A method for fermentative production of aspochalasin d

Abstract

The application discloses a method for fermentatively producing Aspochalasin D and belongs to the technical field of microorganisms and fermentation. The method is characterized in that the liquid fermentation conditions of Aspergillus flavipes are optimized, and a mutant strain ΔaspoA::OEaspoG strain is obtained through aspoA gene knockout and aspoG gene overexpression. The fermentative production of Aspochalasin D by using the method can make the yield of Aspochalasin D reach 812.1 mg / L. The cell relaxation Aspochalasin D obtained through the fermentation of the mutant strain of Aspergillus flavipes has various activities such as antibacterial activity and anticancer activity, and can be applied to the agricultural and pharmaceutical industries.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for fermentative production of Aspochalasin D, and belongs to the field of microorganisms and fermentation technology. BACKGROUND

[0002] Aspochalasin D is a natural product of polyketide-amino acid hybrid, and one of its main sources is Aspergillus flavipes. The compound has typical cytochalasin activity and can specifically bind to actin. In addition, the compound also has antibacterial, anticancer, antifouling and 5-hydroxytryptamine release inhibition activities. Therefore, it has great application prospects in agriculture and drug development.

[0003] At present, Aspochalasin D can be prepared by chemical synthesis and microbial fermentation. The chemical synthesis method has many steps and complex reaction conditions, and it is difficult to efficiently prepare Aspochalasin D. The microbial fermentation method is limited by the limited synthesis capacity of the wild-type strain and the production of other by-products, resulting in low fermentation yield. Therefore, it is urgent to develop a method for efficiently fermenting Aspochalasin D. SUMMARY

[0004] Since the Aspochalasin D yield of the wild-type Aspergillus flavipes is low and there are many by-products, and there is no method to effectively improve the Aspochalasin D yield at present.

[0005] In order to solve the above problems, the present application provides a method for fermenting Aspochalasin D. The method improves the production efficiency of Aspochalasin D of Aspergillus flavipes and reduces the by-products of Aspochalasin D synthesized by Aspergillus flavipes through fermentation process optimization and gene editing technology. The Aspochalasin D yield of Aspergillus flavipes is improved by using the method of the present application.

[0006] The present application provides a recombinant Aspergillus flavipes, which is an expression host of Aspergillus flavipes CGMCC3.17641, the aspoA gene on the genome is knocked out, and an overexpression cassette containing a transcriptional regulator aspoG derived from itself is integrated and expressed.

[0007] In an embodiment of the present application, the nucleotide sequence of the aspoA gene is shown in SEQ ID NO. 1.

[0008] In an embodiment of the present application, the expression cassette is composed of a promoter PgpdA, a terminator TcgrA and an aspoG gene from Aspergillus nidulans, and the nucleotide sequence of the expression cassette is shown in SEQ ID NO. 2.

[0009] The application also provides a method for fermentative production of Aspochalasin D, which comprises adding the Aspergillus flavipes CGMCC 3.17641 or the recombinant Aspergillus flavipes into a fermentation medium to fermentatively produce Aspochalasin D.

[0010] In an embodiment of the application, the fermentation medium comprises 10-50 g / L glucose, 1-5 g / L soybean peptone, 1-3 g / L sodium acetate, 0.5-3 g / L leucine, 1-9 mg / L L-phenylalanine, 20-180 mg / L sodium benzoate, 100-200 mg / L potassium dihydrogen phosphate, 0.5-1.5 mg / L biotin, 3-10 mg / L calcium nitrate, 0.5-1.5 mg / L pyridoxal phosphate, 0.5-1.5 mg / L calcium pantothenate, 0.5-1.5 mg / L thiamine hydrochloride, 1-9 mg / L manganese chloride, 1-3 mg / L ferric chloride, 0.5-1.5 mg / L copper nitrate, 0.5-4.5 mg / L magnesium sulfate, 1-4 mg / L zinc sulfate.

[0011] In an embodiment of the application, the fermentation medium comprises 25.2 g / L glucose, 3 g / L soybean peptone, 1 g / L sodium acetate, 1.4 g / L leucine, 5 mg / L L-phenylalanine, 100 mg / L sodium benzoate, 136 mg / L potassium dihydrogen phosphate, 1 mg / L biotin, 6.5 mg / L calcium nitrate, 1 mg / L pyridoxal phosphate, 1 mg / L calcium pantothenate, 1 mg / L thiamine hydrochloride, 5 mg / L manganese chloride, 2 mg / L ferric chloride, 1 mg / L copper nitrate, 3.6 mg / L magnesium sulfate, 2.5 mg / L zinc sulfate.

