CN112430547B - Acid-resistant aspergillus oryzae strain capable of highly producing acetyl coenzyme A and application thereof - Google Patents

Abstract

The invention belongs to the field of microorganisms, and particularly relates to an acid-resistant aspergillus oryzae strain capable of highly producing acetyl coenzyme A and application thereof. The specific technical scheme is as follows: a kind of Aspergillus oryzae, ITS sequence is shown in SEQ ID No: 1 is shown. The invention provides a brand-new aspergillus oryzae strain, which has strong acid resistance and extremely high acetyl coenzyme A production capacity and wide application prospect.

Description

Acid-resistant aspergillus oryzae strain capable of highly producing acetyl coenzyme A and application thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to an acid-resistant aspergillus oryzae strain capable of highly producing acetyl coenzyme A and application thereof.

Background

Chassis cells refer to cells that can be placed into certain modules of a functionalized biological system to provide specific functionality. For example, the underpan cells can be used for heterologously expressing natural products and derivatives thereof or producing medicaments and the like. The main underpan cells which are most widely used at present are microbial cells such as escherichia coli, streptomycete, yeast, cyanobacteria, pseudomonas putida and the like. However, currently, the existing underpan cells for synthetic biology have two important problems: (1) only a small number of eukaryotic cells are present, which makes it difficult for a large number of genes derived from eukaryotic cells to function normally in these prokaryotic underpan cells. (2) Although eukaryotic cells such as yeast can express genes of other eukaryotes, in vivo metabolic activities can only provide less natural products or synthetic precursors of drug molecules, resulting in lower product concentrations, which is not favorable for the application of synthetic biology in industrial production.

Acetyl-coa is the acetylated form of coa, and plays a critical role in many metabolism as well as a key precursor to the synthesis of numerous natural products and drugs. If a eukaryotic cell capable of producing acetyl-CoA in high yield could be provided, the choice of the underpan cells would be expanded.

Aspergillus oryzae, a filamentous fungus, has been used in food fermentation worldwide for thousands of years and is also certified by the U.S. Food and Drug Administration (FDA) as a food-safe strain (GRAS). Because the aspergillus oryzae has high growth speed and vigorous metabolic activity in vivo, a large amount of natural products or synthetic precursors of drug molecules can be accumulated, the synthesis of a target product with higher concentration is facilitated, and in addition, except kojic acid, the aspergillus oryzae hardly generates secondary metabolites, so that the separation and purification of the product are facilitated. Therefore, Aspergillus oryzae is a underpan cell with great application potential. If Aspergillus oryzae with high acetyl-CoA yield can be provided, the research and application of synthetic biology can be played an important role.

Disclosure of Invention

The invention aims to provide aspergillus oryzae and application thereof.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: the aspergillus oryzae is preserved in China general microbiological culture Collection center (CGMCC) at 11 months and 5 days in 2020, and the preservation address is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, with the deposit number: CGMCC No. 20748.

Correspondingly, the ITS sequence of the Aspergillus oryzae is shown as SEQ ID No: 1 is shown.

Correspondingly, the Aspergillus oryzae is applied to being used as a underpan cell.

Correspondingly, the application of the aspergillus oryzae in preparing acetyl-coenzyme A.

The invention has the following beneficial effects: the invention provides a brand-new aspergillus oryzae strain, which has strong acid resistance and extremely high acetyl coenzyme A production capacity and wide application prospect.

Drawings

FIG. 1 is a colony map of Aspergillus oryzae strain AK 2;

FIG. 2 is a microscopic morphology of encysted shell of Aspergillus oryzae strain AK 2;

FIG. 3 shows the dry weight of A.oryzae strain AK2 under different conditions of initial medium pH.

Detailed Description

The invention provides a novel Aspergillus oryzae strain, Aspergillus oryzae, wherein the Internal Transcribed Spacer (ITS) sequence of the Aspergillus oryzae strain is shown as SEQ ID No: 1 is shown. The aspergillus oryzae has the capability of producing acetyl coenzyme A beyond the average level of the variety, and has wide application prospect in the aspects of being used as a chassis cell and the like.

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

The first embodiment is as follows: screening and identification of Aspergillus oryzae

1. Screening: collecting an environmental sample near a grease factory, and culturing in an enrichment medium. Enrichment medium (g/L): glucose 20, peptone 10, yeast extract powder 5, monopotassium phosphate 3, dipotassium phosphate 1, magnesium sulfate 0.5 and calcium chloride 0.1; then adding 1000mL of distilled water, and adjusting the pH value to 6.5-7.0. Culturing for 3-5 days at 28-32 deg.C and at 150-200 rpm to obtain fermentation liquid.

Diluting the fermentation liquor by 5-20 times, coating the diluted fermentation liquor on a PDA solid culture medium plate, culturing for 3-7 days at 30 ℃, selecting a single colony, inoculating the single colony into a new PDB culture medium, and culturing for 3-7 days at 30 ℃ to obtain a culture of the PDB culture medium. PDA medium (g/L): 200 parts of potato, 20 parts of glucose and 20 parts of agar powder, and additionally 1000mL of distilled water with natural pH. The PDB solid culture medium is formed by adding 25g/L agar powder on the basis of the PDB culture medium.

Taking the culture of PDB culture medium, using sterile glass beads to break up mycelium, adding Nile red dye (Nile red), mixing uniformly, standing for 5 minutes, and measuring the light absorption value at 580nm by using a fluorescence spectrophotometer (excitation wavelength is 485 nm). The absorbance at 580nm is in positive correlation with the content of acetyl coenzyme A in the fungus body. The 3 strains with the highest absorbance at this point were selected, with absorbance values of 0.38, 0.44 and 0.56, respectively.

