CN116478836A - Aspergillus niger engineering strain for liquid spore production and construction method and application thereof - Google Patents

Abstract

The application discloses a liquid spore-producing aspergillus niger engineering strain and a construction method and application thereof, and belongs to the technical field of bioengineering. The liquid spore-producing Aspergillus niger engineering strain comprises the steps of taking original Aspergillus niger as a template, and replacing an original promoter of a brlA gene in the original Aspergillus niger template with a xylose-inducible promoter Pxylp in situ, wherein the gene ID of the brlA gene is ASPNIDRAFT2 _{_} 1171592; gene ID of xylose-inducible promoter Pxylp as PCH _{_} Pc20g07020. The Aspergillus niger engineering strain can utilize xylose-induced promoter Pxylp to control C responsible for asexual spore production of Aspergillus niger ₂ H ₂ The zinc finger transcription factor BrlA is expressed, so that the aim of forcing the aspergillus niger to generate a large amount of asexual conidia in a liquid medium by taking xylose as an induction signal is fulfilled, and the biomass of the asexual conidia can be rapidly improved when the asexual conidia are used for fermentation production of the aspergillus niger.

Description

Aspergillus niger engineering strain for liquid spore production and construction method and application thereof

Technical Field

The application belongs to the technical field of bioengineering, and particularly relates to a liquid spore-forming Aspergillus niger engineering strain, and a construction method and application thereof.

Background

The industrial production strains of the citric acid are generally aspergillus niger and saccharomycetes, wherein the aspergillus niger has strong spore regeneration capability, can ensure sufficient effective bacteria in fermentation, and can utilize cheap renewable carbon sources such as starch and the like as raw materials, thereby becoming one of the best microorganisms for industrial production of the citric acid.

In the related art, aspergillus niger fermentation production generally needs to be inoculated with a large amount of asexual conidia so as to quickly increase biomass, thereby constructing a cell platform for subsequent enzyme production and acid production.

However, the aspergillus niger serving as an ascomycete can only perform asexual spore production in a solid medium and oxygen exposure environment, and needs to spend a great deal of time and effort to prepare a culture medium, inoculate, collect spores and the like, so that the fermentation production of the aspergillus niger has large workload, time and labor consumption and high cost, and is not beneficial to industrial production.

Disclosure of Invention

The invention aims to provide an Aspergillus niger engineering strain for producing spores in liquid state, and a construction method and application thereof, and aims to solve the technical problems that the existing Aspergillus niger strain can only produce spores in solid state, and fermentation production workload caused by solid state spore production is large, time and labor are consumed and cost is high.

In order to achieve the above purpose, the technical scheme of the application is as follows:

the first aspect of the application provides a liquid spore-forming Aspergillus niger engineering strain, which comprises the steps of taking original Aspergillus niger as a template, and enabling an original promoter of a brlA gene in the original Aspergillus niger to be replaced by a xylose-inducible promoter Pxylp in situ;

wherein, the gene ID of the brlA gene is ASPNIDRAFT21171592;

the gene ID of the xylose-inducible promoter Pxylp is PCH _{_} Pc20g07020。

In a second aspect, the present application provides a method for constructing a liquid spore-forming aspergillus niger engineering strain according to the first aspect, the method comprising:

s1: synthesizing an aspergillus niger uracil auxotroph strain;

s2: introducing a pFC330 plasmid into the aspergillus niger uracil auxotroph strain to synthesize a constitutive aspergillus niger background strain expressing Cas 9;

s3: designing an sgRNA targeting point for a gene brlA promoter region of the Aspergillus niger background strain, and designing an upstream primer T7-brlA-F and a downstream primer sgRNA-R for the sgRNA targeting point sequence; amplifying and synthesizing a double-stranded DNA template of the sgRNA from a pX330 plasmid by using PCR high-fidelity enzyme, the upstream primer T7-brlA-F and the downstream primer sgRNA-R, and then transcribing the double-stranded DNA template into corresponding sgRNA by using an RNA in-vitro transcription kit, wherein the nucleotide sequence of the sgRNA targeting point is 5'-CCGTTGCGCCTTGCCACATTCC-3'; the T7-brlA-F contains a T7 promoter and a specific sgRNA targeting sequence, and the nucleotide sequence of the T7-brlA-F is shown as SEQ ID NO. 7; the nucleotide sequence of the sgRNA-R is shown in SEQ ID NO. 8;