[0012] In an embodiment of the application, the Aspergillus flavipes CGMCC 3.17641 or the recombinant Aspergillus flavipes is added in an amount of 10 6 ~ 10 8 spores.

[0013] In an embodiment of the application, the Aspergillus flavipes CGMCC 3.17641 or the recombinant Aspergillus flavipes is added into the fermentation medium, and the fermentation is carried out at a temperature of 21-31℃, an initial pH of 6.0-8.0, and a rotation speed of 100-160 r / min.

[0014] In an embodiment of the application, the Aspergillus flavipes CGMCC 3.17641 or the recombinant Aspergillus flavipes is added into the fermentation medium, and the fermentation is carried out at a temperature of 29℃, an initial pH of 6.0-8.0, and a rotation speed of 135 r / min.

[0015] The application further provides application of the recombinant Aspergillus fl avus or the Aspergillus fl avus CGMCC 3.17641 in preparation of Aspochalasin D or a product containing Aspochalasin D.

[0016] Advantages

[0017] The application optimizes fermentation process parameters such as temperature, time, pH, carbon source, nitrogen source and leucine concentration, and uses the Aspergillus fl avus ΔaspoA::OEaspoG mutant strain obtained by knocking out aspoA gene and overexpressing aspoG gene, so that the yield of Aspochalasin D is greatly improved. The Aspochalasin D produced by the fermentation method of the application can have a yield of up to 812.1 mg / L, and has great application prospects in agriculture and pharmaceutical industry. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a structural diagram of Aspochalasin D.

[0019] Figure 2 It is a plate morphology of the Aspergillus fl avus ΔaspoA::OEaspoG mutant strain.

[0020] Figure 3 It is Aspochalasin D 1 H spectrum.

[0021] Figure 4 It is Aspochalasin D 13 C spectrum.

[0022] Figure 5 It is the effect of temperature on the yield of Aspochalasin D.

[0023] Figure 6 It is the effect of time on the yield of Aspochalasin D.

[0024] Figure 7 It is the effect of initial pH on the yield of Aspochalasin D.

[0025] Figure 8 It is the effect of carbon source on the yield of Aspochalasin D.

[0026] Figure 9 It is the effect of nitrogen source on the yield of Aspochalasin D.

[0027] Figure 10 It is the effect of leucine concentration on the yield of Aspochalasin D.

[0028] Figure 11 Figure 1 is a plasmid map of pXS-ΔaspoA.

[0029] Figure 12 Figure 2 is PCR verification of knockout of aspoA.

[0030] Figure 13 Figure 3 is a plasmid map of pUCgpdA-OEaspoG.

[0031] Figure 14 Figure 4 is PCR verification of overexpression of aspoG.

[0032] Figure 15 Figure 5 is an Aspochalasin D fermentation curve of Aspergillus flavipes and mutant strains under optimized fermentation culture conditions. DETAILED DESCRIPTION

[0033] The application will be further described below in combination with specific examples.

[0034] The structural diagram of Aspochalasin D prepared by the application is shown in Figure 1 Aspochalasin D 1 H spectrum, Aspochalasin D 13 C spectrum, respectively, as shown in Figure 3 and Figure 4 .

[0035] The medium designed in the following examples is as follows:

[0036] S-7 solid medium: 15-20 g / L agar, 20 g / L glucose, 2 g / L soybean peptone, 1 g / L sodium acetate, 5 mg / L L-phenylalanine, 100 mg / L sodium benzoate, 136 mg / L potassium dihydrogen phosphate, 1 mg / L biotin, 6.5 mg / L calcium nitrate, 1 mg / L pyridoxal phosphate, 1 mg / L calcium pantothenate, 1 mg / L thiamine hydrochloride, 5 mg / L manganese chloride, 2 mg / L ferric chloride, 1 mg / L copper nitrate, 3.6 mg / L magnesium sulfate, 2.5 mg / L zinc sulfate.

