CN107955792B

CN107955792B - Lovastatin high-yield monascus and high-throughput screening method thereof

Info

Publication number: CN107955792B
Application number: CN201710222063.5A
Authority: CN
Inventors: 李一婧; 马伟超; 王廷璞; 包文生; 罗爱玉
Original assignee: Tianshui Normal University
Current assignee: Tianshui Normal University
Priority date: 2017-04-06
Filing date: 2017-04-06
Publication date: 2021-05-07
Anticipated expiration: 2037-04-06
Also published as: CN107955792A

Abstract

The invention relates to the technical field of microorganisms, in particular to lovastatin high-yield monascus and a high-throughput screening method thereof. The high-throughput screening method has the characteristics of simple operation steps, high throughput, high accuracy, less equipment investment and the like, and can be used for high-throughput screening of other secondary metabolite production strains by slightly changing the method. The Monascus anka Mon20-2(CCTCC M2015356) has high lovastatin content, and the lovastatin yield can reach 2952.0mg/L in large-scale production by applying the fermentation process disclosed by the invention, which is far superior to that reported in the prior art.

Description

Lovastatin high-yield monascus and high-throughput screening method thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to lovastatin high-yield monascus and a high-throughput screening method thereof.

Background

Monascus (Monascus spp.) is a small filamentous saprophytic fungus belonging to Ascomycotina (Ascomycotina) Ascomycotina (Plectomycetes) Eurotiales (Eurotiales) Monascus (Monacaceae), under which only one genus of Monascus is established in 1884 for Van Tieghem, a French scholar. The main species are monascus (m.anka), monascus smoky (m.fuliginosus), monascus purpureus (m.purpureus), monascus ruber (m.ruber), and the like.

The monascus purpureus can produce various primary metabolites of aromatic substances such as alcohol, vinegar and acid and various hydrolases, so that the fermented food has high-quality aroma and sweet taste, and is also an excellent brewing material. Especially, secondary metabolites such as pigments, antibiotics, cholesterol inhibitors, traditional Chinese medicine components and the like generated by the compound are the hot spots of research of people in recent years. The most important bioactive metabolite is the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor Lovastatin (Lovastatin), also known as Monacolin K. Lovastatin can significantly reduce cholesterol and low density lipoprotein cholesterol level, has good blood lipid reducing effect, has no accumulation of cholesterol substances, does not affect cholesterol amount required by normal function of cell membrane under therapeutic dose, and has less adverse reaction.

Because the production level of wild monascus lovastatin is always low, space mutation breeding becomes one of important contents of space life science research in recent years, and after space mutation, various mutation factors such as strong radiation, zero gravity, high cleanliness, high vacuum and the like of a space environment provide incomparable mutation conditions for human beings. Under the synergistic action of various factors, the biological characteristics are changed, the content of the mutated monascus lovastatin is possibly increased, unchanged or reduced, and therefore, the mutated strains need to be screened. However, most of the existing screening methods for high-yield lovastatin monascus are based on shaking tests, and the method has the disadvantages of large investment in manpower and material resources and low efficiency, and cannot meet the requirements of actual production.

Disclosure of Invention

The invention aims to solve the technical problems of providing a high-throughput screening method of lovastatin high-yield monascus and a high-yield lovastatin process thereof, solving the problems of complex screening process, low screening efficiency and the like after mutagenesis of lovastatin-producing monascus in the prior art and solving the problem of low lovastatin yield of the prior monascus.

In order to solve the technical problem, the application discloses Monascus anka Mon20-2(CCTCC M2015356). The Monascus monascu anka Mon20-2 is obtained by carrying parent Monascus 30192 (purchased from the China agricultural microbial culture collection management center, strain number ACCC 30192) on China eight spaceships, carrying out space mutagenesis, screening, and preserving in the China center for type culture collection (China, Wuhan) at 6-4 days 2015 with the preservation number CCTCC M2015356.

On the other hand, the application also discloses the application of the monascus in the production of lovastatin.