s4: cloning the hph gene fragment to a product site of a vector pEasy-Bluntzero to synthesize a vector P-0-hph, and cloning a Pxylp promoter sequence to a Not1 site of the vector P-0-hph to synthesize P-0-hph-Pxylp; transforming the P-0-hph-Pxylp into escherichia coli DH5 alpha competent cells, inoculating the competent cells into an LB (LB) culture dish containing ampicillin for overnight culture, and then picking up a monoclonal and verifying by colony PCR (polymerase chain reaction) to obtain a brlA control expression plasmid P-0-hph-Pxylp; amplifying a repair template hph-Pxylp containing a micro-homology arm by using the plasmid P-0-hph-Pxylp as a template through PCR, wherein the gene ID of the hph gene is CP059254.1;

s5: co-transferring the Aspergillus niger background strain into the sgRNA and the hph-Pxylp, culturing for 4 days in a transformation culture medium without xylose, selecting white transformant hypha without generating conidium, transferring the white transformant hypha into a PDA culture medium, and continuously culturing for 4 days, wherein diagnosis PCR proves that the hph-Pxylp is correctly integrated in a promoter region of brlA;

s6: inoculating the correctly integrated white transformant mycelium into a PDA solid medium containing uridine, uracil and 5-FOA, incubating for 7 days to obtain a uracil-deficient spore-producing modified strain without pFC330 plasmid, and integrating the uracil-deficient spore-producing modified strain back to the pyrG gene fragment to obtain the liquid spore-producing Aspergillus niger engineering strain.

In an alternative implementation manner of the second aspect, the method for synthesizing the aspergillus niger uracil auxotroph strain includes:

s11: transferring pFC332 plasmid containing Cas9 gene into original Aspergillus niger strain, and obtaining Cas9 carrying transformant through hph screening;

s12: after pyrG knockout sgRNA is designed by taking the Cas9 carrying transformant as a starting strain and synthesized in vitro, the pyrG gene in the Cas9 carrying transformant is knocked out, and then the pyrG knockout transformant is obtained through 5-FOA screening, wherein the gene ID of the pyrG gene is XM _{_} 001395395.2。

S13: culturing the pyrG knockout transformant in a PDA culture medium without hph to obtain a strain with lost pFC332, namely the aspergillus niger uracil auxotroph strain.

In an alternative implementation of the second aspect, the aspergillus niger uracil auxotroph strain is introduced into the pFC330 plasmid using a PEG-mediated transformation method of aspergillus protoplasts;

wherein, the PEG-mediated Aspergillus protoplast transformation method comprises the following steps:

enzymatic hydrolysis of Aspergillus niger strains into protoplasts;

blending the protoplast and the pFC330 plasmid in a PEG solution, and inducing the pFC330 plasmid to enter the protoplast through PEG;

protoplasts transformed into pFC330 plasmid were mixed with medium and the transformants were grown.

In an alternative implementation of the second aspect, the temperature for the enzymolysis of the aspergillus niger strain into protoplasts is 28 ℃, the rotation speed is 80rpm, and the enzymolysis time is 4 hours.

In an alternative implementation of the second aspect, the Pxylp promoter sequence is amplified from the penicillium genome by the primer Zero-Pxylp-F/Zero-Pxylp-R;

wherein the nucleotide sequence of the Zero-Pxylp-F is shown in SEQ ID NO. 9;

the nucleotide sequence of the Zero-Pxylp-R is shown as SEQ ID NO. 10.

In an alternative implementation of the second aspect, the hph gene fragment is amplified from vector pAN7-1 by primer Pro-hph-F/Not 1-hph-R;

wherein the nucleotide sequence of Pro-hph-F is shown as SEQ ID NO. 11;

the nucleotide sequence of Not1-hph-R is shown as SEQ ID NO. 12.

In an alternative implementation manner of the second aspect, the preparation method of the transformation medium includes:

the preparation method of the transformation medium comprises the following steps:

mixing 10g glucose, 218.6g D-sorbitol, 1mL trace element and 50mL 20 Xsalt solution, adding water to 1L, adjusting pH to be=6.5, and sterilizing at 115 ℃ for 30min to obtain the final product;

wherein the salt solution comprises the following components: naNO ₃ 120g/L、KCl10.4g/L、MgSO ₄ ·7H ₂ O10.4g/L and KH ₂ PO ₄ 30.4g/L; the microelements comprise the following components: znSO (ZnSO) ₄ ·7H ₂ O2.20g/100mL、H ₃ BO ₃ 1.10g/100mL、MnCl ₂ ·4H ₂ O0.50g/100mL、FeSO ₄ ·7H ₂ O0.16g/100mL、CoCl ₂ ·5H ₂ O0.16g/100mL、CuSO ₄ ·5H ₂ O0.16g/100mL、(NH4) ₆ Mo ₇ O ₂₄ ·4H ₂ O0.11g/100mL and Na ₄ EDTA5.00g/100mL。

The third aspect of the application provides application of the liquid spore-forming Aspergillus niger engineering strain or the Aspergillus niger engineering strain constructed and synthesized by the method in the second aspect in asexual spore-forming and fermentation in a xylose liquid medium.

