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

Abstract

The present invention provides a high-mossinoic acid-producing engineered bacterium, YAFWY001, and a cultivation method thereof, relating to the technical field of microbial biosynthesis. The present invention provides a mossinoic acid-producing engineered bacterium, prepared by transferring the yARGAorsA expression element into an Aspergillus nidulans strain lacking the pyrG gene. The yARGAorsA expression element is composed, from the 5' end, of the genes Ya5, pyrG, Rib, gbdA(p), OrsA, and Ya3, linked in sequence. The present invention provides a mossinoic acid-producing engineered bacterium. Adding magnesium acetate (YGM) to the YG liquid culture medium significantly increases the mossinoic acid content in the fermentation broth of the mossinoic acid-producing engineered strain. This invention provides a new, low-cost approach to obtaining mossinoic acid.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial biosynthesis, and in particular to the preparation of a high-yield lysine acid-producing engineered bacterium YAFWY001 and a culture method for obtaining lysine acid. Background Art

[0002] Orsellic acid, also known as 2,4-dihydroxy-6-methylbenzoic acid, belongs to the class of aromatic polyketides with the molecular formula C8H8O4 and the CAS number 480-64-8. Orsellic acid is a precursor to orsellic acid derivatives, which are widely found in herbs, lichens, algae, fungi, and bacteria. Over 200 orsellic acid derivatives have been reported, exhibiting promising biological activities such as antiproliferative, cytotoxic, antibacterial, antiatherosclerotic, anti-inflammatory, antiprotozoal, and antioxidant activities. Different fungi have different derivatives of orsellic acid. For example, in lichens, orsellic acid condenses into orsellic acid, while in Beauveria bassiana, orsellic acid derivatives form oosporin, and in Aspergillus nidulans, orsellic acid derivatives form F-9775A and gerfeli. In Stachybotrys, orsellic acid derivatives form grisea folate. In Microsporum microsporum, orsellic acid derivatives form sporormielone A. The mossinoic acid derivatives in Antrodia cinnamomea are antroquinol and benzodioxetine heterocyclic compound SY1.

[0003] Currently, rucic acid has been isolated and identified from herbs, lichens, bacteria, and fungi. Furthermore, rucic acid biosynthesis genes from different fungi have been introduced into Aspergillus nidulans and yeast for heterologous expression. However, low yields, complex fermentation processes, and commercial fermentation production of rucic acid remain challenges. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the present invention provides a preparation and cultivation method of a high-yield lysine acid engineered bacterium YAFWY001, which can obtain a large amount of lysine acid by constructing the engineered bacterium YAFWY001 and optimizing the culture medium for liquid fermentation.

[0005] To achieve the above objectives, the technical solution of the present invention is implemented through the following technical solutions:

[0006] The present invention provides an engineered bacterium that produces orsinoic acid. The engineered bacterium is prepared by transferring a yARGAorsA expression element into an Aspergillus nidulans strain in which the pyrG gene is deleted. The yARGAorsA expression element 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.

[0007] Furthermore, the engineered bacteria producing lysine acid is Aspergillus nidulans YAFWY001, classified as Aspergillus nidulans, and deposited with CCTCC NO: M 2024908.

[0008] Furthermore, the present invention provides a method for preparing the engineered bacteria producing lysine acid, comprising the following steps:

[0009] S1: Construction of yARGAorsA expression vector

[0010] Genes Ya5, pyrG, Rib, gbdA(p), OrsA, and Ya3 were sequentially linked to the linearized plasmid Puc19 using seamless ligation technology to obtain the expression vector yARGAorsA;

[0011] S2: The yARGAorsA expression element was obtained by PCR amplification using a high-fidelity DNA polymerase, and the yARGAorsA expression element was introduced into the Aspergillus nidulans LO8030 strain using a protoplast transformation method to obtain an engineered strain producing lysine.

[0012] Furthermore, the present invention provides a vector containing a yARGAorsA expression element, wherein the yARGAorsA expression element is composed of genes Ya5, pyrG, Rib, gbdA(p), OrsA and Ya3 linked in sequence from the 5' end, 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.

[0013] Furthermore, the present invention provides an engineered bacterium, which contains the vector. In some specific embodiments, this type of engineered bacterium is mainly used for amplification and expression of target genes, such as the Escherichia coli DH5α provided by the present invention.

[0014] Furthermore, the present invention provides the use of the vector and the engineered bacteria in the preparation of Aspergillus nidulans producing lysinoic acid.

[0015] Furthermore, the present invention provides a method for culturing the engineered bacteria to produce high yields of orselloic acid, wherein the fermentation culture is performed using YGM liquid culture medium, wherein each liter of the culture medium contains 0.5 g of yeast extract, 2 g of glucose, and 0.25-4 g of magnesium acetate.

