CN115725632B - Aomsn2 over-expression aspergillus oryzae engineering bacteria and construction method and application thereof - Google Patents
Aomsn2 over-expression aspergillus oryzae engineering bacteria and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses an Aomsn2 over-expression Aspergillus oryzae engineering bacterium, and a construction method and application thereof, wherein the construction method comprises the following steps: designing a corresponding Aomsn2 primer by taking an Aomsn2 gene in an aspergillus oryzae sample as a template, and carrying out PCR (polymerase chain reaction) amplification on the genome of the aspergillus oryzae sample by using the Aomsn2 primer to obtain an Aomsn2 cloned gene, wherein the nucleotide sequence of the Aomsn2 cloned gene is shown as SEQ ID NO.1, and the nucleotide sequence of the Aomsn2 primer is shown as SEQ ID NO. 2-3; performing enzyme digestion and enzyme ligation treatment on the Aomsn2 cloned gene and an expression vector pEX2B to construct an Aomsn2 overexpression vector; and (3) transforming the Aomsn2 over-expression vector into Aspergillus oryzae by adopting a method of mediating protoplast transformation, and constructing and obtaining the Aomsn2 over-expression Aspergillus oryzae engineering bacteria. According to the invention, research shows that the Aomsn2 gene can improve the yield of kojic acid in Aspergillus oryzae by controlling and regulating transcription factors of the kojic acid gene cluster, thereby laying a foundation for developing industrial brewing strains with high yield of kojic acid and providing a theoretical foundation for industrial application of Aspergillus oryzae and kojic acid production.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to an Aomsn2 over-expressed Aspergillus oryzae engineering bacterium, and a construction method and application thereof.
Background
Aspergillus oryzae (Aspergillus oryzae) is a aerobic fungus that has been identified by the United states FDA and WHO as a safe producing strain. Aspergillus oryzae can produce amylase, saccharifying enzyme, cellulase, phytase, etc. in addition to protease. Aspergillus oryzae has powerful post-translational modification functions such as glycosylation and protein folding compared to E.coli and yeast; compared with higher organisms such as plants and insects, the aspergillus oryzae grows rapidly, has low requirements on nutritional environment, and is an ideal host for expressing exogenous proteins. The Aspergillus oryzae exogenous expression system can be used for producing a plurality of important organic acids such as malic acid, kojic acid, fatty acid, lactic acid and the like. As such, many scholars are also increasingly concerned with the study of aspergillus oryzae gene expression.
MSN2 codes for a typical Cys 2 His 2 Zinc alloyThe gene of the protein is a main transcription factor for regulating and controlling external adversity stress response of fungal cells, has important effects in response regulation of hunger stress, salt stress, oxidation stress, high osmotic pressure stress, low temperature stress and the like of the fungal cells, and researches report that MSN2 genes are related to the number of conidia, colony growth and kojic acid yield in aspergillus nidulans and aspergillus flavus.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide an Aomsn2 over-expressed Aspergillus oryzae engineering bacterium, a construction method and application thereof, and aims to solve the problem of low kojic acid yield in the existing secondary metabolite of Aspergillus oryzae.
The technical scheme of the invention is as follows:
a construction method of Aomsn2 over-expression Aspergillus oryzae engineering bacteria comprises the following steps:
designing a corresponding Aomsn2 primer by taking an Aomsn2 gene in an aspergillus oryzae sample as a template, and carrying out PCR (polymerase chain reaction) amplification on the genome of the aspergillus oryzae sample by using the Aomsn2 primer to obtain an Aomsn2 cloned gene, wherein the nucleotide sequence of the Aomsn2 cloned gene is shown as SEQ ID NO.1, and the nucleotide sequence of the Aomsn2 primer is shown as SEQ ID NO. 2-3;
performing enzyme digestion and enzyme ligation treatment on the Aomsn2 cloned gene and an expression vector pEX2B to construct an Aomsn2 overexpression vector;
and (3) transforming the Aomsn2 over-expression vector into Aspergillus oryzae by adopting a method of mediating protoplast transformation, and constructing and obtaining the Aomsn2 over-expression Aspergillus oryzae engineering bacteria.