In an alternative implementation of the third aspect, the composition of the xylose liquid medium is, in units of 100 mL: 1 gD+xylose, 0.1mL trace elements, 5mL20 Xsalt solution;

the salt solution comprises the following components: naNO ₃ 120g/L、KCl10.4g/L、MgSO ₄ ·7H ₂ O10.4g/L and KH ₂ PO ₄ 30.4g/L; the microelements comprise the following components: znSO (ZnSO) ₄ ·7H ₂ O2.20g/100mL、H ₃ BO ₃ 1.10g/100mL、MnCl ₂ ·4H ₂ O0.50g/100mL、FeSO ₄ ·7H ₂ O0.16g/100mL、CoCl ₂ ·5H ₂ O0.16g/100mL、CuSO ₄ ·5H ₂ O0.16g/100mL、(NH4) ₆ Mo ₇ O ₂₄ ·4H ₂ O0.11g/100mL and Na ₄ EDTA5.00g/100mL。

Compared with the prior art, the advantage or beneficial effect of this application includes at least:

according to the liquid spore-producing Aspergillus niger engineering strain provided by the first aspect of the application, the original promoter for regulating and controlling the brlA coding gene of the asexual spore-producing transcription factor of Aspergillus niger is replaced by the xylose-induced promoter Pxylp in situ by taking the original Aspergillus niger as a template, so that the Aspergillus niger engineering strain can utilize the xylose-induced promoter Pxylp to control C responsible for asexual spore production of Aspergillus niger ₂ H ₂ The zinc finger transcription factor BrlA is expressed under the condition, so that the aim of forcing Aspergillus niger to generate asexual conidium in a large quantity in a liquid medium by taking xylose as an induction signal is achieved. When the Aspergillus niger engineering strain is used for Aspergillus niger fermentation production, the workload of preparing culture medium, inoculating, spore collecting and the like can be effectively simplified and saved, the time and the labor are saved, the cost is low, and the application of Aspergillus niger in industrial fermentation production is facilitated.

The example shows that after 20h of liquid PDA shake flask culture, the Aspergillus niger engineering strain is transferred into xylose liquid culture mediumShake flask cultivation is carried out for 5 days to produce 8×10 in culture medium suspension ⁶ The process of obtaining the asexual spores of the aspergillus niger is simpler and faster and the cost is greatly saved.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the description of the embodiments will be briefly described below. It is obvious that the drawings in the following description are only some embodiments described in the present application, and that other drawings may be obtained from these drawings without inventive effort for a person of ordinary skill in the art.

FIG. 1 is a graph showing the growth status of Aspergillus niger uracil-deficient strain ΔpyrG and original Aspergillus niger strain WG120 in PDA and PDA+UU media, respectively, provided in the examples herein;

FIG. 2 is a phenotype diagram of a BrlA promoter-substituted transformant provided in the examples of the present application, wherein FIG. 2A is a phenotype diagram of a BrlA promoter-substituted transformant T; FIG. 2B shows transformant T with substitution of the BrlA promoter with original strain C _n＝1-10 Is a phenotype map of (2);

FIG. 3 shows a transformant T with BrlA promoter substitution according to the examples of the present application _n Is a PCR verification graph of (2);

FIG. 4 shows an Aspergillus niger engineering strain brlA according to an embodiment of the present application ^Pxylp Phenotype map with original strain WG 120;

FIG. 5 shows an original Aspergillus niger strain WG 120/Aspergillus niger engineered strain brlA provided in the examples of the present application ^Pxylp Spore production under glucose/xylose liquid culture.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. It will be apparent that the described embodiments are some, but not all, of the embodiments of the present application. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.

In the following description of the present embodiment, the term "and/or" is used to describe an association relationship of association objects, which means that three relationships may exist, for example, a and/or B may mean: a alone, B alone and both a and B. Wherein A, B may be singular or plural. The character "/" generally indicates that the context-dependent object is an "or" relationship.

In the following description of the present embodiments, the term "at least one" means one or more, and "a plurality" means two or more. "at least one of" or the like means any combination of these items, including any combination of single item(s) or plural items(s). For example, "at least one (individual) of a, b, or c," or "at least one (individual) of a, b, and c" may each represent: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, c may be single or multiple, respectively.