[0037] S-7 liquid fermentation medium: 20 / L glucose, 2 g / L soybean peptone, 1 g / L sodium acetate, 5 mg / L L-phenylalanine, 100 mg / L sodium benzoate, 136 mg / L potassium dihydrogen phosphate, 1 mg / L biotin, 6.5 mg / L calcium nitrate, 1 mg / L pyridoxal phosphate, 1 mg / L calcium pantothenate, 1 mg / L thiamine hydrochloride, 5 mg / L manganese chloride, 2 mg / L ferric chloride, 1 mg / L copper nitrate, 3.6 mg / L magnesium sulfate, 2.5 mg / L zinc sulfate.

[0038] S-7 fermentation medium: 25.2 / L glucose, 3 g / L soybean peptone, 1 g / L sodium acetate, 1.4 g / L leucine, 5 mg / L L-phenylalanine, 100 mg / L sodium benzoate, 136 mg / L potassium dihydrogen phosphate, 1 mg / L biotin, 6.5 mg / L calcium nitrate, 1 mg / L pyridoxal phosphate, 1 mg / L calcium pantothenate, 1 mg / L thiamine hydrochloride, 5 mg / L manganese chloride, 2 mg / L ferric chloride, 1 mg / L copper nitrate, 3.6 mg / L magnesium sulfate, 2.5 mg / L zinc sulfate.

[0039] Geneticin resistance screening medium: 4 g / L PDA, 0.5 g / L agar, 250 mg / L geneticin.

[0040] Zeocin resistance screening medium: 4 g / L PDA, 0.5 g / L agar, 100 mg / L zeocin.

[0041] The buffers used in the following examples are as follows:

[0042] Permeation buffer: 1.42 g disodium hydrogen phosphate and 144 g magnesium sulfate were dissolved in deionized water, 1000 mL of distilled water was used for constant volume; 0.276 g sodium dihydrogen phosphate and 29 g magnesium sulfate were dissolved in deionized water, 200 mL of distilled water was used for constant volume, and the two prepared solutions were mixed to adjust the pH to 5.8.

[0043] STC buffer: 218.6 g sorbitol, 0.47 g calcium chloride, 1000 mL of distilled water was used for constant volume, filtered and sterilized, and stored in a 4°C refrigerator.

[0044] PEG6000 buffer: 250 g PEG6000, 11.098 g calcium chloride, 44.73 g potassium chloride, 1000 mL of distilled water was used for constant volume, filtered and sterilized, and stored in a 4°C refrigerator

[0045] Example 1: Method for preparing Aspochalasin D by Aspergillus flavipes CGMCC 3.17641

[0046] The specific steps are as follows:

[0047] 1. Effect of temperature on Aspochalasin D yield

[0048] (1) Aspergillus flavipes CGMCC 3.17641 was inoculated on S-7 solid medium and placed at 25°C for 7-14 days.

[0049] (2) 100 mL of S-7 liquid fermentation medium was prepared in a 250 mL shake flask and sterilized at 115°C for 15 min, and then cooled for standby.

[0050] (3) Wash the plate of step (1) with sterilized water to prepare a spore suspension, count about 10 7 spores with a hemocytometer, inoculate the shake flask of step (2) with the spores, and place it in a 21°C, 23°C, 25°C, 27°C, 29°C, 31°C, 135 rpm shaker for 5 days. Take an appropriate amount of fermentation liquor for detection.

[0051] The results are shown in Table 1. Figure 5 As shown in Table 1, when the culture temperature is 29°C, the Aspochalasin D yield is the highest, reaching 58.2 mg / L.

[0052] 2, Effect of time on Aspochalasin D yield

[0053] (1) Inoculate Aspergillus flavipes CGMCC 3.17641 on S-7 solid culture medium and place it in a 25°C shaker for 7-14 days.

[0054] (2) Prepare 100 mL S-7 liquid fermentation medium in a 250 mL shake flask, sterilize it at 115°C for 15 min, and cool it for standby.

[0055] (3) Wash the plate of step (1) with sterilized water to prepare a spore suspension, count about 10 7 spores with a hemocytometer, inoculate the shake flask of step (2) with the spores, and place it in a 29°C, 135 rpm shaker for 7 days. Take an appropriate amount of fermentation liquor for detection.

[0056] The results are shown in Table 2. Figure 6 As shown in Table 2, when the culture time is 7 days, the Aspochalasin D yield is the highest, reaching 80.3 mg / L.