On the other hand, the invention discloses a high-throughput screening method of a high-yield lovastatin monascus mutant strain, which comprises the following steps:

scraping the activated mutagenic strain colony in a seed culture medium plate by using an inoculating shovel, inoculating the activated mutagenic strain colony into a deep hole plate containing a primary screening fermentation culture medium, and carrying out shake cultivation at 27 ℃ and 180rpm for 7 days;

centrifuging the deep-hole plate filled with the fermentation liquor at 4000rpm for 5 minutes, taking 0.2ml of supernatant, adjusting the pH to about 3.0, adding equal volume of ethyl acetate, performing water bath shaking extraction at 50 ℃ and 150rpm for 2 hours, centrifuging at 3000rpm for 5 minutes, taking supernatant into a new centrifuge tube, and drying the solvent by using a vacuum drying pump;

dissolving the crystal with 0.5-1mL of methanol, centrifuging, collecting supernatant, diluting properly, and measuring the ultraviolet absorbance at 237 nm;

the primary screening fermentation medium comprises the following components: 3-7% of rice flour, 1-2% of ammonium chloride, 0.15% of dipotassium hydrogen phosphate and 0.05% of MgSO₄·7H₂O。

The deep-hole plate is a 24-hole or 48-hole or 96-hole deep-hole plate.

On the other hand, the application also discloses a fermentation process for high-yield lovastatin. The process comprises the following steps: inoculating Monascus anka Mon20-2 to a fermentation medium containing 3-7% rice flour, 1-2% ammonium chloride, MgSO4 & 7H2O 0.05.05% and potassium dihydrogen phosphate 0.15%, wherein the initial pH is 3.0-7.0, the liquid loading amount in a 250mL triangular flask is 30-50mL, and fermenting at 27 deg.C and 180rpm for 13 days.

In addition, in the present invention, the seed medium is a conventional medium for culturing monascus, such as a malt extract medium.

In the present invention, a nutrient medium containing assimilable carbon sources, nitrogen sources, inorganic salts and the like can be used as the fermentation medium. Examples of the carbon source include glycerol, rice flour, lactose, glucose, and maltose. As the nitrogen source, various inorganic and organic nitrogen-containing compounds such as peptone, yeast extract, soybean powder, ammonium chloride and ammonium sulfate can be used. As the inorganic salt, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, or the like can be used. In addition, micronutrient sources such as biotin, thiamine, and vitamin B6 may be added as necessary.

The high-throughput screening method has the characteristics of simple operation steps, high throughput, high accuracy, less equipment investment and the like, and can be used for high-throughput screening of other secondary metabolite production strains by slightly changing the method. The lovastatin produced by the monascus of the invention has high content, and the lovastatin yield can reach 2952.0mg/L in large-scale production by applying the fermentation process disclosed by the invention, which is far superior to that reported in the prior art. The fermentation process disclosed by the invention can be amplified step by step and is used for producing functional red rice powder.

Drawings

FIG. 196 correlation validation of well plates with Shake flask culture

FIG. 2 rescreening results of mutagenized strains

FIG. 3 Effect of different carbon sources on the production of lovastatin

FIG. 4 Effect of carbon Source content on lovastatin production

FIG. 5 Effect of different Nitrogen sources on lovastatin production

FIG. 6 Effect of different initial pH on lovastatin production

FIG. 7 Effect of liquid loading on lovastatin production

FIG. 8 Effect of fermentation time on lovastatin production

Detailed Description

The invention is further illustrated below with reference to specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Test materials

Strain: the starting strain is monascus 30192, purchased from China agricultural microorganism culture preservation management center (ACCC 30192), carried by China eight spaceship and subjected to space mutagenesis.

Reagent: malt extract, ammonium chloride, ammonium sulfate, potassium sulfate, magnesium sulfate, sodium hydroxide, potassium dihydrogen sulfate, etc. are analytically pure.

Test equipment: a Thermo U-3000HPLC system, an ultraviolet visible spectrophotometer, an electric heating constant temperature incubator, a DZF-6030 type vacuum drier, a multi-channel pipettor, a Multipette M4 dispenser, a 96-hole deep-hole plate (2 ml).

The lovastatin determination method comprises the following steps: the high throughput screening was measured spectrophotometrically using a varioskann (tm) lux multi-functional microplate reader. The product detection in the optimization process of the fermentation process adopts liquid chromatography and is measured by a Thermo U-3000HPLC system.