It should be understood by those skilled in the art that, in the following description of the embodiments of the present application, the sequence number does not mean that the sequence of execution is not sequential, and some or all of the steps may be executed in parallel or sequentially, and the execution sequence of each process should be determined by its functions and inherent logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

The terminology used in the embodiments of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application in the examples and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The terms "commercialized vector pFC330 plasmid", "commercialized vector pEasy-Bluntzero plasmid", "RNA in vitro transcription kit" and the like in the following examples are all commercially available, and the RNA in vitro transcription kit is preferably MEGAscript T7 kit. The specific model and source of the reagents and equipment are not particularly limited in this application; meanwhile, the related gene editing techniques in the construction method are all implemented according to methods known in the art, and the embodiments of the present application are not specifically described.

Example 1

The embodiment provides an Aspergillus niger engineering strain brlA for producing spores in liquid state ^Pxylp Specifically, the construction method comprises the following steps S1-S6.

S1-Synthesis of Aspergillus niger uracil auxotroph Strain ΔpyrG, specifically including S11-S13.

S11: transferring a commercialized vector pFC332 plasmid containing a Cas9 gene into an original Aspergillus niger strain WG120, and screening to obtain a transformant WG120 carried by the Cas9 by taking hph (hygromycin resistance screening gene) as a screening marker ^pFC332 . To confirm that the commercialized vector pFC332 plasmid was introduced, after designing primer Hei-CAS9-yz-F/pFC-CAS9-R for pFC332 plasmid based on the characteristic that pFC332 plasmid is episomal plasmid, cas9 carrying transformant WG120 was extracted ^pFC332 Performing PCR verification by taking genome of (2) as a template;

wherein the nucleotide sequence of the Hei-cas9-yz-F is shown as SEQ ID NO. 1; the nucleotide sequence of the pfc-cas9-R is shown in SEQ ID NO. 2.

S12: cas9 synthesized in step S11 carries transformant WG120 ^pFC332 After pyrG is designed for knocking out sgRNA and synthesized in vitro for the starting strain, the strain is knocked out by a PEG-mediated Aspergillus protoplast transformation method to carry transformant WG120 of Cas9 ^pFC332 The pyrG gene in the strain is screened by taking 5-FOA as a screening marker to obtain pyrG knockout transformant WG120 ^{pFC332 ΔpyrG} . To confirm that the pyrG gene was knocked out, pyrG knockdown transformant WG120 was extracted after designing primer pyrG-yz-F/pyrG-An-cas9-R for pyrG knockdown position ^pFC332ΔpyrG Performing PCR verification by taking genome as a template;

wherein the nucleotide sequence of pyrG-yz-F is shown as SEQ ID NO. 3; the nucleotide sequence of pyrG-An-cas9-R is shown as SEQ ID NO. 4.

S13: based on the property of the commercial vector pFC332 plasmid that is easily lost, the pyrG knockout transformant WG120 synthesized in step S12 is ^pFC332ΔpyrG Streaking in PDA culture medium without hph (hygromycin resistance screening gene) to obtain strain with pFC332 plasmid loss, namely the Aspergillus niger uracil auxotroph strainΔpyrG；

The preparation method of the PDA culture medium comprises the following steps: cutting 200g of potato into pieces, adding 1000mL of water, boiling, heating, preserving heat for 30min, filtering with double-layer gauze to obtain clear liquid, adding 20g of glucose, dissolving completely, and adding water to constant volume of 1L.

To verify the pyrG gene knockout of the A.niger uracil auxotroph strain ΔpyrG, the example inoculates the A.niger uracil auxotroph strain ΔpyrG and the original A.niger strain WG120 into PDA and PDA+UU media, respectively, and cultures overnight at 37℃and observed the strain growth, the results are shown in FIG. 1. In which FIG. 1 shows a comparison of the growth status of Aspergillus niger uracil-deficient strain ΔpyrG and of the original Aspergillus niger strain WG120 on PDA and PDA+UU media, respectively.

As can be seen from FIG. 1, the original A.niger strain WG120 grew well, whereas uracil auxotroph strain ΔpyrG was unable to grow normally in normal PDA medium without UU, indicating that the pyrG gene of uracil auxotroph strain ΔpyrG was confirmed to be knocked out.