[0057] 3, Effect of initial pH on Aspochalasin D yield

[0058] (1) Inoculate Aspergillus flavipes CGMCC 3.17641 on S-7 solid culture medium and place it in a 25°C shaker for 7-14 days.

[0059] (2) Prepare 100 mL S-7 liquid fermentation medium with initial pH of 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 in a 250 mL shake flask, sterilize it at 115°C for 15 min, and cool it for standby.

[0060] (3) Wash the plate of step (1) with sterilized water to prepare a spore suspension, count about 10 7 spores with a hemocytometer, inoculate the shake flask of step (2) with the spores, and place it in a 29°C, 135 rpm shaker for 7 days. Take an appropriate amount of fermentation liquor for detection.

[0061] Results as shown in Table 1, when the initial pH is 8.0, Aspochalasin D production is highest up to 82.1 mg / L. Figure 7

[0062] 4, Effect of carbon source on Aspochalasin D production

[0063] (1) Aspergillus flavipes CGMCC 3.17641 was inoculated on S-7 solid medium and placed at 25°C for 7-14 days.

[0064] (2) Fermentation medium was prepared as follows:

[0065] The carbon source 20 g / L glucose in 100 mL S-7 liquid fermentation medium with initial pH 8.0 was adjusted to fructose, maltose, sucrose, starch, respectively, with a concentration of 20 g / L.

[0066] The above fermentation medium was placed in a 250 mL flask and sterilized at 115°C for 15 min, and cooled for standby.

[0067] (3) The plate of step (1) was washed with sterile water to prepare a spore suspension, and about 10 7 spores were inoculated in the flask of step (2) and placed in a 29°C, 135 rpm shaker for 7 days, and an appropriate amount of fermentation broth was taken for detection.

[0068] Results as shown in Table 1, when the initial pH is 8.0, Aspochalasin D production is highest up to 82.1 mg / L. Figure 8

[0069] 5, Effect of nitrogen source on Aspochalasin D production

[0070] (1) Aspergillus flavipes CGMCC 3.17641 was inoculated on S-7 solid medium and placed at 25°C for 7-14 days.

[0071] (2) Fermentation medium was prepared as follows:

[0072] The nitrogen source 2 g / L soybean peptone in 100 mL S-7 liquid fermentation medium with initial pH 8.0 was adjusted to yeast extract, tryptone, sodium nitrate, ammonium sulfate, urea, respectively, with a concentration of 2 g / L.

[0073] The above liquid fermentation medium was placed in a 250 mL flask and sterilized at 115°C for 15 min, and cooled for standby.

[0074] (3) The plate of step (1) was washed with sterile water to prepare a spore suspension, and about 10 7 ​​Spores were inoculated in shake flasks and incubated at 29°C, 135 rpm for 7 days. Appropriate amount of fermentation broth was taken for detection.

[0075] The results are shown in Table 1. Figure 9 As shown in Table 1, the Aspochalasin D yield was the highest, reaching 84.2 mg / L when the nitrogen source was soybean peptone.

[0076] 6. Effect of leucine concentration on Aspochalasin D yield

[0077] (1) Aspergillus flavipes CGMCC 3.17641 was inoculated on S-7 solid medium and placed at 25°C for 7-14 days.

[0078] (2) Fermentation medium was prepared:

[0079] 0 g / L leucine in 100 mL S-7 liquid fermentation medium with initial pH of 8.0 was adjusted to 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 g / L, respectively.

[0080] The above liquid fermentation medium was sterilized at 115°C for 15 min in 250 mL shake flasks and cooled for standby.

[0081] (3) The plate of step (1) was washed with sterilized water to prepare a spore suspension, which was counted with a hemocytometer to about 10 7 Spores were inoculated in shake flasks and incubated at 29°C, 135 rpm for 7 days. Appropriate amount of fermentation broth was taken for detection.

[0082] The results are shown in Table 1. Figure 10 As shown in Table 1, the Aspochalasin D yield was the highest, reaching 84.2 mg / L when the nitrogen source was soybean peptone.

[0083] 7. Optimization of liquid medium by response surface method

[0084] Box-Behnken design commonly used in response surface method was selected, and Design-Expert software was used for experimental design and result analysis. Three factors and three levels were selected for experimental design and optimization, including glucose, soybean peptone and leucine concentration. Experiments were designed according to the software, and the experimental results were input into the software for analysis to establish a mathematical model.