The construction method of the Aomsn2 over-expression Aspergillus oryzae engineering bacteria comprises the step of arranging a kana resistance marker and a uracil guanosine nutritional marker on an Aomsn2 over-expression vector.
The Aomsn2 over-expression Aspergillus oryzae engineering bacteria are prepared by adopting the construction method of the Aomsn2 over-expression Aspergillus oryzae engineering bacteria.
The Aomsn2 over-expression Aspergillus oryzae engineering bacteria prepared by the construction method are used for producing kojic acid.
The beneficial effects are that: according to the invention, the Aomsn2 over-expression Aspergillus oryzae engineering bacteria and the Aomsn2 knockout engineering strain are constructed, and comparison analysis shows that the Aomsn2 gene has a larger influence on the growth of Aspergillus oryzae and the synthesis of secondary metabolite kojic acid thereof, and the Aomsn2 gene can control and regulate transcription factors of the kojic acid gene cluster in Aspergillus oryzae to improve the yield of kojic acid, thereby laying a foundation for developing industrial brewing strains with high yield of kojic acid and providing a theoretical foundation for industrial application of Aspergillus oryzae and kojic acid production.
Drawings
FIG. 1 is a flow chart of a construction method of an Aomsn2 over-expressed Aspergillus oryzae engineering bacterium.
FIG. 2 is a schematic representation of the digestion electrophoresis of pPTRII-Cas 9, wherein lanes 1-2 are linearized pPTRII-Cas 9; lane 3 is a control pPTRII-Cas 9.
FIG. 3 is an electrophoretically contrasted view of Pu6-Aomsn2 fragment, pu6-Aomsn2-sgRNA-Tu6 fragment and Aomsn2-sgRNA-TU6 fragment, wherein lane 1 is Pu6-Aomsn2 fragment; lanes 2,3 are fusion fragment Pu6-Aomsn2-sgRNA-Tu6; lane 4 is the Aomsn2-sgRNA-TU6 fragment control.
FIG. 4 is a graph showing the results of electrophoresis verification of two fragments of Pu6-Aomsn2 and Aomsn2-sgRNA-Tu6, wherein lanes 1-4 are verified for 4 monoclonal Pu6-Aomsn 2; lanes 5-8 were verified for 4 monoclonal Aomsn2-sgRNA-Tu 6.
FIG. 5 is a cut gel electrophoresis of pEX 2B.
FIG. 6 is a graph showing the results of the verification of Aomsn2 overexpressing bacteria.
FIG. 7 is a graph showing the screening results of Aomsn2 over-expressed positive strains, wherein A is a protoplast transformation panel; b is a map of transformant cultures picked up in protoplast transformation plates.
FIG. 8 is a diagram showing the results of PCR verification of Aomsn2 overexpressing strain, wherein A is a diagram showing the electrophoresis verification, and lane 1 is a control of Aomsn2 overexpressing plasmid; lanes 2-6 are transformants picked by DNA digestion verification; b is an analysis result graph of the expression pattern of the Aomsn2 over-expression strain.
FIG. 9 is a graph showing the results of screening transformants of Aomsn2 knockout strains, wherein A is a protoplast transformation panel; b is a map of transformant cultures picked up in protoplast transformation plates.
FIG. 10 is a graph showing the results of Aomsn2 sequencing homozygosity.
FIG. 11 is a graph showing the results of analysis of the expression pattern of Aomsn2 knockout strains.
FIG. 12 is a graph showing growth conditions of Aomsn2 overexpressing strains, wherein A is a graph showing results of culturing WT and Aomsn2 overexpressing strains at 30℃for 3 days, and B is a strain growth diameter analysis; c: analyzing the spore number of the strain; d: strain biomass analysis, (. Times.p < 0.05; times.p < 0.01).
FIG. 13 is a graph showing growth of Aomsn2 knockout strain, wherein A is a graph showing results of culturing WT and Aomsn2 knockout strain at 30℃for 3 days, and B is a strain growth diameter analysis; c: analyzing the spore number of the strain; d: strain biomass analysis, (. Times.p < 0.05; times.p < 0.01).