S2-Synthesis of A.niger background Strain WG120 constitutively expressing Cas9 ^pFC330 The method specifically comprises the following steps:

the commercial vector pFC330 plasmid contains screening marker genes of original Ptef-Cas9-TtrPC and pyrG for constitutive expression of Cas9 enzyme, so that 1 mu m commercial vector pFC330 plasmid is transferred into protoplast obtained by enzymolysis of Aspergillus niger uracil auxotroph strain delta pyrG synthesized in step S1 by using PEG-mediated Aspergillus protoplast transformation method, thus synthesizing Aspergillus niger background strain WG120 for constitutive expression of Cas9 ^pFC330 . To confirm that the pFC330 plasmid was introduced, primers Cas9-pyrG1-F/Cas9-wn-R were designed for the pFC330 plasmid and WG120 was extracted ^pFC330 Performing PCR verification by taking genome as a template;

wherein the Cas9-pyrG1-F sequence is shown in SEQ ID NO. 5; the Cas9-wn-R sequence is shown as SEQ ID NO. 6.

The specific method for transferring 1 μm plasmid into protoplast digested by Aspergillus niger uracil auxotroph strain ΔpyrG synthesized in step S1 by PEG-mediated transformation method of Aspergillus protoplast comprises S21-S23:

s21: and (3) carrying out enzymolysis on the Aspergillus niger uracil auxotroph strain delta pyrG synthesized in the step (S1) into protoplasts by using conventional enzyme solutions such as snailase, lyase and the like, wherein the enzymolysis conditions are as follows: and (3) performing enzymolysis for 4 hours at 28 ℃ at 80 rpm.

S22: the protoplast obtained in S21 and the commercialized vector pFC330 plasmid were blended in PEG solution, and the pFC330 plasmid was induced into the protoplast by PEG.

S23: mixing the protoplast obtained in the step S22 and transferred into the pFC330 plasmid with a culture medium, and obtaining the Aspergillus niger background strain WG120 which constitutively expresses Cas9 after the transformant grows ^pFC330 。

S3-the sgRNA required for in vitro synthesis of CRISPR/Cas9 system comprises S31-S33:

s31: is Aspergillus niger background strain WG120 ^pFC330 Designing a sgRNA targeting point for a gene brlA promoter region, and designing an upstream primer T7-brlA-F containing a T7 promoter and a specific sgRNA targeting sequence and a nonspecific downstream primer sgRNA-R for the sgRNA targeting point sequence;

wherein, the sgRNA targeting sequence is 5'-CCGTTGCGCCTTGCCACATTCC-3'; the sequence of T7-brlA-F is shown in SEQ ID NO. 7; the sgRNA-R sequence is shown as SEQ ID NO. 8.

S32: double-stranded DNA templates of the sgRNA are amplified from a commercialized vector pX330 plasmid by using PCR high-fidelity enzyme, an upstream primer T7-brlA-F and a downstream primer sgRNA-R, and transcribed into corresponding sgRNA by using a MEGAscript T7 kit (LifeTechnologies, cat.no.AM1333) and stored at the temperature of minus 80 ℃ for standby.

The repair template required by S4-PCR synthesis CRISPR/Cas9 system specifically comprises S41-S43:

s41: amplifying Pxylp promoter sequences of xylanase coding genes from the genome of penicillium (Penicilliumrubens Wisconsin-1255) by using primers Zero-Pxylp-F/Zero-Pxylp-R respectively, and amplifying hph (hygromycin resistance screening gene) fragments from a commercial vector pAN7-1 by using primers Pro-hph-F/Not 1-hph-R;

wherein the Zero-Pxylp-F sequence is shown in SEQ ID NO. 9; the Zero-Pxylp-R sequence is shown as SEQ ID NO. 10; the Pro-hph-F sequence is shown as SEQ ID NO. 11; the Not1-hph-R sequence is shown in SEQ ID NO. 12.

S42: cloning the hph gene fragment to the product site of the vector pEasy-Bluntzero to synthesize vector P-0-hph, and cloning the Pxylp promoter sequence to the Not1 site of the vector P-0-hph to synthesize vector P-0-hph-Pxylp, wherein the gene ID of the hph gene is CP059254.1.

S43: after the vector P-0-hph-Pxylp is transformed into E.coli DH5 alpha competent cells, the cells are inoculated into LB culture dishes containing 100 mug/mL ampicillin, cultured overnight at 37 ℃, and monoclonal is selected and verified by colony PCR to obtain the brlA control expression plasmid P-0-hph-Pxylp.

S44: the plasmid P-0-hph-Pxylp is used as a template, and primers BrlA-hph-F/BrlA-xylp-R are used for amplifying the hph and Pxylp into a repair template hph-Pxylp containing micro homology arms (homologous to brlA sequences at two sides of an sgRNA targeting region) through PCR.