[0085] According to the mathematical model prediction, when the glucose concentration in the S-7 liquid fermentation medium is 25.2 g / L, the soybean peptone concentration is 3.0 g / L, and the leucine concentration is 1.4 g / L, the predicted Aspochalasin D yield is the highest, which is 202.5 mg / L; that is, the S-7 liquid fermentation medium is: 25.2 / L glucose, 3 g / L soybean peptone, 1 g / L sodium acetate, 1.4 g / L leucine, 5 mg / L L-phenylalanine, 100 mg / L sodium benzoate, 136 mg / L potassium dihydrogen phosphate, 1 mg / L biotin, 6.5 mg / L calcium nitrate, 1 mg / L pyridoxal phosphate, 1 mg / L calcium pantothenate, 1 mg / L thiamine hydrochloride, 5 mg / L manganese chloride, 2 mg / L ferric chloride, 1 mg / L copper nitrate, 3.6 mg / L magnesium sulfate, and 2.5 mg / L zinc sulfate.

[0086] The software optimal conditions were verified, and the results are shown in Figure 15 Aspochalasin D yield of 199.2 mg / L on the 7th day, which is consistent with the software prediction.

[0087] Example 2: Construction of Aspergillus flavipes ΔaspoA mutant strain

[0088] (1) Preparation of Aspergillus flavipes protoplast

[0089] Aspergillus flavipes was inoculated on S-7 plates, and the plates were placed in a 25°C constant temperature incubator for 7-14 days. The plates were washed with sterile water to prepare a spore suspension, which was inoculated into a 250 mL flask containing 90 mL of S-7 liquid fermentation medium, and placed in a constant temperature shaker at 29°C and 135 rpm for 18 h. The bacterial solution was centrifuged at 8000 rpm for 15 min to collect the bacterial bodies. The bacterial bodies were washed with an appropriate amount of osmotic buffer, then centrifuged at 8000 rpm for 15 min, and repeated 2-3 times. 10 mL of osmotic buffer containing 200 mg of lywallzyme was added, and incubated at 29°C and 135 rpm in a constant temperature shaker for 3 h. The incubated bacterial solution was filtered with 5-7 layers of sterile gauze, the filtrate was added with an equal volume of STC buffer, and centrifuged at 5000 rpm for 15 min, and the supernatant was discarded. Add 2 times the volume of STC buffer, centrifuge at 6000 rpm for 8 min, and discard the supernatant. Add an appropriate amount of STC buffer, and count the number of protoplasts using a hemocytometer to ensure that the number of protoplasts is 10 6 million per milliliter, and finally stored at -80°C.

[0090] (2) Preparation of knockout fragments

[0091] The aspoA gene reading frame was found from the Aspergillus flavipes genome, and the 2000 bp fragment upstream and downstream of the aspoA gene reading frame was cloned, and the upstream and downstream sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4, and the geneticin resistance expression cassette sequence is shown in SEQ ID NO. 5. According to the upstream sequence, the geneticin resistance expression cassette and the downstream sequence, the vector pXS (published in Liu J, Chai X, Guo T, Wu J, Yang P, Luo Y, Zhao H, Zhao W, Nkechi O, Dong J, Bai J, Lin Q (2019) Disruption of the ergosterol biosynthetic pathway results in increased membrane permeability, vausing overproduction and secretion of extracellular Monascus Pigments in submerged fermentation. J Agric Food Chem 67 (49): 13673-13683 doi: https: / / doi.org / 10.1021 / acs.jafc.9b05872) was integrated to obtain the complete plasmid pXS-ΔaspoA Figure 11 ). The complete plasmid pXS-ΔaspoA was used as a template, and primers pXS-D-F (5'-GCGAATTGGGTACCGGTCGAC-3') and pXS-D-R (5'-TTGCTGGCCTTTTGCTCACATGT-3') were used for PCR amplification of the knockout fragment containing the upstream and downstream sequences and the geneticin resistance gene. The PCR reaction used a 50 μL system: 2 μL forward primer, 2 μL reverse primer, 1 μL template, 20 μL double distilled water and 25 μL 2x Prime Star Max premix (Japan, Takara). According to the manufacturer's instructions, the PCR program was set as follows: denaturation, 98°C, 10s; annealing, 58°C, 10s; extension, 72°C, 30s, repeated for 30 cycles, and finally extended at 72°C for 10 min. The amplified knockout fragment was purified and recovered for later use.

