CN117025638A - Gene Aokap3 for improving kojic acid yield of aspergillus oryzae, method and application - Google Patents
Gene Aokap3 for improving kojic acid yield of aspergillus oryzae, method and application Download PDFInfo
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Abstract
The invention provides a gene Aokap3 for improving the yield of aspergillus oryzae kojic acid, a method and application, wherein the Aokap3 gene is a novel gene for improving the content of aspergillus oryzae kojic acid, and the yield of aspergillus oryzae kojic acid can be obviously improved by destroying the biological function of the protein encoded by the Aokap3 gene. The invention discovers that the nucleotide sequence from 390 th to 409 th in the Aokap3 gene of aspergillus oryzae is taken as a target sequence, the fixed-point knockout of the Aokap3 gene can be realized efficiently, and the coded protein of the Aokap3 gene can be damaged due to insertion or deletion from 390 th to 409 th of the sequence shown as SEQ ID NO.2, thereby improving the yield of the aspergillus oryzae kojic acid, and experiments prove that the engineering strain with high kojic acid yield can be prepared by knocking out the Aokap3 gene, and has very important application value in the production of the kojic acid.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a gene Aokap3 for improving aspergillus oryzae kojic acid yield, a method and application.
Background
Kojic acid, chemical name 5-hydroxy-2-hydroxymethyl-1, 4-pyrone, is an organic acid synthesized by microorganisms during fermentation. Kojic acid has various effects including antibacterial, antioxidant, antiviral, antitumor, freshness improving and whitening effects. Therefore, kojic acid has wide application value in the fields of food, medicine, cosmetics and the like. At present, the kojic acid is usually produced by adopting a method of Aspergillus oryzae fermentation. Since kojic acid yield of Aspergillus oryzae strain obtained directly from nature is low, high kojic acid yield strain should be screened by mutagenesis, but current researches show that the effect of improving kojic acid yield by mutagenesis is not obvious. Therefore, the genes for improving the synthesis amount of the aspergillus oryzae kojic acid are excavated, the production path of the kojic acid is optimized by utilizing the genetic engineering technology and the metabolic engineering technology, and the high-yield kojic acid strain is constructed, so that the yield and the quality of the kojic acid are improved, the production cost is reduced, and the application and the development of the kojic acid are promoted.
Disclosure of Invention
In order to solve the technical problems, the invention provides a gene Aokap3 for improving the yield of aspergillus oryzae kojic acid, a method and application.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a gene Aokap3 for increasing kojic acid yield of aspergillus oryzae, the encoded protein of the gene Aokap3 having any one of the following amino acid sequences:
(1) An amino acid sequence as shown in SEQ ID NO. 1;
(2) Amino acid sequence related to kojic acid yield obtained by substituting, inserting or deleting one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1;
(3) An amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO. 1; preferably, the homology is at least 90%; more preferably 95%.
The amino acid sequence shown in SEQ ID No.1 is the encoding protein sequence of the Aspergillus oryzae Aokap3 gene, and one skilled in the art can substitute, delete and/or add one or more amino acids according to the conventional technical means in the art such as the disclosed amino acid sequence and conservative substitution of amino acids under the premise of not affecting the activity of the protein, so as to obtain the mutant with the same activity as the encoding protein of the Aspergillus oryzae Aokap3 gene disclosed by the invention.
In a second aspect, the present invention provides a gene Aokap3 for increasing kojic acid yield of aspergillus oryzae, said gene Aokap3 having any one of the following nucleotide sequences:
(1) A nucleotide sequence as shown in SEQ ID NO. 2;
(2) The nucleotide sequence shown as SEQ ID NO.2 is obtained by replacing, inserting or deleting one or more nucleotides to obtain the nucleotide sequence encoding the same functional protein.
The gene Aokap3 of the invention can be any nucleotide sequence capable of encoding the encoded protein of the gene Aokap 3. In view of the degeneracy of codons and the preferences of codons of different species, one skilled in the art can use codons appropriate for expression of a particular species as desired.