Wherein the sequence of BrlA-hph-F is shown in SEQ ID NO. 13; the BrlA-xylp-R sequence is shown as SEQ ID NO. 14;

the synthesis of S5-brlA conditional expression strain specifically comprises S51-S53:

s51: WG120 of constitutive expression Cas9 enzyme synthesized in step S2 ^pFC330 As a background strain, 4. Mu.g of sgRNA synthesized in step S3 and 5. Mu.g of hph-Pxylp synthesized in step S4 were co-transformed by a protoplast transformation method to obtain a BrlA promoter-substituted transformant T, and inoculated into a transformation medium for 4 days, and the growth of the BrlA promoter-substituted transformant T was observed, as a result of which it was shown in FIG. 2A. Among them, FIG. 2A shows a phenotype diagram of transformant T with replacement of the BrlA promoter.

As can be seen from FIG. 2A, the promoter replacement transformation experiment of Aspergillus niger succeeded in growing multiple transformants.

The preparation method of the transformation medium comprises the following steps:

mixing 10g glucose, 218.6g D-sorbitol, 1mL trace element and 50mL 20 Xsalt solution, adding water to 1L, adjusting pH to be=6.5, and sterilizing at 115 ℃ for 30min to obtain the final product;

wherein the salt solution comprises the following components: naNO ₃ 120g/L、KCl10.4g/L、MgSO ₄ ·7H ₂ O10.4g/L and KH ₂ PO ₄ 30.4g/L; the microelements comprise the following components: znSO (ZnSO) ₄ ·7H ₂ O2.20g/100mL、H ₃ BO ₃ 1.10g/100mL、MnCl ₂ ·4H ₂ O0.50g/100mL、FeSO ₄ ·7H ₂ O0.16g/100mL、CoCl ₂ ·5H ₂ O0.16g/100mL、CuSO ₄ ·5H ₂ O0.16g/100mL、(NH ₄ ) ₆ Mo ₇ O ₂₄ 4H2O0.11g/100mL and Na ₄ EDTA5.00g/100mL。

S52: white transformants which did not give rise to conidia on the transformation plates (BrlA promoter-replaced transformant T were picked _n＝1-10 ) And an original strain C (WG 120), after continuous cultivation in PDA medium for 4 days, white transformants were observed (BrlA promoter-replaced transformant T _n＝1-10 ) The results are shown in FIG. 2B, compared with the growth of the original strain C (WG 120). Wherein FIG. 2B shows transformant T with original strain C replaced with BrlA promoter _n＝1-10 Is a phenotype of (a).

As can be seen from FIG. 2B, original strain C grew normally in the normal PDA medium, whereas the transformant Tn with the BrlA promoter was unable to produce spores in the normal solid medium, indicating that the BrlA promoter was successful in replacing the original promoter.

The correct integration of hph-Pxylp in the promoter region of brlA was confirmed by diagnostic PCR (primer BrlA-yz-F/BrlA-yz-R) and the results are shown in FIG. 3. Among them, FIG. 3 shows a PCR verification diagram of the BrlA promoter-substituted transformant Tn.

Wherein the BrlA-yz-F sequence is shown as SEQ ID NO. 15; the sequence of BrlA-yz-R is shown as SEQ ID NO. 16;

as can be seen from FIG. 3, under the same pair of verification primers, the gene band of the BrlA promoter-replaced transformant Tn was longer than that of the original strain C, indicating that the BrlA promoter was successful in replacing the original promoter.

The preparation method of the PDA culture medium comprises the following steps:

cutting 200g of potato into pieces, adding 1000mL of water, boiling, heating, preserving heat for 30min, filtering with double-layer gauze to obtain clear liquid, adding 20g of glucose, dissolving completely, and adding water to constant volume of 1L.

The complementation of the S6-pyrG gene fragment specifically includes S61-S62:

s61: inoculating correctly integrated white transformant hyphae into PDA solid medium containing 5mM uridine, 10mM uracil and 1mM5-FOA, and incubating for 7 days to obtain uracil-deficient spore-forming modified strain without pFC330 plasmid;

s62: amplifying a gene pyrG fragment from the genome of an original Aspergillus niger strain WG120 by using a primer pyrG-F/pyrG-R, and supplementing the gene pyrG fragment back into a uracil-deficient spore-producing modified strain which does not contain a pFC330 plasmid to obtain a brlA conditional expression strain brlA ^Pxylp Namely the Aspergillus niger engineering strain brlA for producing the spore in liquid state ^Pxylp 。

Meanwhile, in order to understand the growth condition, the embodiment uses the engineering strain brlA ^Pxylp Inoculating with original strain WG120 into common PDA culture medium, culturing, and observing engineering strain brlA ^Pxylp The results are shown in FIG. 4, which shows the growth of the original strain WG120 in PDA medium. Wherein FIG. 4 shows the engineering strain brlA ^Pxylp Phenotype map with original strain WG 120.