The 3 strains were inoculated into a new PDB medium, and cultured at 30 ℃ and 200rpm for 5 to 7 days. Then filtered at 4 ℃ to obtain the mycelium. And (4) filtering again after washing with sterile water to obtain clean mycelia. Liquid nitrogen was poured into the mycelium and the mycelium was ground in a grinder. The Acetyl-CoA content of each mycelial disruption solution was measured using an Acetyl CoA Assay kit (Abcam Co., Ltd.). And simultaneously measuring the dry weight of the mycelium in unit volume, thereby calculating the acetyl coenzyme A content of the unit dry weight of the mycelium. The acetyl coenzyme A content of 3 strains is respectively determined as follows: 9.3nmol/mg, 4.2nmol/mg and 3.8 nmol/mg. Selecting the bacterium with the highest acetyl coenzyme A content, and naming the bacterium as: strain AK 2. Meanwhile, the acetyl-CoA content of the commonly used A.oryzae strain NSAR1 was measured to be 4.1nmol/mg using the same method.

2. And (3) identification: the strains were identified using the Internal Transcribed Spacer (ITS) sequence. Firstly, collecting mycelium of a strain AK2, and extracting genomic DNA as a PCR template; then an ITS fragment of the strain AK2 is obtained by PCR cloning through a forward primer (the sequence is shown as SEQ ID No: 2) and a reverse primer (the sequence is shown as SEQ ID No: 3), and an ITS sequence (the sequence is shown as SEQ ID No: 1) is obtained after sequencing. Homology analysis is carried out on the ITS sequence and the sequence of a GenBank database, the sequence homology of the strain AK2 and an Aspergillus oryzae strain is higher than 99 percent, and the strain AK2 is Aspergillus oryzae and Aspergillus oryzae from the perspective of molecular evolution; hereinafter collectively referred to as Aspergillus oryzae AK 2.

3. Physiological and biochemical characteristics: after Aspergillus oryzae strain AK2 was cultured on PDA medium at 30 deg.C for 5 days, the colony appeared circular, and was light yellow overall, the mycelium had loose texture, and the surface had wrinkled ridges, as shown in FIG. 1. Microscopic morphology observation shows that hyphae have a diaphragm, the molecular spore stalks are rough and have a somewhat convex shape, the top sacs are expanded into a spherical shape, the diameter is about 50 mu m, and the small stalks are double-layered. Conidia are clustered and spherical, the diameter of each conidia is 4.6-5.3 mu m, and the closed capsule shells are spherical and are dispersed among the mycelia, as shown in figure 3. The fact that the strain AK2 was Aspergillus oryzae was demonstrated by morphological evidence.

4. And (4) preservation: is preserved in China general microbiological culture Collection center (CGMCC) at 11/5/2020 with the preservation addresses as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, with the deposit number: CGMCC No. 20748.

Example two: acid resistance application of aspergillus oryzae AK2

Aspergillus oryzae AK2 was inoculated on a slant of CMP solid medium and cultured at 30 ℃ for 5 days. CMP solid medium (g/L): maltose 20, peptone 10, leaching powder 3, sodium nitrate 3, dipotassium phosphate 1, magnesium sulfate 0.5, potassium chloride 0.5, ferrous sulfate 0.01 and agar powder 25. Followed by a solution containing 0.5% (v/v) of vomitThe Aspergillus oryzae spores on the slant were eluted with sterile water at-80 deg.C to obtain a spore suspension. Measuring spore concentration by cytometry using a hemocytometer, and diluting spore solution to 1.0 × 10⁵～5.0×10⁵In the concentration range of one/mL. The spore liquid was inoculated at an inoculum size of 5% (v/v) into CMP liquid media (reduced agar powder based on CMP solid media) at different initial phs ( pH 2, 3, 4, 5, 6, 7) and cultured at 200rpm at 30 ℃ for 7 days. The results are shown in fig. 3, when the initial pH is 2.0 and 3.0, the dry weight of aspergillus oryzae strain AK2 reaches 8.7 and 8.5g/L respectively, which is higher than the culture conditions of other initial pH, indicating that aspergillus oryzae strain AK2 has higher acid resistance and wide application prospect.

Example three: application of aspergillus oryzae in production of acetyl coenzyme A

Preparation of 1.0X 10 as described in example two⁵～5.0×10⁵Spore liquid of Aspergillus oryzae strain AK2 at a concentration of one/mL. The spore liquid was inoculated at an inoculum size of 5% (v/v) into a CMP liquid culture having an initial pH of 3.0, and cultured at 200rpm at 30 ℃ for 7 days. Aspergillus oryzae mycelia were collected by filtration through filter cloth. Measured as described in example one: the dry weight of the mycelia was 8.7g/L, the acetyl-CoA content was 9.3nmol/mg, and the calculated acetyl-CoA yield was 80.9. mu. mol/L (65.5 mg/L).

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes, modifications, alterations, and substitutions which may be made by those skilled in the art without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Sequence listing

<110> university of agriculture in Huazhong

<120> acid-resistant aspergillus oryzae strain with high acetyl coenzyme A yield and application thereof

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gggctctcag ccccgggccc gcgcccgccg gagacaccac gaactcagtc tgatctagtg 180

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tcctccgctt attgatatgc 20

Claims (2)

1. Aspergillus oryzae AK2 (Aspergillus oryzae) The culture medium is characterized by being preserved in China general microbiological culture Collection center at 11 months and 5 days in 2020, and the preservation addresses are as follows: beijing & Chao Xilu No. 1Hospital No. 3, accession number: CGMCC No. 20748.

2. Use of Aspergillus oryzae according to claim 1 for the preparation of acetyl-CoA.

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