In a third aspect, the present invention provides an inhibitor of the above gene Aokap3, which is a nucleic acid capable of disrupting the biological function of the encoded protein of gene Aokap 3.
Preferably, the nucleic acid is sgRNA or interfering RNA.
Preferably, the target sequence of the sgRNA is a nucleotide sequence of the gene Aokap3 in the form of XXXNGG, wherein XXX is a 19-20bp nucleic acid sequence, and N is any one base of A, T, G, C.
More preferably, the target sequence of the sgRNA is from position 390 to position 409 of the sequence shown in SEQ ID NO.2.
In a fourth aspect, the invention provides a sgRNA for mutating the gene Aokap3, wherein the target sequence of the sgRNA is 390 th to 409 th of the sequence shown in SEQ ID NO.2.
Preferably, the sgRNA comprises a sequence as shown in SEQ ID NO. 4.
The sgRNA can be matched with a CRISPRP/Cas 9 gene editing tool to realize the efficient fixed-point knockout of the Aspergillus oryzae Aokap3 gene and destroy the biological function of the encoded protein of the Aspergillus oryzae Aokap3 gene.
Experiments prove that the Aspergillus oryzae strain with the Aspergillus oryzae Aokap3 gene mutation can be obtained efficiently by using the sgRNA to carry out CRISPRP/Cas 9 mediated gene editing, and the biological functions of the Aspergillus oryzae Aokap3 gene are destroyed due to insertion or deletion at 390 th to 409 th positions of a sequence shown as SEQ ID NO.2, so that the Aspergillus oryzae shows the character of improving the kojic acid yield, and the construction efficiency of the high-yield kojic acid strain based on the Aspergillus oryzae Aokap3 mutation is further improved effectively.
In a fifth aspect, the invention also provides a biological material comprising the sgRNA for the mutant gene Aokap3, the biological material comprising an expression cassette, a vector, a host cell, an engineering bacterium or a transgenic plant cell line.
In a sixth aspect, the invention provides an application of the gene Aokap3, its coding protein or inhibitor in regulating and controlling aspergillus oryzae kojic acid synthesis or constructing engineering bacteria with high kojic acid yield.
Preferably, in the above application, the kojic acid yield is increased by disrupting the biological function of the encoded protein of the gene Aokap 3.
The application of the gene Aokap3, the encoded protein or the inhibitor thereof can be applied in the form of the gene Aokap3, the encoded protein or the inhibitor thereof, or in the form of an expression cassette, a vector or a host cell containing the gene Aokap3 or the inhibitor thereof.
In a seventh aspect, the present invention provides a method for controlling aspergillus oryzae kojic acid synthesis or constructing a high yield kojic acid engineering strain, comprising: by disrupting the biological function of the encoded protein of the gene Aokap3, the kojic acid yield of aspergillus oryzae is improved.
The biological function of the protein encoded by the disrupted gene Aokap3 can be achieved by conventional technical means in the art.
Preferably, the CRISPRP/Cas 9 technology is utilized to destroy the biological function of the encoded protein of the gene Aokap 3; the CRISPRP/Cas 9 technology uses sgRNA with target sequence from 390 th to 409 th of sequence shown as SEQ ID NO.2.
More preferably, the sgRNA comprises the sequence shown as SEQ ID NO. 4.
The CRISPRP/Cas 9 technology can be utilized to cut a nucleotide sequence in the form of XXXNGG in the Aokap3 gene of aspergillus oryzae at a position 3-4 bp upstream of NGG to generate DNA double-strand break, so that insertion deletion of the nucleotide sequence is introduced, further, translation of the gene is terminated in advance or protein conformation is changed, and finally the biological function of protein encoded by the gene is destroyed; wherein XXXNGG is a 19-20bp nucleic acid sequence and N is A, T, G, C.