FIG. 14 is a graph showing growth of Aomsn2 overexpressing strain under various treatment conditions, wherein A is a graph showing results of culturing WT and Aomsn2 overexpressing strain at 30℃for 3 days under various treatments; b is a graph of the analysis results of the growth diameters of the strains under different treatments, wherein P is less than 0.05, and P is less than 0.01.
FIG. 15 is a graph showing the change of kojic acid when the Aomsn2 gene is knocked out, wherein A is a state diagram of solid chromogenic medium of each strain cultured for 3 days at 30 ℃, and red indicates that the strain secretes kojic acid; b is fermentation color development liquid of each strain; c is a kojic acid yield analysis chart of each strain; d is an analysis chart of the expression pattern of the kojA gene of each strain; e is an analysis chart of the expression pattern of the kojR gene of each strain, and F is an analysis chart of the expression pattern of the kojT gene of each strain (P < 0.05; P < 0.01).
Detailed Description
The invention provides an Aomsn2 over-expression Aspergillus oryzae engineering bacterium, a construction method and application thereof, and further detailed description of the invention is provided below for the purpose, technical scheme and effect of the invention to be clearer and more definite. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Referring to fig. 1, fig. 1 is a flowchart of a method for constructing an Aomsn2 over-expressed aspergillus oryzae engineering bacterium, which includes the following steps:
s10, designing a corresponding Aomsn2 primer by taking an Aomsn2 gene in an Aspergillus oryzae sample as a template, and carrying out PCR (polymerase chain reaction) amplification on the genome of the Aspergillus oryzae sample by using the Aomsn2 primer to obtain an Aomsn2 cloned gene, wherein the nucleotide sequence of the Aomsn2 cloned gene is shown as SEQ ID NO.1, and the nucleotide sequence of the Aomsn2 primer is shown as SEQ ID NO. 2-3;
s20, carrying out enzyme digestion and enzyme linking treatment on the Aomsn2 cloned gene and an expression vector pEX2B to construct an Aomsn2 over-expression vector;
s30, converting the Aomsn2 over-expression vector into the Aspergillus oryzae by adopting a method of mediating protoplast conversion, and constructing and preparing the Aomsn2 over-expression Aspergillus oryzae engineering bacteria.
Specifically, the invention constructs the Aomsn2 over-expression Aspergillus oryzae engineering bacteria and the Aomsn2 knockout engineering strain, and finds that the Aomsn2 gene has larger influence on the growth of Aspergillus oryzae and the synthesis of secondary metabolite kojic acid thereof through comparative analysis, and the Aomsn2 gene can improve the yield of kojic acid in Aspergillus oryzae by controlling and regulating transcription factors of kojic acid gene clusters, thereby laying a foundation for developing industrial brewing strains for producing kojic acid in high yield and providing a theoretical foundation for industrial application of Aspergillus oryzae and production of kojic acid.
In some embodiments, an Aomsn2 over-expressed aspergillus oryzae engineering bacterium is also provided, which is prepared by adopting the construction method of the Aomsn2 over-expressed aspergillus oryzae engineering bacterium.
In some embodiments, an application of the Aomsn2 over-expressed Aspergillus oryzae engineering bacteria is also provided, wherein the Aomsn2 over-expressed Aspergillus oryzae engineering bacteria prepared by the construction method is used for producing kojic acid.
The invention is further illustrated by the following examples:
example 1
Construction of Aomsn2 knockout vector:
based on the CRISPR/Cas9 system, the CRISPR/Cas9 system comprises two components: one part is Cas9 endonuclease and the other part is single stranded guide RNA (sgRNA). Wherein the Cas9 endonuclease is an RNA-guided DNA lyase, the Cas9 endonuclease is controlled by the gpdA promoter, and the DNA that the Cas9 endonuclease can target to recognize is determined by a guide RNA sequence of 20bp in length; the sgrnas may function as guides, guiding Cas9 enzymes to recognize and cleave target sequences upstream of the protospacer motif (PAM). In this example, the Aomsn2 knockout vector uses ppthii as a parent vector, and the vector ppthii-Cas 9 is obtained after transformation of the vector, where the vector ppthii-Cas 9 is used for constructing the Aomsn2 knockout vector, contains a resistance selection marker of pyrithione, and the vector ppthii-Cas 9 is digested with smai, and whether the vector is cut or not is detected using agarose gel, and the result is shown in fig. 2.