[0092] (3) Aspergillus flavipes protoplast transformation

[0093] Take 150 μL of Aspergillus flavipes protoplast, add an equal volume of STC buffer dissolved with 20 μg of knock-out fragment, mix well, and incubate on ice for 1 h. Add 1.25 mL of PEG6000 buffer, mix well, and incubate in a constant temperature shaker at 29℃ and 135 rpm for 20 min. Add 6 mL of 1.2 mol / L sorbitol PDB, and incubate in a constant temperature shaker at 29℃ and 135 rpm for about 12 h. Centrifuge at 7500 rpm for 15 min, discard the excess supernatant, and spread on a plate containing geneticin.

[0094] (4) Recombinant screening

[0095] The transformants screened from the resistant plate were liquid cultured, and then the mycelium was collected to extract the genome. The genome of the transformant was used as a template to perform PCR verification with primers testA-F (5'-GCTTTCTCTTCCTGGCTCGTAGAT-3') and testA-R (5'-TCGGTGTGATCATCCATACAGCAACA-3'). The PCR reaction used a 20 μL system: 1 μL forward primer, 1 μL reverse primer, 1 μL template, 7 μL double-distilled water, and 10 μL 2x ES Taq MasterMix (China, Kangwei Century). The PCR program was set according to the manufacturer's instructions: denaturation, 98℃, 10 s; annealing, 58℃, 10 s; extension, 72℃, 90 s, repeated for 30 cycles, and finally extended at 72℃ for 10 min. The PCR product was analyzed by agarose gel electrophoresis, and the results are shown in FIG. 2. As compared with the wild type Aspergillus flavipes CGMCC 3.17641, the amplified fragment of the positive transformant was about 2000 bp, which was smaller than that of the wild type, proving that the aspoA gene in the transformant was successfully replaced by the geneticin resistance expression cassette. Figure 12

[0096] The Aspergillus flavipes CGMCC 3.17641ΔaspoA mutant strain was prepared.

[0097] Example 3: Fermentation production of Aspochalasin D by Aspergillus flavipes CGMCC 3.17641ΔaspoA mutant strain

[0098] The specific steps are as follows:

[0099] (1) The Aspergillus flavipes CGMCC 3.17641ΔaspoA mutant strain prepared in Example 2 was inoculated on S-7 solid culture medium and placed at 25℃ for growth for 7-14 days.

[0100] (2) An optimized 100 mL S-7 liquid culture medium was prepared in a 250 mL shake flask, sterilized at 115℃ for 15 min, and cooled for standby use. ​

[0101] The optimized fermentation medium for S-7 is: 25.2 g / L glucose, 3 g / L soybean peptone, 1 g / L sodium acetate, 1.4 g / L leucine, 5 mg / L L-phenylalanine, 100 mg / L sodium benzoate, 136 mg / L potassium dihydrogen phosphate, 1 mg / L biotin, 6.5 mg / L calcium nitrate, 1 mg / L pyridoxal phosphate, 1 mg / L calcium pantothenate, 1 mg / L thiamine hydrochloride, 5 mg / L manganese chloride, 2 mg / L ferric chloride, 1 mg / L copper nitrate, 3.6 mg / L magnesium sulfate, and 2.5 mg / L zinc sulfate.

[0102] (3) Wash the plate from step (1) with sterile water to prepare a spore suspension, and count approximately 10 spores using a hemocytometer. 7 One spore was inoculated into the shake flask of step (2) and placed in a shaker at 29°C and 135 rpm for culture. The fermentation broth was then taken for testing.

[0103] The results are as follows Figure 15 As shown, the Aspergillus flavus CGMCC 3.17641ΔaspoA mutant strain achieved the highest yield of 325.3 mg / L on day 7 under optimized fermentation culture conditions.

[0104] Example 4: Construction of Aspergillus flavus ΔaspoA::OEaspoG mutant strain

[0105] (1) Construction of overexpression plasmid

[0106] The complete aspoG gene reading frame was cloned from the Aspergillus flavus genome. The aspoG gene sequence is shown in SEQ ID NO.6. It was then integrated into the vector pUC (deposited in the laboratory of Jiangnan University) in the order of bleomycin resistance expression cassette, promoter PgpdA, aspoG gene, and terminator TcgrA to form the complete plasmid pUCgpdA-OEaspoG. Figure 13 The bleomycin resistance expression cassette sequence is shown in SEQ ID NO.7, the promoter PgpdA sequence from Aspergillus nidulans is shown in SEQ ID NO.8, and the terminator TcgrA sequence from Aspergillus nidulans is shown in SEQ ID NO.9.