According to fig. 4, the engineering strain brlazylp has the same growth vigor as the original strain WG120 except for spore production, which indicates that the original promoter can be replaced by the xylose-induced promoter Pxylp in situ to change the spore production performance of the strain without affecting the normal growth of the strain.

Wherein the nucleotide sequence of pyrG-F is shown as SEQ ID NO. 17; the nucleotide sequence of pyrG-R is shown as SEQ ID NO. 18;

the preparation method of the PDA solid medium comprises the following steps:

adding 15g agar into PDA culture medium, and sterilizing at 115deg.C for 30 min;

the gene pyrG sequence comprises its own promoter and terminator, is 2332bp in length, and has a gene ID of XM_001395395.2.

Example 2

The embodiment provides a liquid-state spore-producing Aspergillus niger engineering strain brlA ^Pxylp Asexual production in xylose liquid mediumThe application of spore and fermentation specifically comprises:

s201: the Aspergillus niger engineering strain brlA synthesized in example 1 was introduced ^Pxylp Spore liquid (1×10) ⁸ And then inoculated into 50mL of PDA liquid medium, and cultured for 20h at the temperature of 28 ℃ and the rotating speed of 200rpm, so that the spores germinate and grow hyphae.

S202: discarding the PDA liquid culture medium in the step S201, inoculating mycelium into the xylose liquid culture medium, and culturing for 5 days at 28 ℃ and a rotating speed of 200rpm to obtain obvious spore liquid;

s203: microscopic observation shows that the Aspergillus niger engineering strain brlA ^Pxylp After the sporulation yield in the xylose liquid culture medium, the production of the black conidium with the yield can be observed in the xylose liquid culture medium after shaking bottle culture for 2 days; after 5 days of culture, the culture broth was filtered through nylon cloth and centrifuged to obtain spores in the filtrate.

The composition of the xylose liquid medium (100 mL) in this example was: 1 gD+xylose, 0.1mL trace elements, 5mL20 Xsalt solution;

wherein the salt solution comprises the following components: naNO ₃ 120g/L、KCl10.4g/L、MgSO ₄ ·7H ₂ O10.4g/L and KH ₂ PO ₄ 30.4g/L; the microelements comprise the following components: znSO (ZnSO) ₄ ·7H ₂ O2.20g/100mL、H ₃ BO ₃ 1.10g/100mL、MnCl ₂ ·4H ₂ O0.50g/100mL、FeSO ₄ ·7H ₂ O0.16g/100mL、CoCl ₂ ·5H ₂ O0.16g/100mL、CuSO ₄ ·5H ₂ O0.16g/100mL、(NH ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O0.11g/100mL and Na ₄ EDTA5.00g/100mL；

The preparation method of the PDA culture medium in the embodiment comprises the following steps: cutting 200g of potato into pieces, adding 1000mL of water, boiling, heating, preserving heat for 30min, filtering with double-layer gauze to obtain clear liquid, adding 20g of glucose, dissolving completely, and adding water to constant volume of 1L.

The preparation method of the PDA solid state medium in the embodiment comprises the following steps: adding 15g of agar into the PDA culture medium, and sterilizing at 115 ℃ for 30 min.

To quantify the Aspergillus niger engineered strain brlA ^Pxylp In this example, 1X 108 spores obtained in example 2 were inoculated again into 50mL of liquid PDA medium, and after 20 hours, 5mL of mycelia were inoculated into xylose liquid medium, and the liquid spores were induced by culturing in the same manner as in example 2, resulting in 8X 106/mL of conidia.

In addition, in order to verify that the xylose liquid medium of the present example was against the Aspergillus niger engineering strain brlA ^Pxylp Influence of spore production, comparative examples were provided to replace xylose liquid medium in example 2 with glucose liquid medium, i.e. xylose in the medium components with glucose, and the remaining operations and parameters were kept unchanged, and original Aspergillus niger strain WG120 and Aspergillus niger engineering strain brlA were observed ^Pxylp The results of the spore production are shown in FIG. 5. Wherein FIG. 5 shows the original Aspergillus niger strain WG120 and the Aspergillus niger engineered strain brlA ^Pxylp Spore production under glucose/xylose liquid culture.

