Preparation and culture method of high-yield serotonic acid engineering bacterium YAFWY001

Abstract

The invention provides a high-yield lichen acid engineering bacterium YAFWY001 and a culture method thereof, and relates to the technical field of microbial biosynthesis. The invention provides an engineering bacterium capable of producing serotonic acid, the engineering bacterium is prepared by transferring a yARGAorsA expression element into a pyrG gene-deleted aspergillus nidulans strain, the yARGAorsA expression element is formed by linking genes Ya5, pyrG, Rib, gbdA (p), OrsA and Ya3 in sequence from the 5end, and the engineering bacterium capable of producing serotonic acid provided by the invention is characterized in that magnesium acetate (YGM) is added into a YG liquid culture medium, so that the engineering bacterium capable of producing serotonic acid is obtained. The content of the lichen acid in the fermentation liquor of the lichen acid producing engineering strain can be obviously increased, and the method is a new way for obtaining the lichen acid at low cost.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial biosynthesis, and particularly to the preparation of a high-yield orsellic acid engineering bacterium YAFWY001 and a culture method for obtaining orsellic acid. Background Art

[0002] Orsellic acid, alias: 2,4-dihydroxy-6-methylbenzoic acid, belongs to aromatic polyketide compounds with the molecular formula: C8H8O4 and the CAS number: 480-64-8. Orsellic acid is the precursor of orsellic acid derivatives, which are widely present in herbaceous plants, lichens, algae, fungi, and bacteria. Currently, more than 200 orsellic acid derivatives have been reported, and orsellic acid derivatives exhibit good biological activities, such as anti-cell proliferation, cytotoxicity, antibacterial, anti-atherosclerosis, anti-inflammatory, anti-protozoal, antioxidant and other activities. Orsellic acid has different derivatives in different fungi. For example, in lichens, orsellic acid condenses into erythrinic acid, while in Beauveria bassiana, the orsellic acid derivative is oosporein, and in Aspergillus nidulans, the orsellic acid derivatives are F-9775A and gerfeli. In Stachybotrys fungi, the orsellic acid derivative is griseofolic acid. In Microdochium, the orsellic acid derivative is sporormielone A. And in Antrodia cinnamomea, the orsellic acid derivatives are androquinol and benzodioxane compound SY1.

[0003] Currently, orsellic acid has been isolated and identified from herbaceous plants, lichens, bacteria, and fungi, and the orsellic acid biosynthesis genes from different fungal sources have been introduced into Aspergillus nidulans and yeast to achieve heterologous expression to obtain orsellic acid. However, there are still problems such as low yield, complex fermentation process, and the failure to achieve commercial fermentation production of orsellic acid. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides a preparation and culture method for a high-yield orsellic acid engineering bacterium YAFWY001, and a large amount of orsellic acid is obtained through constructing the engineering bacterium YAFWY001 and optimizing the liquid fermentation of the culture medium.

[0005] To achieve the above objectives, the technical solution of the present invention is realized through the following technical solutions: The present invention provides an engineering bacterium for producing usnic acid. The engineering bacterium is obtained by transferring the yARGAorsA expression element into an Aspergillus nidulans strain with a deleted pyrG gene. The yARGAorsA expression element is composed of genes Ya5, pyrG, Rib, gbdA(p), OrsA, and Ya3 linked in sequence from the 5'-end. The base sequence of Ya5 is as shown in SEQ ID NO:1, the base sequence of pyrG is as shown in SEQ ID NO:2, the base sequence of Rib is as shown in SEQ ID NO:3, the base sequence of gbdA(p) is as shown in SEQ ID NO:4, the base sequence of OrsA is as shown in SEQ ID NO:5, and the base sequence of Ya3 is as shown in SEQ ID NO:6.

[0006] Further, for the engineering bacterium for producing usnic acid, the engineering bacterium is Aspergillus nidulans YAFWY001, classified and named as Aspergillus nidulans, and the preservation number is CCTCC NO: M 2024908.