[0107] (2) Preparation of protoplasts from Aspergillus flavus CGMCC 3.17641ΔaspoA mutant strain

[0108] The Aspergillus flavus CGMCC 3.17641ΔaspoA mutant prepared in Example 2 was inoculated on S-7 plates, and the plates were placed in a constant temperature incubator at 25°C for 7-14 days. The plates were washed with sterile water to prepare a spore suspension, and the spore suspension was inoculated in 250 mL shake flasks containing 90 mL of S-7 liquid fermentation medium, and placed in a constant temperature shaker at 29°C, 135 rpm for 18 h. The bacterial liquid was centrifuged in a centrifuge tube at 8000 rpm for 15 min to collect the bacterial cells. The bacterial cells were washed with an appropriate amount of osmotic buffer, and then centrifuged at 8000 rpm for 15 min, repeated 2-3 times. 10 mL of osmotic buffer containing 200 mg of lywallzyme was added, and incubated at 29°C, 135 rpm for 3 h. The incubated bacterial liquid was filtered with 5-7 layers of sterile gauze, and the filtrate was added with an equal volume of STC buffer, centrifuged at 5000 rpm for 15 min, and the supernatant was discarded. 2 times the volume of STC buffer was added, centrifuged at 6000 rpm for 8 min, and the supernatant was discarded. An appropriate amount of STC buffer was added, and the number of protoplasts was counted using a hemocytometer to ensure that the number of protoplasts was 10 6

[0109] (3) Protoplast transformation of Aspergillus flavus CGMCC 3.17641ΔaspoA mutant

[0110] Take 150 μL of Aspergillus flavus CGMCC 3.17641ΔaspoA mutant protoplasts, add an equal volume of STC buffer containing 10 μg of plasmid pUCgpdA-OEaspoG, mix well, and incubate on ice for 1 h. Add 1.25 mL of PEG6000 buffer, mix well, and incubate in a constant temperature shaker at 29°C, 135 rpm for 20 min. Add 6 mL of 1.2 mol / L sorbitol PDB, and incubate in a constant temperature shaker at 29°C, 135 rpm for about 12 h. Centrifuge at 7500 rpm for 15 min, discard the excess supernatant, and spread on plates containing bleomycin.

[0111] (4) Screening of recombinant strains

[0112] ​The transformants screened by resistant plates were liquid cultured, and then the mycelium was collected to extract the genome. The genome of the transformants was used as a template to perform PCR verification with primers OEaspoG-F (5'-GTCAGTCCTGCTCCTCGGCCAC-3') and OEaspoG-R (5'-TGCATGGCAGACACTGAAGCAACAGC-3'), and the PCR reaction was performed in a 20 μL system: 1 μL forward specific primer, 1 μL reverse specific primer, 1 μL template, 7 μL double-distilled water, and 10 μL 2x ES Taq MasterMix (China, Kangwei Century). The PCR program was set according to the manufacturer's instructions: denaturation, 98°C, 10 s; annealing, 58°C, 10 s; extension, 72°C, 120 s, repeated for 30 cycles, and finally extended at 72°C for 10 min. The PCR product was analyzed by agarose gel electrophoresis, and the results are shown in Figure 14 Figure 2. There was no amplified fragment for the wild type Aspergillus flavipes CGMCC 3.17641 and ΔaspoA, and the amplified fragment of the positive transformants was about 3500 bp, proving that the aspoG overexpression cassette was successfully integrated into the genome of the transformants.

[0113] The Aspergillus flavipes ΔaspoA::OEaspoG mutant strain was prepared (the plate morphology is shown in Figure 2

[0114] Example 5: Fermentation of Aspergillus flavipes ΔaspoA::OEaspoG mutant strain to produce Aspochalasin D

[0115] The specific steps are as follows:

[0116] (1) The Aspergillus flavipes ΔaspoA::OEaspoG mutant strain was inoculated on S-7 solid culture medium and placed at 25°C for 7-14 days.

[0117] (2) The optimized 100 mL S-7 liquid culture medium was prepared in a 250 mL shake flask, sterilized at 115°C for 15 min, and cooled for standby.