Example 1 high throughput screening of high-yielding lovastatin-producing strains of Monascus purpureus by mutagenesis in space

Weighing a proper amount of monascus lyophilized powder, diluting by 50 times with peptone sodium chloride activating solution, coating on malt extract agar culture medium, and culturing for 5-7 days at 30 ℃. After activation, the monascus colonies in the plate are scraped by an inoculation shovel and inoculated into a 96-hole deep-hole plate containing 1ml of primary screening fermentation medium, and the plate is subjected to shake cultivation at 27 ℃ and 180rpm for 7 days. The primary screening fermentation medium comprises the following components: 7% rice flour, 1.5% ammonium chloride, 0.15% dipotassium hydrogen phosphate, 0.05% MgSO₄·7H₂O, pH is natural.

Centrifuging a 96-well plate filled with fermentation liquor at 4000rpm for 5 minutes, taking 0.2mL of supernatant by using a multi-channel pipette, adjusting the pH value to be about 3.0 by using phosphoric acid, adding equal volume of ethyl acetate, extracting for 2 hours by using a water bath shaker at 50 ℃ and 150rpm, centrifuging for 5 minutes at 3000rpm, taking the supernatant into a new centrifuge tube, pumping out a solvent by using a vacuum drying pump, dissolving crystals by using 0.5mL of methanol, centrifuging to take the supernatant, appropriately diluting, and measuring the ultraviolet absorption value at 237 nm.

Randomly selecting 5 separated strains as research strains, respectively inoculating the research strains into a 96-pore plate and a 250ml shake flask for fermentation culture, stopping fermentation on the 7 th day, respectively detecting the ultraviolet absorption values, and respectively detecting the results as shown in figure 1.

The results of the high-throughput screening are shown in Table 1, the obtained results are subjected to SPSS 16.0 cluster analysis, the initially screened 53 strains can be divided into two categories, and 6 strains with higher lovastatin production are obtained by screening and are respectively named as 37-2, B11-1, 20-3, 20-2, A25 and B40.

TABLE 1 mutant strains high throughput screening results

Strain numbering	OD237	Strain numbering	OD237
				37—2	0.886±0.013	A23	0.167±0.017
B11-1	0.86±0.007	1—8	0.165±0.046
				20—2	0.672±0.018	B1-1	0.165±0.005
B40	0.618±0.036	24—5	0.161±0.012
				20—3	0.562±0.006	21—3	0.149±0.018
A25	0.535±0.024	17—2	0.139±0.024
				B10	0.485±0.013	B18-2	0.133±0.044
B3	0.483±0.01	26—1	0.126±0.002
				20—1	0.469±0.003	36—2	0.115±0.039
A9	0.408±0.046	36—1	0.112±0.041
				B24-6	0.327±0.021	4-1	0.097±0.012
B24-3	0.301±0.031	B21	0.093±0.016
				37—1	0.292±0.042	25-2	0.089±0
B22-1	0.292±0.045	43-1	0.087±0.012
				B24-2	0.243±0.013	26-2	0.084±0.049
35—1	0.239±0.038	B5	0.081±0.017
				25—1	0.233±0.047	B1-2	0.078±0.026
41—2	0.228±0.033	10-1	0.074±0.029
				48—1	0.22±0.013	A26	0.07±0.04
A15	0.215±0.004	B18-1	0.07±0.015
				B24-4	0.204±0.039	17-1	0.068±0.02
41—1	0.199±0.016	C2	0.067±0.028
				B24-1	0.193±0.035	B11-2	0.06±0.023
B22-2	0.184±0.004	21-1	0.055±0.032
				B15	0.181±0.002	21-2	0.048±0.039
26—3	0.177±0.028	9-1	0.038±0.004
				B22-1	0.176±0.036

Example 2 rescreening of aerospace Monascus 30192 high-yield lovastatin-producing strains

6 strains such as 37-2, B11-1, 20-3, 20-2, A25, B40 and the like are selected for shake flask rescreening. The seed solution of the above strain was inoculated at an inoculum size of 7% into a 250mL Erlenmeyer flask containing 40mL of fermentation medium, and shake-cultured at 27 ℃ and 180 rpm. The fermentation medium comprises 7% rice flour, 1.5% ammonium chloride, 0.15% dipotassium hydrogen phosphate, and 0.05% MgSO₄·7H₂O, pH is natural. The lovastatin production was measured 7 days after fermentation.