[0007] Further, the present invention provides a preparation method for the engineering bacterium for producing usnic acid, comprising the following steps: S1: Construction of the yARGAorsA expression vector The genes Ya5, pyrG, Rib, gbdA(p), OrsA, and Ya3 are sequentially linked to the linearized plasmid Puc19 using seamless ligation technology to obtain the expression vector yARGAorsA; S2: The yARGAorsA expression element is obtained by PCR amplification using a high-fidelity DNA polymerase, and the yARGAorsA expression element is introduced into the Aspergillus nidulans LO8030 strain by the protoplast transformation method to obtain the engineering strain for producing usnic acid.

[0008] Further, the present invention provides a vector containing the yARGAorsA expression element. The yARGAorsA expression element is composed of genes Ya5, pyrG, Rib, gbdA(p), OrsA, and Ya3 linked in sequence from the 5'-end. The base sequence of Ya5 is as shown in SEQ ID NO:1, the base sequence of pyrG is as shown in SEQ ID NO:2, the base sequence of Rib is as shown in SEQ ID NO:3, the base sequence of gbdA(p) is as shown in SEQ ID NO:4, the base sequence of OrsA is as shown in SEQ ID NO:5, and the base sequence of Ya3 is as shown in SEQ ID NO:6.

[0009] Furthermore, the present invention provides a genetically engineered bacterium, which contains the said vector. In some specific embodiments, such genetically engineered bacteria are mainly used for the amplification and expression of target genes, such as Escherichia coli DH5α provided by the present invention.

[0010] Furthermore, the present invention provides the application of the said vector and the said genetically engineered bacterium in the preparation of Aspergillus nidulans producing orsellinic acid.

[0011] Furthermore, the present invention provides a culture method for the said genetically engineered bacterium to produce high-yield orsellinic acid, which uses YGM liquid medium for fermentation culture. Each liter of the medium contains 0.5 g yeast extract, 2 g glucose, and 0.25 - 4 g magnesium acetate.

[0012] For the said culture method, the culture conditions are as follows: after culturing on a shaker at 37°C and 150 rpm for 24 hours, the temperature is changed to 30°C, and the shaker speed remains at 150 rpm for continuous culturing for 72 hours.

[0013] Furthermore, the present invention provides the application of magnesium acetate in improving the yield of orsellinic acid in the culture of Aspergillus nidulans YAFWY001. Aspergillus nidulans YAFWY001, classified and named as Aspergillus nidulans, with the deposit number of CCTCC NO: M2024908.

[0014] Furthermore, the present invention provides a production method of orsellinic acid, which includes the following steps: (1) Fermenting and culturing Aspergillus nidulans YAFWY001 to obtain a fermentation broth. Aspergillus nidulans YAFWY001, classified and named as Aspergillus nidulans, with the deposit number of CCTCC NO: M 2024908; (2) Filtering the mycelium in the fermentation broth to obtain a bacterial liquid, then adding ethyl acetate to the bacterial liquid at a volume ratio of 1:1.5, performing ultrasonic treatment, standing for layering, and extracting the supernatant. After multiple extractions, the combined extracted supernatants are condensed and refluxed with a rotary evaporator and dried to obtain a crude extract of the fermentation broth of strain YAFWY001; (3) Purifying the crude extract to obtain high-purity orsellinic acid.

[0015] The present invention provides a preparation and culture method for a genetically engineered bacterium with high-yield orsellinic acid. Compared with the prior art, the advantages are as follows: First, the present invention uses the Aspergillus nidulans LO8030 strain with a low background of secondary metabolites as the host, which can greatly simplify the subsequent separation and purification of orsellinic acid; second, the expression element yARGAorsA used in the present invention carries the strong promoter gbdA, which can efficiently initiate the expression of the orsellinic acid synthase-encoding gene orsA; third, the present invention provides a liquid culture containing magnesium acetate, which can greatly promote the large-scale directional synthesis of orsellinic acid by the high-yield orsellinic acid engineering strain YAFWY001, control the entire fermentation time within 96 hours, and the separation and purification are simple and easy to perform, greatly reducing the cost.