As a preferable scheme of the invention, the method for regulating and controlling the synthesis of the aspergillus oryzae kojic acid or constructing the engineering strain with high kojic acid yield comprises the following steps:
1) Constructing CRISPRP/Cas 9 gene editing plasmid containing sgRNA shown in SEQ ID NO. 4;
2) Transferring the CRISPRP/Cas 9 gene editing plasmid constructed in the step 1) into aspergillus oryzae;
3) The material for improving the yield of the kojic acid is obtained through screening and identification.
Preferably, in the step 1), the CRISPRP/Cas 9 gene editing plasmid is a type II CRISPR system;
preferably, in the step 2), the CRISPRP/Cas 9 gene editing plasmid constructed in the step 1) is transferred into aspergillus oryzae in a protoplast mode.
Preferably, in the step 3), the identifying method specifically includes: amplifying and sequencing the converted Aspergillus oryzae Aokap3 gene fragment by using a specific primer of the Aspergillus oryzae Aokap3 gene, and if the 390 st to 409 th nucleic acid sequence of the sequence shown as SEQ ID NO.2 is subjected to insertion or deletion mutation, the biological function of the Aspergillus oryzae Aokap3 gene is destroyed.
The host cell of the invention is a filamentous fungal host cell Aspergillus oryzae, preferably a filamentous fungal host cell of the genus Aspergillus, including but not limited to Aspergillus oryzae.
The invention has the beneficial effects that:
(1) The invention provides a novel gene for improving the kojic acid content of aspergillus oryzae: aokap3 gene. The yield of kojic acid of Aspergillus oryzae can be remarkably improved by disrupting the biological function of the protein encoded by the Aokap3 gene. Experiments prove that the engineering strain with high kojic acid yield can be prepared by knocking out the Aokap3 gene, and has very important application value in the production of kojic acid.
(2) The invention further utilizes CRISPRP/Cas 9 technology to carry out genome targeting modification of Aokap3 genes, thereby realizing efficient fixed-point knockout of Aokap 3. The invention discovers that the nucleotide sequence from 390 th to 409 th in the Aokap3 gene of Aspergillus oryzae is taken as a target sequence, the site-directed knockout of the Aokap3 gene can be efficiently realized, and the coded protein of the Aokap3 gene can be destroyed due to insertion or deletion from 390 th to 409 th of the sequence shown as SEQ ID NO.2, thereby improving the yield of Aspergillus oryzae kojic acid and effectively improving the construction efficiency of engineering strains of high yield kojic acid based on Aokap3 gene mutation.
Drawings
FIG. 1 type of mutation of the nucleotide sequence of Aokap3 gene in Aokap3 gene homozygous mutant strains in the wild type Aspergillus oryzae 3.042 background.
FIG. 2 mutation types of amino acid sequences of Aokap3 genes in Aokap3 gene homozygous mutant strains in the wild type Aspergillus oryzae 3.042 background.
FIG. 3 analysis of kojic acid yield in wild type Aspergillus oryzae 3.042, aokap3 gene homozygous mutant strains.
Detailed Description
In order to more clearly illustrate the present invention, the present invention will be described in further detail below with reference to examples and with reference to the accompanying drawings. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.
Examples
The relevant primers used below are shown in Table 1.
TABLE 1 primer information
The main steps of this embodiment are as follows:
1. construction of A.oryzae Aokap3 knockout Strain Using CRISPR technology
1. Sequence analysis and target sequence screening of Aspergillus oryzae Aokap3 gene
The sequence of the Aspergillus oryzae Aokap3 coding gene is shown in SEQ ID NO.2. Sequence analysis showed that the sequence shown in SEQ ID NO.2 (CDS sequence of Aokap3 gene) consists of 663 nucleotides, encoding the protein shown in SEQ ID NO. 1. The sequence shown in SEQ ID NO.3 consists of 1204 nucleotides, comprises UTR, exon and intron of the Aokap3 gene, and the 161 th to 208 th, 259 th to 403 th and 467 th to 936 th positions of the sequence shown in SEQ ID NO.3 are respectively three exons of the Aokap3 gene. The sequences of the three exons of the Aokap3 gene in SEQ ID NO.3 are sequentially connected to obtain SEQ ID NO.2.