[0016] The culture method comprises the following culture conditions: culture at 37° C. and a shaking platform at 150 rpm for 24 hours, and then culture at 30° C. and a shaking platform at 150 rpm for a further 72 hours.

[0017] Furthermore, the present invention provides the use of magnesium acetate in increasing the yield of orsellinic acid in the culture of Aspergillus nidulans YAFWY001, the Aspergillus nidulans YAFWY001 is classified and named Aspergillus nidulans, and its deposit number is CCTCC NO: M2024908.

[0018] Furthermore, the present invention provides a method for producing orsellinic acid, comprising the following steps:

[0019] (1) Aspergillus nidulans YAFWY001 was fermented to obtain a fermentation broth. Aspergillus nidulans YAFWY001 was classified and named Aspergillus nidulans, with a deposit number of CCTCC NO: M 2024908.

[0020] (2) The mycelia in the fermentation broth were separated to obtain a bacterial liquid, and 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 separation and the supernatant was extracted. After multiple extractions, the combined extracted supernatants were condensed and refluxed on a rotary evaporator and dried to obtain a crude extract of the fermentation broth of the YAFWY001 strain;

[0021] (3) Purify the crude extract to obtain high-purity tricosic acid.

[0022] The present invention provides a method for preparing and culturing a high-yielding engineered bacterium of lysine, which has the following advantages over the prior art:

[0023] First, the present invention uses the Aspergillus nidulans LO8030 strain with a low secondary metabolite background as a host, which can greatly simplify the later separation and purification of orsellinic acid; second, the expression element yARGAorsA used in the present invention carries the strong gbdA promoter, 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 high-yield orsellinic acid engineered bacteria YAFWY001 to directional synthesize a large amount of orsellinic acid, control the entire fermentation time to 96 hours, simplify and easily separate and purify, and greatly reduce costs.

[0024] The present invention is suitable for large-scale liquid fermentation production. The YAFWY001 engineered strain is used to carry out fermentation using the above-mentioned culture medium and culture conditions, and the fermentation products are separated and extracted. The YAFWY001 engineered strain can produce orsellinic acid in large quantities with a yield of 650 mg / L, which can effectively solve the problem of the source 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

[0025] Figure 1 This is a schematic diagram of the yARGAorsA expression element in Example 1 of the present invention;

[0026] Figure 2 This is a picture of the hyphae growth of Aspergillus nidulans YAFWY001;

[0027] Figure 3 This is a TLC analysis of the difference in orsellinic acid production in crude extracts at different magnesium acetate concentrations. Samples 1: 0.25g magnesium acetate / 100ml; 2: 0.5g magnesium acetate / 100ml; 3: 1g magnesium acetate / 100ml; and 4: 1g magnesium acetate / 100ml. Red represents orsellinic acid; larger red dots indicate higher content.

[0028] Figure 4 HPLC detection results of orselloic acid in YAFWY001;

[0029] Figure 5 UV absorption spectrum of moss acid

[0030] Figure 6 1H NMR spectrum of mossinoic acid

[0031] Figure 7 This is the 13C nuclear magnetic resonance spectrum of mossinoic acid. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the embodiments of the present invention. For purchased commodities in the test method, if no specific conditions are specified, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they can all be conventional products purchased from the market.

[0033] The high-yield lysine acid engineering bacteria provided by the present invention has a preservation name: Aspergillus nidulans YAFWY001 Aspergillus nidulans YAFWY001; classification name: Aspergillus nidulans, Deposit number: CCTCC NO: M 2024908; depositor: China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China; the culture was received by the Collection on May 11, 2024, and registered. According to the request, it was preserved for thirty years from that date. The viability of the culture was tested by the Collection on May 18, 2024, and the result was viable.

[0034] Example 1 Preparation method of engineered bacteria producing lysine

[0035] 1. Preparation of yARGAorsA expression element

[0036] First, DNA was extracted from Aspergillus nidulans and Gaoligong Antrodia cinnamomea. Using the A. nidulans DNA as a template, the upstream region of the A. nidulans yA gene, Ya5 (SEQ ID NO: 1), the gbdA(p) promoter (SEQ ID NO: 4), and the downstream region of the yA gene, Ya3 (SEQ ID NO: 6), were amplified. Furthermore, using the Gaoligong Antrodia cinnamomea DNA as a template, the Rib (SEQ ID NO: 3), pyrG (SEQ ID NO: 2), and OrsA (SEQ ID NO: 5) gene fragments of Gaoligong Antrodia cinnamomea were amplified. Finally, the amplified fragments were sequentially ligated into the linearized plasmid Puc19 using a seamless cloning kit (Order NO. B632219). The ligation products were transformed into Escherichia coli DH5α, and positive clones were obtained after overnight culture. The expression vector, yARGAorsA, was confirmed by PCR and sequencing. After extracting the yARGAorsA vector plasmid using a plasmid extraction kit, the yARGAorsA expression element was obtained by PCR amplification using a high-fidelity DNA polymerase using the yARGAorsA vector plasmid as a template, and the yARGAorsA expression element was purified using a PCR product recovery kit for later use.