[0016] The present invention is suitable for large-scale liquid fermentation production. Using the YAFWY001 engineering strain to ferment with the above-mentioned culture medium and culture conditions, separating and extracting the fermentation products, the YAFWY001 engineering strain can produce a large amount of orsellinic acid, with a yield of 650 mg / L, which can effectively solve the source problem of orsellinic acid and provide sufficient raw materials for the synthesis of natural products containing the orsellinic acid skeleton structure and related anti-cancer drugs. Brief Description of the Drawings

[0017] Figure 1 Schematic diagram of the yARGAorsA expression element in Example 1 of the present invention; Figure 2 Growth diagram of Aspergillus nidulans YAFWY001 mycelium; Figure 3 Difference diagram of orsellinic acid production in the crude extract under different magnesium acetate concentration conditions detected by TLC; where 1: magnesium acetate 0.25 g / 100 ml; 2: magnesium acetate 0.5 g / 100 ml; 3: magnesium acetate 1 g / 100 ml; 4: magnesium acetate 1 g / 100 ml. Red is orsellinic acid, and the larger the red dot, the higher the content.

[0018] Figure 4 Result diagram of HPLC detection of orsellinic acid in YAFWY001; Figure 5 UV absorption spectrum diagram of orsellinic acid Figure 6 1H nuclear magnetic resonance spectrum diagram of orsellinic acid Figure 7 13C nuclear magnetic resonance spectrum diagram of orsellinic acid. Detailed Embodiments

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. For purchased commodities in the test methods, if the specific conditions are not indicated, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0020] The high-yield usnic acid engineering bacterium provided by the present invention has a deposit name: Aspergillus nidulans YAFWY001 Aspergillus nidulans YAFWY001; classified and named as: Aspergillus nidulans, Deposit number: CCTCC NO: M 2024908; depositary institution: China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China; the culture was received by the depositary institution on May 11, 2024 and registered. Upon request, it will be deposited for thirty years starting from that date. The viability of the culture was detected by the depositary institution on May 18, 2024, and the result was viable.

[0021] Example 1 Preparation method of the usnic acid-producing engineering bacterium 1. Preparation of the yARGAorsA expression element First, extract the DNA of Aspergillus nidulans and Antrodia cinnamomea var. kanoi. Then, using the Aspergillus nidulans DNA as a template, amplify to obtain the upstream region Ya5 (SEQ ID NO: 1) of the yA gene of Aspergillus nidulans, the gbdA(p) promoter (SEQ ID NO: 4), and the gene fragment Ya3 (SEQ ID NO: 6) of the downstream region of the yA gene. Next, using the Antrodia cinnamomea var. kanoi DNA as a template, amplify to obtain the Rib (SEQ ID NO: 3), pyrG (SEQ ID NO: 2), and OrsA (SEQ ID NO: 5) gene fragments of Antrodia cinnamomea var. kanoi. Finally, use a seamless cloning kit (Order NO. B632219) to sequentially ligate the amplified fragments onto the linearized plasmid Puc19. Transform the ligation product into Escherichia coli DH5α, and after overnight culture, obtain positive clones. Verify by PCR and sequencing to determine the obtained expression vector yARGAorsA. After extracting the yARGAorsA vector plasmid with a plasmid extraction kit, use the yARGAorsA vector plasmid as a template, and use a high-fidelity DNA polymerase to perform PCR amplification to obtain the yARGAorsA expression element, and purify the yARGAorsA expression element with a PCR product recovery kit for standby.

