculating the lethality and screening strains;

(5) and (3) strain breeding: the strain breeding model is a 2-aminovaleric acid resistance model. In the 2-aminopentanoic acid resistance model, 2-aminopentanoic acid is a structural analogue of a side chain biosynthesis precursor of Rakicidin H, can be used for feedback inhibition and catalysis of key enzymes for Rakicidin H side chain biosynthesis, can be used for removing the feedback inhibition of 2-aminopentanoic acid, can effectively improve the fermentation titer of Rakicidin H, and can be used for greatly improving the probability of breeding Rakicidin H high-yield strains by adopting 2-aminopentanoic acid as a resistance breeding standard. The standard for screening concentration of the 2-aminopentanoate resistance model is shown in table 1 below.

TABLE 12 Aminopentanoic acid tolerance structure

2-amino-pentanoic acid concentration (%)	Growth conditions ^*
		0	++++
0.2	++++
		0.4	+++
0.6	++
		0.8	+
1.0	-

Excellent in +++; good, if not, then; medium ++, medium; +: difference; no growth.

Inoculating the bacterial suspension obtained by mutagenesis in a small amount of seed culture medium for amplification and propagation, then uniformly distributing and inoculating the grown seed liquid to a seed culture medium containing 1.0% of 2-aminovaleric acid in a 96-deep-hole culture plate according to the inoculation amount of 5%, wherein each hole plate is filled with about 0.5ml of the seed culture medium which is equivalent to an independent screening and enriching system, and selecting the well-grown bacterial liquid for carrying out slant plate transfer and fermentation plate after culturing for 48 hours at 28 ℃. The inclined plate is a 96-deep-well plate, a small amount of aspartyl solid culture medium is added into the plate, and the solid culture medium is cultured at the constant temperature of 28 ℃ for later use. The fermentation plate is a 96-deep-well plate containing 1.0% of 2-aminopentanoic acid fermentation medium, and is subjected to shaking fermentation at the constant temperature of 28 ℃ for 96 hours. And after fermentation, adding 3 times volume of methanol into the fermentation liquor, uniformly mixing, performing ultrasonic treatment for 30min, centrifuging at 10000rpm, taking supernatant, performing HPLC (high performance liquid chromatography) determination, and selecting strains with high Rakicidin H titer and good components for natural separation and passage re-screening. An electron microscope scanning image of a high-yield mutant strain with the serial number of Micromonospora sp, FIM-R181103 is obtained through multiple rounds of compound mutagenesis and mass breeding, and the mutant strain FIM-R181103 is shown in figure 1.

Example 2 mutant stability Studies

After mutagenesis, the obtained high-yield strain micromonospora FIM-R181103 is subjected to slant continuous passage for 6 generations, and no obvious change is found in the fermentation titer of Rakicidin H. The strain is stored in a seed preserving cabinet at 20 ℃ and is subjected to passage once every 2 months for fermentation verification, and the Rakicidin H fermentation titer of the strain is not obviously changed within 12 months. The conclusion shows that the mutant strain FIM-R181103 has high yield and stable heredity, and is suitable for industrial production. The obtained strain, namely the micromonospora FIM-R181103, is stored in the common microorganism center of the China general microbiological culture Collection center with the preservation number of CGMCC No.17014 and the preservation date of 2018, 12 months and 19 days.

EXAMPLE 3 comparison of starting FIM02523 strain and mutant FIM-R181103 strain in flask fermentation

The freshly cultured marine micromonospora FIM02523 and mutant FIM-R181103 slant spores are respectively scraped to prepare bacterial suspensions, the bacterial suspensions are inoculated into shake flask seed culture, the culture is carried out at 32 ℃ and 250rpm for 48 hours, the bacterial suspensions are inoculated into shake flask fermentation medium according to the inoculum size of 10 percent, the culture is carried out at 30 ℃ and 250rpm for 120 hours, then the culture medium is placed in a bottle, and the fermentation product is measured by HPLC (the HPLC detection refers to Chinese patent CN 108586380A).

Preparing a seed culture medium formula (mass fraction): 2.0 percent of soluble starch, 1.0 percent of glucose, 2.0 percent of yeast powder, MgSO4 & 7H₂O 0.05％，KH₂PO₄ 0.05％，CaCO₃0.2 percent of tap water, adjusting the pH value to 7.0-7.5, sterilizing for 30min at 121 ℃, cooling, inoculating a certain amount of spore suspension, and culturing the spore suspension in a seed solution at 32 ℃.

Preparing a fermentation medium formula (mass fraction): 5.0 percent of soluble starch, 0.8 percent of cane sugar, 3.0 percent of cold-rolled soybean cake powder, 0.5 percent of ammonium sulfate, 0.3 percent of alanine, and MgSO₄·7H₂O 0.04％， FeSO₄·7H₂O 0.005％，CuSO₄·5H₂O 0.005％，CoCl₂·6H₂O 0.0005％，CaCO₃0.5 percent of the seed culture broth is prepared by tap water, the pH value is adjusted to 7.5, the seed culture broth is sterilized at 121 ℃ for 30min, and the seed culture broth is inoculated into the cultured seed liquid according to the inoculum size of 10 percent after being cooled and cultured at 32 ℃.

The content of Rakicidin H in the fermentation liquor is detected by HPLC in the fermentation process, the fermentation titer of the original strain FIM02523 is about 63mg/L after the fermentation is finished, the shake flask fermentation titer of the mutant strain FIM-R181103 is 232mg/L, and the improvement is 368.2%.