The sequence on the third exon of the Aspergillus oryzae Aokap3 gene is an Aokap3 target sequence based on a CRISPR/Cas9 technology Aspergillus oryzae Aokap3 gene fixed-point knockout method.
Through a large number of screening, the invention determines that the 390 th bit to 409 th bit of the sense strand of the third exon of the Aspergillus oryzae Aokap3 gene is targeted by using the CRISPR/Cas9 technology as a target sequence, and the target sequence is shown as SEQ ID NO. 4.
Construction of pPRTII-Cas9-Aokap3 knockout vector
2.1 construction of sgRNA expression cassettes containing Aokap3 Gene targeting sequences
The genome DNA of Aspergillus oryzae RIB40 strain is used as template, and the primers PU6-F and PU6-Aokap3-R (the reverse primer contains the targeting sequence of Aokap 3) are used to amplify the U6 promoter (PU 6), the amplification system is shown in Table 2, and the PU6-Aokap3 fragment is obtained.
The sgRNA and the U6 terminator sequence are directly synthesized by a gene synthesis method, and PCR amplification is carried out by taking the sgRNA and the U6 terminator sequence as templates and TU6-Aokap3-F (taking the targeting sequence of the Aokap3 target gene as a primer adapter) and TU6-R as primers (the amplification system is shown in Table 2) to obtain the Aokap3-sgRNA-TU6.
TABLE 2 50. Mu.l amplification System for the U6 promoter
Amplification conditions: pre-denaturation at 95℃for 30s, denaturation at 95℃for 10s, annealing at 55℃for 30s, extension at 72℃for 5min,35 cycles, and thorough extension at 72℃for 5min.
mu.L of PU6-Aokap3 and Aokap3-sgRNA-TU6PCR products were used as templates and PU6-F and TU6-R were used as primers for overlap PCR amplification (the overlap PCR amplification system is shown in Table 3).
TABLE 3 overlapping PCR amplification System
Amplification conditions: pre-denaturation at 95℃for 30s, denaturation at 95℃for 10s, annealing at 55℃for 30s, and extension at 72℃for 5min. After 5 cycles, 4. Mu.l of the primer (PU 6-F and TU6-R mixture (10. Mu.M)) was added and amplification was continued for 30 cycles to obtain the sgRNA expression cassette PU6-Aokap3-TU6 containing the Aokap3 gene targeting sequence.
2.2 recombinant ligation of pPRTII-Cas9-Aokap3 knockout vector
Linearization of 2.2.1pPRTII-Cas9 vector
pPRTII-Cas9 vector: specific description please refer to patent "a method for directionally genetically modifying Aspergillus oryzae gene based on CRISPR technology (publication No. CN110592073A, publication No. 2019, 12, 20)" filed in 2019 by applicant "
The pPTRII-Cas9 plasmid was digested with SmaI (see Table 4 for the cleavage system) to obtain linearized pPTRII-Cas9 fragment.
TABLE 4 pPRTII-Cas9 vector cleavage System
Incubate at 37℃for 1 hour.
2.2.2 recombinant ligation of linearization vectors to PCR products
The linearized pPTRII-Cas9 fragment is recombined and connected with a PU6-Aokap3-TU6 expression cassette by utilizing recombinase to obtain a recombinant plasmid pPTRII-Cas9-Aokap3 (the recombination connection reaction system is shown in Table 5).
TABLE 5 recombination ligation reaction System
The reaction was carried out at 37℃for 30min, after which the temperature was reduced to 4℃or immediately cooled on ice.