[0022] 2. Obtaining of the high-yield usnic acid engineering bacterium YAFWY001 According to the method for transforming protoplasts of Aspergillus nidulans in the article (Hu Meimei, Chi Yujie. Establishment of a transformation method for the MnP gene of Lenzites gibbosa in Aspergillus nidulans [J]. Journal of Northeast Forestry University, 2013, 41 (07): 129-133.), the yARGAorsA expression element was transferred into the Aspergillus nidulans strain lacking the pyrG gene. In this invention, the specific strain used was Aspergillus nidulans LO8030 (the pyrG gene of Aspergillus nidulans LO8030 is deleted and it cannot grow in a medium without uracil. It was purchased from Bioscience, and the purchase link is http: / / www.bio-sci.com.cn / index.php?id=2577). Through nutritional deficiency (introducing the pyrG gene of Antrodia camphorata), the high-yield usnic acid engineering strain YAFWY001 was obtained by screening.

[0023] Example 2: Effects of adding different concentrations of magnesium acetate on the yield of usnic acid 1. After activating the Aspergillus nidulans YAFWY001 strain, spores were obtained by cultivation. 2. 1 ml of the spore suspension (spore concentration: 106 / ml) was respectively inoculated into 100 ml of YG liquid medium containing (0.25 g, 0.5 g, 1 g, 2 g, 4 g of magnesium acetate). After culturing on a shaker at 37°C and 150 rpm for 24 hours, the temperature was changed to 30°C and cultured on a shaker at 150 rpm for 72 hours. The YG formula is: 0.5 g / L yeast extract; 2 g / L glucose.

[0024] 3. After filtering the mycelium in the fermentation broth to obtain the bacterial liquid, ethyl acetate was added to the bacterial liquid at a volume ratio of 1:1.5. After ultrasonic treatment, it was allowed to stand for stratification and the supernatant was extracted. After extraction 3 times, the extraction liquids were combined and condensed and refluxed and dried using a rotary evaporator to obtain the crude extract of YG liquid culture containing different concentrations of magnesium acetate.

[0025] 4. The effects of different concentrations of magnesium acetate on the production of usnic acid by Aspergillus nidulans YAFWY001 were detected by TLC (Figure 3). The red color is usnic acid, and the larger the red spot, the higher the content. Therefore, 2 g of magnesium acetate can significantly increase the content of usnic acid in the fermentation broth of Aspergillus nidulans YAFWY001. By comparing with the usnic acid standard product through HPLC, it was calculated that for the YG liquid culture of Aspergillus nidulans YAFWY001 with 2 g of magnesium acetate added, the yield of usnic acid was 650 mg / L (Figure 4).

[0026] Example 3: Preparation method of usnic acid 1. Filter the mycelium in the fermentation broth to obtain the broth, then add ethyl acetate to the broth at a volume ratio of 1:1.5. After ultrasonic treatment, let it stand for stratification and extract the supernatant. After extraction 3 times, combine the extracts and use a rotary evaporator for condensation reflux and drying to obtain the crude extract of the fermentation broth of the high-yield orsellinic acid engineering strain YAFWY001.

[0027] 2. First, use AB-8 type (weak polarity) macroporous resin to roughly fractionate the fermentation broth extract. Flush the column successively with pure water, 30% ethanol, 60% ethanol, 90% ethanol and ethyl acetate (each eluent is flushed for 6 column volumes) for fractionation, and then use TLC to judge the fraction where orsellinic acid is located, and combine the fractions containing orsellinic acid. 3. Then use a LC3050 type preparative chromatograph for gradient elution to finely fractionate orsellinic acid. 4. Finally, after using TLC to judge the location of orsellinic acid, finally use an Agilent 1260 high performance liquid chromatograph for preparation under the detection of 254 nm wavelength to obtain the pure product (purity > 98%). Determine that the separated pure product is orsellinic acid through the UV absorption spectrum and nuclear magnetic resonance ( Figure 5 ,6,7).