Firstly, taking Aspergillus oryzae 3.042 strain genome DNA as a template, and amplifying a U6 promoter by using a primer containing a target gene sequence of Aomsn2 of 20bp to obtain a Pu6-Aomsn2 sequence, wherein the primer is shown in a table 1, and the corresponding nucleotide sequence is shown in SEQ ID No. 4-7.
TABLE 1 primer sequences
| PU6-Aomsn2-R | CACTCCAGGACTGGCGGGGTACTTGTTCTTCTTTACAATGATTTATTTA |
| TU6-Aomsn2-F | ACCCCGCCAGTCCTGGAGTGGTTTTAGAGCTAGAAATAGCAAGTTAAA |
| PU6-F | CGACTCTAGAGGATCCCCGGGTAATGCCGGCTCATTCAAA |
| TU6-R | AATTCGAGCTCGGTACCCGGGAGCAGCTCTATATCACGTGACG |
The U6 terminator sequence and the sgRNA sequence were synthesized by the company (i.e., sgRNA-Tu 6). The targeting sequence of Aomsn2 is amplified and connected to the sgRNA-Tu6 by using the sgRNA-Tu6 as a template to obtain the Aomsn2-sgRNA-Tu6 sequence. Two fragments of Pu6-Aomsn2 and Aomsn2-sgRNA-Tu6 were fused to obtain a fused fragment Pu6-Aomsn2-sgRNA-Tu6, as shown in FIG. 3. The fusion fragment Pu6-Aomsn2-sgRNA-Tu6 was recombinantly ligated to the linearized pPTRII-Cas 9 vector and transformed by E.coli host. And (3) selecting monoclonal shaking bacteria, performing bacterial liquid PCR verification, and respectively verifying two fragments Pu6-Aomsn2 and Aomsn2-sgRNA-Tu6 by bacterial liquid PCR to determine that two ends are fused together and successfully transformed, wherein the constructed recombinant plasmid is named as pPTRII-Cas 9-Aomsn2, and the result is shown in figure 4. And (3) converting the pPTRII-Cas 9-Aomsn2 plasmid into Aspergillus oryzae by a PEG (polyethylene glycol) -mediated protoplast conversion method, and constructing the Aomsn2 knockout engineering strain.
Example 2
Construction of Aomsn2 over-expression Aspergillus oryzae engineering bacteria:
the pEX2B vector is used for constructing an Aomsn2 gene over-expression vector, and comprises a kana resistance marker and a uracil guanosine nutritional marker. The vector pEX2B was digested with both the fast-cutting enzymes AflII and BamHI, the DsRed fragment was excised, and the vector was checked for cleavage using agarose gel, the results of which are shown in FIG. 5. The genome DNA of aspergillus oryzae 3.042 is used as a template, the Aomsn2 fragment is amplified through a designed Aomsn2 primer, the linearized pEX2B vector and the Aomsn2 fragment are cut into gel, recovered, recombined and connected, transformed into Trans10, and subjected to single-clone shaking bacteria selection, and bacterial liquid PCR verification, and the result is shown in figure 6. Sequencing the positive strain in a sequencing company, carrying out secondary plasmid extraction after sequencing is correct, and converting the plasmid (Aomsn 2 over-expression vector) into Aspergillus oryzae by adopting a PEG-mediated protoplast conversion method to construct the Aomsn2 over-expression Aspergillus oryzae engineering bacteria.