3. Transfer of the pPTRII-Cas9-Aokap3 knockout vector into Aspergillus oryzae 3.042 Strain by protoplast transformation
(1) Aspergillus oryzae 3.042 spores were inoculated into 100ml DPY broth (2% glucose,1% peptone,0.5% yeastex, 0.5% KH) 2 PO 4 ，0.05％MgSO 4 ·7H 2 O, pH 5.5), mycelia were collected after culturing for 16-20 hours, and the mycelia were washed with an enzyme buffer (50 mM maleic acid, 0.6M (NH) 4 ) 2 SO 4 Ph 5.5) one wash;
(2) Protoplasts were prepared by lysing the mycelium with 1% Yatalase (TaKaRa) and 1.5% cell lysis enzyme (Sigma); (3) With washing buffer (1.2M sorbitol, 50mM NaCl) 2 ·2H 2 O，35mMNaCl，10mMTris-HCl，pH
7.5 Resuspension of protoplasts, centrifugation at 1,000rpm for 8min, collection of protoplasts, resuspension of protoplasts with wash buffer, and placement on ice;
(4) Mu.g of pPTRII-Cas9-Aokap3 was mixed with 200. Mu.l of protoplasts and incubated on ice for 30min. Then divided into three times (250
Mu.l, 250. Mu.l, 850. Mu.l) were added to PEG buffer (60% PEG4000, 50mM NaCl) 2 ·2H 2 O，10mMTris-HCl，
pH 7.5), gently mixing, and standing at room temperature for 10-20min;
(5) Dilution of PEG-treated Prostrates with washing bufferProtoplasts were collected by centrifugation at 1,000rpm for 8min at 4℃and then added to M+Met medium (0.2% NH) containing 1.2M sorbitol and 0.5% agar 4 Cl，0.1％(NH 4 ) 2 SO 4 ，0.05％KCl，0.05％NaCl，0.1％KH 2 PO 4 ，0.05％MgSO 4 ·7H 2 O，0.002％FeSO 4 ·7H 2 O，2％
glucose,0.15% methionine, l.2 Msorbol, pH 5.5) by mixing, and spreading on a gel containing 1.2M sorbitol
1.5% agar M+Met medium plate, 30 ℃ culture for 3-5 days.
4. Aokap3 targeting sequence analysis of transformants
The hyphae grown in the MM medium were picked up and transferred to CD medium (2% glucose,0.2% NaNO) containing 0.1. Mu.g/ml pyridine thiamine 3 ，0.1％KH 2 PO 4 ，0.05％MgSO 4 ，0.05％KCl，0.05％NaCl，0.002％FeSO 4 Ph 5.5). After 3-5 days of incubation at 30℃the mycelium after screening was picked up and placed in a 1.5ml centrifuge tube, 100. Mu.l of 25mM NaOH solution was added and heated at 100℃for 20min. 2 μl of supernatant was used as a template, CRISPR-S-Aokap3-F/R was used as a primer, and KODDNA polymerase (TOYOBO) was used to amplify the Aokap3 targeting sequence, and the Aokap3 targeting sequence was analyzed by sequencing.
50 μl of Aokap3 targeting sequence amplification System
As a result, as shown in FIGS. 1 and 2, 2 homozygous mutant strains of Aokap3 gene were successfully obtained in the background of Aspergillus oryzae 3.042: ΔAokap3-1 and ΔAokap3-2. Aokap3 gene targeting sequences in the ΔAokap3-1 and ΔAokap3-2 mutant strains appeared to have deletions of 11 bases and 31 bases, respectively (FIG. 1), which resulted in premature termination of Aokap3 gene translation (FIG. 2).