[0037] 2. Obtaining high-yield lysine acid engineered bacteria YAFWY001

[0038] According to the Aspergillus nidulans protoplast transformation method described in the article (Hu Meimei, Chi Yujie. Establishment of a transformation method for the MnP gene of Lederma lucidum in Aspergillus nidulans [J]. Journal of Northeast Forestry University, 2013, 41 (07): 129-133.), the yARGAorsA expression element was transferred into the pyrG gene-deficient Aspergillus nidulans strain. Specifically used in the present invention was the Aspergillus nidulans LO8030 strain (the Aspergillus nidulans LO8030 strain had a pyrG gene deficiency and could not grow in a culture medium without uracil. Purchase from Baosai Bio, purchase link http: / / www.bio-sci.com.cn / index.php?id=2577). The high-yield moss acid engineered strain YAFWY001 was obtained by screening through nutrient deficiency (introduction of the Gaoligong Antrodia cinnamomea pyrG gene).

[0039] Example 2 Effect of adding different concentrations of magnesium acetate on the yield of rucic acid

[0040] 1. After activating the Aspergillus nidulans YAFWY001 strain, culture and obtain spores;

[0041] 2. Inoculate 1 ml of the spore suspension (spore concentration: 106 / ml) into 100 ml of YG liquid medium containing 0.25 g, 0.5 g, 1 g, 2 g, and 4 g of magnesium acetate. Incubate at 37°C, 150 rpm, and shake for 24 hours. Then, incubate at 30°C, 150 rpm, and shake for 72 hours. The YG formulation is: 0.5 g / L yeast extract; 2 g / L glucose.

[0042] 3. The mycelium in the fermentation broth was filtered to obtain a bacterial liquid, and 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 separation and the supernatant was extracted. After extraction three times, the combined extracts were condensed and refluxed on a rotary evaporator and dried to obtain crude extracts containing different concentrations of magnesium acetate YG liquid culture.

[0043] 4. TLC analysis of the effects of varying magnesium acetate concentrations on the production of orsellinic acid by Aspergillus nidulans YAFWY001 was performed (Figure 3). Red indicates orsellinic acid, and larger red dots indicate higher content. Therefore, 2g of magnesium acetate significantly increased the orsellinic acid content in the fermentation broth of Aspergillus nidulans YAFWY001. HPLC analysis compared the results with orsellinic acid standards, indicating that the orsellinic acid production by Aspergillus nidulans YAFWY001 in YG liquid culture supplemented with 2g of magnesium acetate was 650mg / L (Figure 4).

[0044] Example 3: Preparation of tricholic acid

[0045] 1. The mycelium in the fermentation broth was filtered to obtain a bacterial liquid, 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 separation and the supernatant was extracted. After extraction three times, the combined extracts were condensed and refluxed on a rotary evaporator and dried to obtain a crude extract of the fermentation broth of the high-yielding lysine engineered strain YAFWY001.

[0046] 2. First, use AB-8 type (weak polarity) macroporous resin to crudely fractionate the fermentation broth extract, and then flush the column with pure water, 30% ethanol, 60% ethanol, 90% ethanol and ethyl acetate in sequence (each eluent flushes 6 column volumes) to segment. Then use TLC to determine the segment where the rucic acid is located, and merge the segments containing rucic acid.

[0047] 3. Then, the LC3050 preparative chromatograph was used for gradient elution to subdivide the tricholonic acid.

[0048] 4. Finally, after using TLC to determine the location of orsellinic acid, the preparation was carried out using an Agilent 1260 high performance liquid chromatograph at a wavelength of 254 nm to obtain a pure product (purity> 98%). The isolated pure product was confirmed to be orsellinic acid by UV absorption spectrum and nuclear magnetic resonance (NMR). Figure 5 ,6,7).

[0049] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A lysine acid-producing engineered bacterium, characterized in that: The engineered bacteria is Aspergillus nidulans YAFWY001, which is classified as Aspergillus nidulans , the deposit number is CCTCC NO: M 2024908.

2. A method for culturing the engineered bacteria for high production of orsellinic acid according to claim 1, 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.

3. The culture method according to claim 2, wherein 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.

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