Example 3
After the Aomsn 2-overexpressing Aspergillus oryzae engineering bacteria constructed in example 2 were cultured for 3 to 4 days, transformants grown from the medium layers were picked up and transferred onto CD medium as shown in FIG. 7. After 2-3 days of culture, the outermost hyphae were picked with a medium-sized gun head, 50uLTE medium-boiled DNA was added as a template, PCR was performed with primers on the vector, and Aomsn2 was amplified with pEX2B-Aomsn2 plasmid as a template as a control, and agarose gel electrophoresis was used to examine whether the vector was transferred into A.oryzae, and the results are shown in FIG. 8. Two over-expressed strains were obtained, designated OE-Aomsn2-1 and OE-Aomsn2-2, respectively. And analyzing the expression level of Aomsn2 by using RT-PCR by using two overexpression strains and WT extract RNA, and the result shows that: the expression level of the Aomsn2 gene in two Aomsn2 over-expression strains is obviously higher than that in wild strain. Two over-expression strains are spread on KA culture medium to enlarge and culture the conidium, and glycerol is added for sterilization (bacterial liquid: 50% glycerol=1:1) and stored in a refrigerator at-80 ℃ for later use.
The Aomsn2 knockout engineering strain constructed in example 1 was cultured, and after the transformant grew, the transformant was transferred to a cd+pt medium containing 0.1ug/mL thiamine pyridine, as shown in fig. 9. After 2-3 days of culture, the outermost hyphae were picked with a medium-sized gun head, 50uLTE medium-boiling DNA was added as a template, the Aomsn2 fragment was amplified with primers containing the targeting sequence for verification, detection by agarose gel electrophoresis, and the correct target band was cut and sequenced. The sequencing results are shown in FIG. 10, wherein the exon of one knocked-out strain is inserted into one base A to cause the frame shift mutation of the following base, the sequence is named as delta Aomsn2-1, the exon of the other strain is deleted by 20bp to cause premature translation termination, the sequence is named as delta Aomsn2-2, and the sequencing results are all homozygous. In FIG. 11, the expression level of Aomsn2 gene in both ΔAomsn2-1 and ΔAomsn2-2 knockout strains was significantly down-regulated compared to the wild type. The ΔAomsn2-1 and ΔAomsn2-2 knockout strains are spread on KA medium for expansion culture of conidia, and glycerol is added for sterilization (bacterial liquid: 50% glycerol=1:1) and stored in a refrigerator at-80 ℃ for later use.
Example 4
Phenotype analysis is carried out on two Aomsn2 over-expression engineering bacteria and two Aomsn2 knockout strains:
preparing a CD solid culture medium, respectively coating spores of the Aomsn2 over-expression Aspergillus oryzae engineering bacteria and the Aomsn2 knockout engineering bacteria in the example 3 at the temperature of minus 80 ℃ on KA culture medium for activation, then using 0.9% NaCl solution for conidium, storing the collected spore liquid in a refrigerator at the temperature of 4 ℃ for 10-15 days, taking 3uL of the same amount of spore liquid for spotting, and culturing in an incubator at the temperature of 30 ℃ for 3 days. The medium was treated differently with 120ug/mL Congo red, 60uM K3, 1M NaCl, respectively. In the application process of aspergillus oryzae, the living environment becomes bad due to the living and metabolism, for example, oxidation damage is easy to occur under the oxidation condition, and congo red and vitamin K3 oxidizing agent are used for researching the function of Aomsn2 in the research, so that the oxidation condition is created. It is also very necessary to explore a physiological condition of aspergillus oryzae under NaCl salt stress by fermenting foods with aspergillus oryzae in industry.
1. Determination of diameter and spore count of each strain:
after the solid medium of CD inoculated with spore liquid was cultured in an incubator at 30℃for 3 days, the growth diameter thereof was measured (crisscross method), and data were recorded.
The spores were rinsed with 1500 μl of water (0.25% triton100 added), vigorously vortexed using a vortexing shaker to break up the sporangia thoroughly, and counted using a hemocytometer, if the concentration was too high, diluted appropriately.
2. Determination of the dry weight of each strain:
100uL of bacterial liquid is coated on a CD culture medium paved with cellophane, and is cultured for 3 days at 30 ℃. Scraping, drying and weighing.
3. Determination of growth diameter under different treatments:
the different strains were cultured in an incubator at 30℃for 3 days, and then their growth diameters were measured (crisscross method) to record data.