The lovastatin content is detected by using a liquid chromatography, and the specific steps are as follows: centrifuging the fermentation liquor at 4000rpm for 5 minutes, taking 4mL of supernatant, adjusting the pH value to 3 with phosphoric acid, adding equal volume of ethyl acetate, extracting for 2 hours at 50 ℃ and 150rpm in a water bath shaker, centrifuging at 3000rpm for 3 minutes, taking supernatant to a new centrifuge tube, pumping out the solvent by using a vacuum drying pump, dissolving crystals by using 4mL of methanol, filtering the crystals by using a 0.22 mu m filter membrane, and detecting by liquid chromatography. HPLC analysis was carried out using a Thermo U-3000HPLC system, and the column was a Thermo Hypersil GOLD C18 column (250 mm. times.4.6 mm. times.5 μm). The HPLC conditions were as follows: mobile phase methanol: water: 385 parts of phosphoric acid: 115: 0.14 (volume fraction), flow rate: 1.0 ml/min; column temperature: 28 +/-1 ℃; sample introduction amount: 20 μ l. DAD detector (DAD-3000), detection wavelength 238 nm.

As shown in FIG. 2, the yield of lovastatin produced by strain Mon20-2 was higher than that of other strains, and reached 546.7 mg/l.

Example 3 Effect of carbon source and carbon Source content on lovastatin production

7 percent of carbon source, 1.5 percent of peptone, 0.2 percent of ammonium chloride and MgSO₄·7H₂0.05% of O and 0.15% of monopotassium phosphate are used as fermentation culture media, the influence of glycerol, rice flour, lactose, glucose and maltose as carbon sources on the yield of lovastatin is detected, the inoculation amount is 7%, the liquid loading amount of a 250mL triangular flask is 40mL, and the lovastatin content in fermentation liquid is detected after shaking fermentation at 27 ℃ and 180rpm for 7 days. The detection method was the same as in example 2.

As shown in FIG. 3, the yield of lovastatin was the highest when glycerol was used as the carbon source, and the yields were 590mg/L and 567.5mg/L, respectively, after that of rice flour. Glycerol is not only used as a nutrient substance for the absorption of Monascus, but also, due to its molecular structure, it may serve as a precursor to promote the synthesis of lovastatin. The rice flour is characterized in that various natural components such as vitamins in the rice flour provide a good nutrition system, and the rice flour is easy to attach and absorb microorganisms in a granular state, so that the yield is high. No significant difference exists between the two analysis methods, so that the rice flour is preferably selected as the carbon source for economic reasons.

The optimum contents of carbon sources were investigated with the rice flour contents of 1%, 3%, 5%, 7%, 9% and 11%, respectively, and the results are shown in fig. 4, where the rice flour content of 5% resulted in a lovastatin yield of 979 mg/L.

Example 4 influence of Nitrogen Source on the production of lovastatin

1.5 percent of nitrogen source, 5 percent of rice flour and MgSO₄·7H₂Preparing a culture medium by 0.05 percent of O and 0.15 percent of monopotassium phosphate, and detecting the influence on the yield of lovastatin when peptone, yeast extract, soybean meal, ammonium chloride and ammonium sulfate are used as nitrogen sources. Taking a mixed nitrogen source with peptone 1.5% and ammonium chloride 0.2% as a reference, wherein the inoculation amount is 7%, the liquid loading amount of a 250mL triangular flask is 40mL, and the lovastatin content in the fermentation liquid is detected after shaking culture for 7 days at 27 ℃ and 180 rpm. The detection method was the same as in example 2. As a result, as shown in FIG. 5, the nitrogen source was 1.5% ammonium chlorideThe yield is obviously higher than other nitrogen sources, and reaches 1474 mg/L.