[0118] The S-7 optimized fermentation medium was as follows: 25.2 / L glucose, 3 g / L soybean peptone, 1 g / L sodium acetate, 1.4 g / L leucine, 5 mg / L L-phenylalanine, 100 mg / L sodium benzoate, 136 mg / L potassium dihydrogen phosphate, 1 mg / L biotin, 6.5 mg / L calcium nitrate, 1 mg / L pyridoxal phosphate, 1 mg / L calcium pantothenate, 1 mg / L thiamine hydrochloride, 5 mg / L manganese chloride, 2 mg / L ferric chloride, 1 mg / L copper nitrate, 3.6 mg / L magnesium sulfate, and 2.5 mg / L zinc sulfate.

[0119] ​(3) The plate of step (1) was washed with sterilized water to prepare a spore suspension, and about 10 7 spores were inoculated into a flask and cultured at 29°C, 135 rpm in a shaker. The fermentation broth was detected.

[0120] The results are shown in Table 1. Figure 15 The yield of the ΔaspoA::OEaspoG mutant strain of Aspergillus flavipes reached 812.1 mg / L on the 7th day under the optimized fermentation culture conditions.

[0121] Although the present application has been disclosed in the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A recombinant Aspergillus flavus, characterized in that, The recombinant Aspergillus flavus is based on Aspergillus flavus CGMCC. 3.17641 was used as the expression host. The aspoA gene in its genome was knocked out, and an overexpression cassette containing the transcriptional regulatory factor aspoG from Aspergillus flavus was integrated and expressed. The nucleotide sequence of the aspoA gene is shown in SEQ ID NO.1; The nucleotide sequence of the overexpression cassette containing the transcriptional regulatory factor aspoG derived from Aspergillus flavus is shown in SEQ ID NO.

2.

2. A method for fermenting and producing Aspochalasin D, characterized in that, The method involves adding the recombinant Aspergillus flavus of claim 1 to a fermentation medium and fermenting to prepare Aspochalasin D.

3. The method according to claim 2, characterized in that, The fermentation medium comprises: 10-50 g / L glucose, 1-5 g / L soybean peptone, 1-3 g / L sodium acetate, 0.5-3 g / L leucine, 1-9 mg / L L-phenylalanine, 20-180 mg / L sodium benzoate, 100-200 mg / L potassium dihydrogen phosphate, 0.5-1.5 mg / L biotin, 3-10 mg / L calcium nitrate, 0.5-1.5 mg / L pyridoxal phosphate, 0.5-1.5 mg / L calcium pantothenate, 0.5-1.5 mg / L thiamine hydrochloride, 1-9 mg / L manganese chloride, 1-3 mg / L ferric chloride, 0.5-1.5 mg / L copper nitrate, 0.5-4.5 mg / L magnesium sulfate, and 1-4 mg / L zinc sulfate.

4. The method according to claim 2 or 3, characterized in that, The fermentation medium consisted of: 25.2 g / L glucose, 3 g / L soybean peptone, 1 g / L sodium acetate, 1.4 g / L leucine, 5 mg / L L-phenylalanine, 100 mg / L sodium benzoate, 136 mg / L potassium dihydrogen phosphate, 1 mg / L biotin, 6.5 mg / L calcium nitrate, 1 mg / L pyridoxal phosphate, 1 mg / L calcium pantothenate, 1 mg / L thiamine hydrochloride, 5 mg / L manganese chloride, 2 mg / L ferric chloride, 1 mg / L copper nitrate, 3.6 mg / L magnesium sulfate, and 2.5 mg / L zinc sulfate.

5. The method according to claim 4, characterized in that, The amount of the recombinant Aspergillus flavus added is: 10 6 ~10 8 One spore.

6. The method according to claim 5, characterized in that, The recombinant Aspergillus flavus was added to the fermentation medium and fermented at a temperature of 21-31°C, an initial pH of 6.0-8.0, and a rotation speed of 100-160 r / min.

7. The method according to claim 6, characterized in that, The recombinant Aspergillus flavus was added to the fermentation medium and fermented at a temperature of 29°C, an initial pH of 6.0-8.0, and a rotation speed of 135 r / min.

8. The use of the recombinant Aspergillus flavus according to claim 1 in the preparation of Aspochalasin D or products containing Aspochalasin D.

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