Example 4 fermentation of mutant strain FIM-R150103 on a 20L fermenter

Seed medium and fermentation medium were prepared according to the recipe in example 3.

The seed culture medium is shake flask seeds, the liquid loading capacity of each 500ml shake flask is 80ml, and the seeds are cultured for 48 hours at the temperature of 28 ℃ and the rpm of 250; then inoculating the strain into a 20L fermentation tank (actual liquid loading is 13L) according to the inoculation amount of 10 percent for fermentation, culturing at 28 ℃, controlling the tank pressure to be 0.03-0.05Mpa and the initial rotating speed to be 200rpm, gradually adjusting to 400rpm according to the change of fermentation parameters DO after 48 hours, and adjusting the aeration quantity to be 1: 1.2vvm, about 96-120 hours to the end of fermentation.

In the fermentation process, the Rakicidin H content in the fermentation liquor is detected by HPLC, and the final fermentation titer is 256 mg/L.

Example 5

The fermentation process of this example is the same as that of example 4, except that 1.0% 2-aminovaleric acid solution was fed 15 hours after the start of the fermentation: namely continuously adding 2-aminovaleric acid solution with the total volume of 1.0 percent of the volume of the fermentation liquid at a constant speed between 16 hours and 72 hours of fermentation. In the fermentation process, the Rakicidin H content in the fermentation liquor is detected by HPLC, and the final fermentation titer is 432 mg/L.

Example 6

The fermentation process of this example was the same as that of example 4 except that the amount of the solution in the 100L fermenter was 70L and the inoculum amount was 10%, and that 1.0% 2-aminovaleric acid solution was fed in 15 hours after the start of the fermentation: namely continuously adding 2-aminovaleric acid solution with the total volume of 1.0 percent of the volume of the fermentation liquid at a constant speed between 15 hours and 72 hours of fermentation. In the fermentation process, the Rakicidin H content in the fermentation liquor is detected by HPLC, and the final fermentation titer is 430 mg/L.

Example 7

The fermentation process of this example was the same as that of example 4 except that the amount of the liquid in the 1000L fermenter was 720L, and 1.0% 2-aminopentanoic acid solution was fed 15 hours after the start of the fermentation: namely continuously adding 2-aminovaleric acid solution with the total amount of 1.0 percent of the volume of the fermentation liquid at a constant speed between 15 hours and 72 hours of fermentation. The fermentation process is characterized in that the Rakicidin H content in the fermentation liquor is detected by HPLC, the highest fermentation titer is 410mg/L, and the fermentation process curve is shown in figure 2.

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

1. Marine micromonospora strain, its classification and name areMicromonosporaFIM-R181103, which has been preserved in China general microbiological culture Collection center (CGMCC), with the preservation number of CGMCC No. 17014.

2. Use of a marine micromonospora strain according to claim 1 for the fermentative production of a Rakicidins compound.

3. Use according to claim 2, wherein the compound Rakicidins is Rakicidin H.

4. A method for producing Rakicidin H by fermentation, which comprises the step of inoculating the marine micromonospora strain of claim 1 in a suitable fermentation medium for fermentation culture.

5. Process for the fermentative production of Rakicidin H according to claim 4, wherein the fermentation medium comprisesThe composition comprises the following components in percentage by mass: 3.0-5.0% of soluble starch, 0.8-1.5% of cane sugar, 2.0-4.0% of cold-rolled soybean cake powder, 0.2-0.8% of ammonium sulfate, 0.1-0.5% of alanine and MgSO₄·7H₂O 0.03-0.06%，FeSO₄·7H₂O 0.004-0.008%，CuSO₄·5H₂O 0.004-0.008%，CoCl₂·6H₂O 0.0004-0.0008%，CaCO₃0.4-0.8 percent of the total weight of the mixture, and adjusting the pH value to 7.0-7.5.

6. A method for fermentative production of Rakicidin H according to claim 5, wherein the conditions of the fermentation step are: inoculating the strain with an inoculum size of 5.0-15.0%, controlling the rotation speed of 200-500rpm, ventilating at 0.5-1.8vvm, controlling the DO value in the fermentation process to be more than 20%, and performing fermentation culture at 28-36 ℃.

7. A method for fermentative production of Rakicidin H according to any of claims 4-6, further comprising the step of feeding 2-aminopentanoic acid to the fermentation broth during fermentation.

8. A method for fermentative production of Rakicidin H according to claim 7, further comprising the step of inoculating the strain in a seed culture medium for seed broth culture, said seed culture medium comprising the following components in mass: 1.0-3.0% of soluble starch, 0.5-1.5% of glucose, 1.0-3.0% of yeast powder, MgSO4 & 7H₂O 0.04-0.08%，KH₂PO₄ 0.02-0.06%，CaCO₃0.1-0.3 percent of the total nutrient solution is prepared by tap water, the pH value is adjusted to 7.0-7.5, and the seed solution culture is carried out at 28-35 ℃ after sterilization.

9. The method for fermentative production of Rakicidin H according to claim 8, further comprising the step of inoculating the strain in a solid slant culture medium of aspartokinase for activation culture, wherein the solid slant culture medium comprises the following components in parts by mass: 1-3% of soluble starch, 0.04-0.06% of L-aspartyl and KNO₃ 0.08-0.12%，K₂HPO₄·3H₂O 0.04-0.06%，NaCl 0.04-0.06%，MgSO₄·7H₂O 0.04-0.06%，CaCO₃0.08-0.12% of agar and 1.2-2.2% of agar, and the pH value is adjusted to 7.5.