Example 5 Effect of initial pH on lovastatin production

Adding 5% of rice flour, 1.5% of ammonium chloride and MgSO 5 into a 250mL triangular flask₄·7H₂Fermentation medium of 0.05% O, 0.15% potassium dihydrogen phosphate, and 40ml of deionized water adjusted to

pH

3, 5, 7, 9 and 11 were added to the above medium, and the effect of different initial pH on lovastatin production was examined. The inoculum size is 7%, and the lovastatin content in the fermentation liquid is detected after fermentation for 7 days at 27 ℃ and 180rpm in a shaking table. The detection method was the same as in example 2. As shown in FIG. 6, the maximum lovastatin production was 1829mg/L at an initial pH of 5.0.

Example 6 Effect of liquid Loading on lovastatin production

The liquid contents in a 250mL triangular flask were 30, 40, 50, 60, 70, and 80mL, respectively, in terms of rice flour 5%, ammonium chloride 1.5%, and MgSO₄·7H₂Preparing a culture medium by 0.05 percent of O and 0.15 percent of monopotassium phosphate, wherein the inoculation amount is 7 percent, and detecting the lovastatin content in the fermentation liquor after fermentation is carried out for 7 days at 27 ℃ and 180rpm by a shaking table. The detection method was the same as in example 2. As shown in FIG. 7, the yield reached as high as 1833mg/L when the liquid loading was 40 mL.

Example 7 Effect of fermentation time on lovastatin production

According to the weight percentage of 5 percent of rice flour, 1.5 percent of ammonium chloride and MgSO₄·7H₂Preparing a culture medium by 0.05 percent of O and 0.15 percent of monopotassium phosphate, wherein the inoculation amount is 7 percent, loading the culture medium into a 250mL triangular flask by 40mL, fermenting the culture medium by a shaking table at 27 ℃ and 180rpm, and detecting the lovastatin content in the fermentation liquid when the fermentation liquid is fermented for 7d, 10d, 13d, 16d, 19d, 22d and 25d respectively, wherein the detection method is the same as that in example 2. As shown in FIG. 8, the maximum yield of lovastatin reached 2187mg/L at a fermentation time of 13 days.

Example 8 fermentation Condition optimization (orthogonal test)

The factors and levels of the orthogonality test are shown in Table 2, using L₉(3⁴) The orthogonal test further optimizes the fermentation conditions. The other component composition of the culture medium is 0.05% MgSO₄·7H₂O、0.15％Potassium dihydrogen phosphate, 250mL triangular flask, 27 ℃, 180rpm shake fermentation 13 days later to detect the lovastatin content in the fermentation broth, the detection method is the same as example 2.

TABLE 2 fermentation condition orthogonal experiment design factor horizon

As shown in Table 3, the effect of the four factors on the yield of lovastatin in the fermentation broth is most significant in terms of the liquid loading capacity, and the primary and secondary relationships of the four factors are the liquid loading capacity in turn>Initial pH>Rice flour content>The optimum factor level combination of ammonium chloride content is A₂B₃C₁D₂Namely 5% rice flour, 2% ammonium chloride, a liquid containing amount of 30ml and a pH value of 5.0. When the strain Mon20-2 is cultured by using the culture medium, the yield of lovastatin in fermentation liquor can reach 2952.0mg/L after 13 days under the conditions of 27 ℃ and 180rpm, and compared with 2186.8mg/L before optimization, the yield of lovastatin is improved by 1.35 times.

TABLE 3 results and analysis of orthogonal experiments

The scope of the invention is not to be limited by the specific embodiments described, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Each of the above-mentioned references is incorporated herein by reference in its entirety.

Claims

1. Monascus anka Mon20-2 with biological preservation number of CCTCC M2015356.

2. Use of Monascus anka Mon20-2 to produce lovastatin as claimed in claim 1.

3. A fermentation process for high yield of lovastatin comprises the following steps: inoculating Monascus anka Mon20-2 of claim 1 to a solution containing 3-7% rice flour, 1-2% ammonium chloride, MgSO₄·7H₂0.05% of O and 0.15% of monopotassium phosphate, the initial pH value is 3.0-7.0, the liquid loading amount in a 250mL triangular flask is 30-50mL, and the fermentation is carried out for 13 days at 27 ℃ and 180 rpm.