2. Kojic acid content determination of Aspergillus oryzae Aokap3 knockout strain
Spore solutions of Aspergillus oryzae 3.042, aokap3 knockout bacteria ΔAokap3-1 and ΔAokap3-2 (10) 5 personal/mL) connectorSeed to liquid kojic acid fermentation medium (10% glucose,0.1% K) 2 HPO 4 ，0.05％MgSO 4 0.05% kcl,0.1% yeastex) to produce kojic acid. Fermentation conditions: 30℃at 200rpm. The fermentation time was 7 days. The method for measuring the yield of the kojic acid is FeCl 3 Color development. The method comprises the following steps: taking 2mL of fermentation broth fermented to 7 days, centrifuging at 10,000rpm for 2min, taking 0.5mL of fermentation supernatant to a 2mL centrifuge tube, adding 0.5mL of ddH 2 O and 1mLFECl 3 -HCl solution (0.06 MFeCl 3 And 0.27m hcl), vortex mix well. And (3) taking water instead of fermentation supernatant as a control, measuring the absorbance of the mixed solution at the wavelength of 500nm by using an enzyme-labeled instrument, and further calculating the kojic acid yield by using a drawn kojic acid standard curve. The results are shown in FIG. 3A, feCl 3 The color development method shows that the fermentation liquid of the wild strain 3.042 has red color, and the Aokap3 knocked-out bacteria fermentation liquid shows deeper red color, which indicates that the Aokap3 knocked-out bacteria fermentation liquid promotes the synthesis of aspergillus oryzae kojic acid. The kojic acid content measurement shows that the yield of the Aokap3 knockout kojic acid is 1.6 times that of the wild type 3.042 bacteria, and the average content reaches 21.62 mg/ml.g, as shown in figure 3B.
It should be understood that the foregoing examples of the present invention are merely illustrative of the present invention and not limiting of the embodiments of the present invention, and that various other changes and modifications can be made by those skilled in the art based on the above description, and it is not intended to be exhaustive of all the embodiments of the present invention, and all obvious changes and modifications that come within the scope of the invention are defined by the following claims.
Claims (10)
1. A gene Aokap3 for improving the yield of kojic acid of Aspergillus oryzae, wherein the encoded protein of the gene Aokap3 has any one of the following amino acid sequences:
(1) An amino acid sequence as shown in SEQ ID NO. 1;
(2) Amino acid sequence related to kojic acid yield obtained by substituting, inserting or deleting one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1;
(3) An amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO. 1.
2. The gene Aokap3 of claim 1, wherein the gene Aokap3 has any one of the following nucleotide sequences:
(1) A nucleotide sequence as shown in SEQ ID NO. 2;
(2) The nucleotide sequence shown as SEQ ID NO.2 is obtained by replacing, inserting or deleting one or more nucleotides to obtain the nucleotide sequence encoding the same functional protein.
3. The inhibitor of gene Aokap3 of claim 1 or 2, which is a nucleic acid capable of disrupting the biological function of the encoded protein of gene Aokap 3.
4. The inhibitor according to claim 3, wherein the nucleic acid is sgRNA or interfering RNA; the target sequence of the sgRNA is a nucleotide sequence of XXXNGG form in the gene Aokap3, wherein XXX is a 19-20bp nucleic acid sequence, and N is any one base of A, T, G, C.
5. An sgRNA for mutating the gene Aokap3 according to claim 1 or 2, said sgRNA having the target sequence as set forth in SEQ ID no
No.2 shows the 390 st to 409 th positions of the sequence.
6. The sgRNA of claim 5, wherein the sgRNA comprises a sequence as set forth in SEQ ID No. 4.
7. A biological material comprising the sgRNA of claim 5, the biological material comprising an expression cassette, a vector, a host cell, an engineering bacterium, or a transgenic plant cell line.
8. The use of the gene Aokap3, its encoded protein or inhibitor according to claim 1 or 2 in regulating aspergillus oryzae kojic acid synthesis or constructing engineering bacteria with high kojic acid yield.
9. A method for regulating and controlling aspergillus oryzae kojic acid synthesis or constructing a high-yield kojic acid engineering strain, comprising the following steps: the kojic acid yield is increased by disrupting the biological function of the encoded protein of the gene Aokap3 according to claim 1 or 2.
10. The method of claim 9, wherein the biological function of the protein encoded by the aspergillus oryzae aokappa 3 gene is disrupted using CRISRP/Cas9 technology; the target sequence of sgRNA used by the CRISPRP/Cas 9 technology is shown as SEQ
390 th to 409 th bits of the sequence shown in ID No.2.
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