[0028] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An engineered bacterium producing lysine acid, characterized in that: The engineering bacteria are prepared by transferring the yARGAorsA expression element into a pyrG gene-deficient Aspergillus nidulans strain, wherein the yARGAorsA expression element is sequentially linked from the 5' end by genes Ya5, pyrG, Rib, gbdA(p), OrsA and Ya3, wherein the Ya5 base sequence is shown in SEQ ID NO:1, the pyrG base sequence is shown in SEQ ID NO:2, the Rib base sequence is shown in SEQ ID NO:3, the gbdA(p) base sequence is shown in SEQ ID NO:4, the OrsA base sequence is shown in SEQ ID NO:5, and the Ya3 base sequence is shown in SEQ ID NO:

6.

2. The engineered bacteria producing lysine acid according to claim 1, characterized in that: The engineered bacteria is Aspergillus nidulans YAFWY001, which is classified as Aspergillus nidulans and has a preservation number of CCTCC NO: M 2024908.

3. A method for preparing the engineered bacteria producing lysine acid as claimed in claim 1 or 2, characterized in that: The following steps are involved: S1: Construction of yARGAorsA expression vector Genes Ya5, pyrG, Rib, gbdA(p), OrsA and Ya3 were linked to the linearized plasmid Puc19 in sequence using seamless linking technology to obtain the expression vector yARGAorsA; S2: The yARGAorsA expression element was obtained by PCR amplification using high-fidelity DNA polymerase, and the yARGAorsA expression element was introduced into the Aspergillus nidulans LO8030 strain by protoplast transformation to obtain an engineered strain producing lysine.

4. A carrier, characterized in that The vector contains a yARGAorsA expression element, which is composed of genes Ya5, pyrG, Rib, gbdA(p), OrsA and Ya3 linked in sequence from the 5' end, the Ya5 base sequence is shown in SEQ ID NO:1, the pyrG base sequence is shown in SEQ ID NO:2, the Rib base sequence is shown in SEQ ID NO:3, the gbdA(p) base sequence is shown in SEQ ID NO:4, the OrsA base sequence is shown in SEQ ID NO:5, and the Ya3 base sequence is shown in SEQ ID NO:

6.

5. An engineered bacterium, characterized in that: The engineered bacteria contains the vector according to claim 4.

6. Use of the vector according to claim 4 or the engineered bacteria according to claim 5 in the preparation of Aspergillus nidulans producing lyssinoic acid.

7. A method for culturing an engineered bacterium for high production of orsuccinic acid according to claim 1 or 2, characterized in that: YGM liquid culture medium is used for fermentation culture, and each liter of culture medium contains 0.5g yeast extract, 2g glucose, and 0.25-4g magnesium acetate.

8. The culture method according to claim 7, characterized in that: The culture conditions were 37°C, 150 rpm shaking incubator for 24 hours, then changed to 30°C, 150 rpm shaking incubator for further 72 hours.

9. The use of magnesium acetate in increasing the yield of lysine in the culture of Aspergillus nidulans YAFWY001, characterized in that: Aspergillus nidulans YAFWY001 is classified as Aspergillus nidulans and its deposit number is CCTCC NO: M 2024908.

10. A method for producing orsuccinic acid, characterized in that: The following steps are involved: (1) fermenting Aspergillus nidulans YAFWY001 to obtain a fermentation broth, Aspergillus nidulans YAFWY001 is classified and named Aspergillus nidulans, and its deposit number is CCTCC NO: M 2024908; (2) After the mycelium in the fermentation broth was filtered, a bacterial liquid was obtained, and then ethyl acetate was added to the bacterial liquid at a volume ratio of 1:1.

5. After ultrasonic treatment, the liquid was allowed to stand for stratification and then the supernatant was extracted. After multiple extractions, the supernatants were combined and condensed, refluxed and dried using a rotary evaporator to obtain a crude extract of the fermentation broth of the YAFWY001 strain; (3) Purifying the crude extract to obtain high-purity lysine.

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