The test results are shown in fig. 12-13, and it can be seen from the results of fig. 12-13 that the over-expression of Aomsn2 gene reduces the growth diameter and biomass of the strain and increases the spore number; and knockout of the Aomsn2 gene can increase the growth diameter and biomass of the strain and reduce the number of spores. It was demonstrated that the Aomsn2 gene has an important role in the growth of Aspergillus oryzae.
4. The influence of Aomsn2 gene on Aspergillus oryzae growth under stress was investigated:
respectively using Congo red and microorganism K 3 And respectively treating the engineering strains by sodium chloride. The use process is the same as that described above, and the result is shown in fig. 14. The results show that the Aomsn2 gene knockout does not exist for the growth of Aspergillus oryzae stress environmentThe Aomsn2 gene is obviously influenced, and the over-expression of the Aomsn2 gene makes the Aspergillus oryzae more sensitive to external stress, which indicates that the Aomsn2 gene is a response factor for growth under the stress of the Aspergillus oryzae.
5. The influence of the Aomsn2 gene on kojic acid, a secondary metabolite of aspergillus oryzae, was explored and the content of kojic acid was determined using the method reported by Bentley:
mu.L of each engineering strain was plated on a solid medium of CD kojic acid and a 3mmol/L FeCl3 plate, and cultured at 30℃for 3 days, followed by observation.
100uL of each engineering strain was inoculated into 40mLCD kojic acid culture medium broth (the fermentation bottle was a 250mL conical flask), and fermented for 7 days at 30℃with shaking table 200 rpm.
2mL of the fermentation broth was centrifuged and the supernatant was taken as a chromogenic reagent (1% FeCl3, ddH2O in 10g FeCl) 3 ·6H 2 O, 22.5mL of concentrated hydrochloric acid was added to volume 1L) =1: 2, generally taking 500uL of supernatant, 1000uL of developing solution in a clean 2.0mL centrifuge tube, and adding 500uL of ddH2O. The absorbance at 500nm was measured. Wherein the control group was formulated with 1mL ddH2O and 1mL developer (1% FeCl 3). Substituting the obtained data into a kojic acid standard curve, and calculating the kojic acid yield of each strain. Knocking out the Aomsn2 gene was found to inhibit aspergillus oryzae kojic acid synthesis. WT and Aomsn2 knockout strains were fermented in liquid kojic acid CD medium for 3 days, aspergillus oryzae mycelia were collected, RNA was extracted, and RT-PCR analysis was performed, and the knockout of Aomsn2 inhibited the expression of kojic acid synthesis-related genes kojA and kojT, and had no effect on kojR, and the results are shown in FIG. 15.
It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.
Claims (3)
1. The construction method of the Aomsn2 over-expression Aspergillus oryzae engineering bacteria is characterized by comprising the following steps:
designing a corresponding Aomsn2 primer by taking an Aomsn2 gene in an aspergillus oryzae sample as a template, and carrying out PCR (polymerase chain reaction) amplification on the genome of the aspergillus oryzae sample by using the Aomsn2 primer to obtain an Aomsn2 cloned gene, wherein the nucleotide sequence of the Aomsn2 cloned gene is shown as SEQ ID NO.1, and the nucleotide sequence of the Aomsn2 primer is shown as SEQ ID NO. 2-3;
performing enzyme digestion and enzyme ligation treatment on the Aomsn2 cloned gene and an expression vector pEX2B to construct an Aomsn2 overexpression vector;
transforming the Aomsn2 over-expression vector into Aspergillus oryzae by adopting a method of mediating protoplast transformation, and constructing and preparing Aomsn2 over-expression Aspergillus oryzae engineering bacteria; the Aomsn2 over-expression vector is provided with a kana resistance marker and a uracil guanosine nutritional marker.
2. The Aomsn2 over-expression Aspergillus oryzae engineering bacterium is characterized in that the Aomsn2 over-expression Aspergillus oryzae engineering bacterium is prepared by a construction method of the Aomsn2 over-expression Aspergillus oryzae engineering bacterium.
3. The application of the Aomsn2 over-expression Aspergillus oryzae engineering bacteria is characterized in that the Aomsn2 over-expression Aspergillus oryzae engineering bacteria prepared by the construction method of claim 1 are used for producing kojic acid.
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