As can be seen from FIG. 5, the Aspergillus niger engineering strain brlA was cultivated in a glucose liquid medium ^Pxylp No conidiophoresis with the original aspergillus niger strain WG 120; however, when cultured in xylose liquid medium, the A.niger engineered strain brlA ^Pxylp A large amount of conidia were produced, but the original Aspergillus niger strain WG120 did not sporulate, indicating that the Aspergillus niger engineering strain brlA ^Pxylp Can start the asexual propagation process in xylose liquid culture medium.

Various embodiments in this specification are described in an incremental manner, and identical or similar parts of the various embodiments are referred to each other, with each embodiment focusing on differences from the other embodiments.

The above embodiments are only for illustrating the technical solution of the present application, and not for limiting the present application; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced with equivalents; such modifications and substitutions do not depart from the spirit of the corresponding technical solutions.

Claims (10)

1. The liquid-state spore-producing Aspergillus niger engineering strain is characterized by comprising the step of taking original Aspergillus niger as a template to enable an original promoter of a brlA gene in the original Aspergillus niger to be replaced by a xylose-induced promoter Pxylp in situ;

wherein the brlA gene has a gene ID of ASPNIDRAFT2 _{_} 1171592；

The gene ID of the xylose-inducible promoter Pxylp is PCH _{_} Pc20g07020。

2. The construction method of the liquid spore-forming aspergillus niger engineering strain is characterized by comprising the following steps:

s1: synthesizing an aspergillus niger uracil auxotroph strain;

s2: introducing a pFC330 plasmid into the aspergillus niger uracil auxotroph strain to synthesize a constitutive aspergillus niger background strain expressing Cas 9;

s3: designing an sgRNA targeting point for a gene brlA promoter region of the Aspergillus niger background strain, and designing an upstream primer T7-brlA-F and a downstream primer sgRNA-R for the sgRNA targeting point sequence; amplifying and synthesizing a double-stranded DNA template of the sgRNA from a pX330 plasmid by using PCR high-fidelity enzyme, the upstream primer T7-brlA-F and the downstream primer sgRNA-R, and then transcribing the double-stranded DNA template into corresponding sgRNA by using an RNA in-vitro transcription kit, wherein the nucleotide sequence of the sgRNA targeting point is 5'-CCGTTGCGCCTTGCCACATTCC-3'; the T7-brlA-F contains a T7 promoter and a specific sgRNA targeting sequence, and the nucleotide sequence of the T7-brlA-F is shown as SEQ ID NO. 7; the nucleotide sequence of the sgRNA-R is shown in SEQ ID NO. 8;

s4: cloning the hph gene fragment to a product site of a vector pEasy-Bluntzero to synthesize a vector P-0-hph, and cloning a Pxylp promoter sequence to a Not1 site of the vector P-0-hph to synthesize P-0-hph-Pxylp; transforming the P-0-hph-Pxylp into escherichia coli DH5 alpha competent cells, inoculating the competent cells into an LB (LB) culture dish containing ampicillin for overnight culture, and then picking up a monoclonal and verifying by colony PCR (polymerase chain reaction) to obtain a brlA control expression plasmid P-0-hph-Pxylp; amplifying a repair template hph-Pxylp containing a micro-homology arm by using the plasmid P-0-hph-Pxylp as a template through PCR, wherein the gene ID of the hph gene is CP059254.1;

s5: co-transferring the Aspergillus niger background strain into the sgRNA and the hph-Pxylp, culturing for 4 days in a transformation culture medium without xylose, selecting white transformant hypha without generating conidium, transferring the white transformant hypha into a PDA culture medium, and continuously culturing for 4 days, wherein diagnosis PCR proves that the hph-Pxylp is correctly integrated in a promoter region of brlA;

s6: inoculating the correctly integrated white transformant mycelium into a PDA solid medium containing uridine, uracil and 5-FOA, incubating for 7 days to obtain a uracil-deficient spore-producing modified strain without pFC330 plasmid, and integrating the uracil-deficient spore-producing modified strain back to the pyrG gene fragment to obtain the liquid spore-producing Aspergillus niger engineering strain.

3. The construction method according to claim 2, wherein the method for synthesizing the strain auxotrophic for aspergillus niger comprises:

s11: transferring pFC332 plasmid containing Cas9 gene into original Aspergillus niger strain, and obtaining Cas9 carrying transformant through hph screening;

s12: after pyrG knockout sgRNA is designed by taking the Cas9 carrying transformant as a starting strain and synthesized in vitro, the pyrG gene in the Cas9 carrying transformant is knocked out, and then the pyrG knockout transformant is obtained through 5-FOA screening, wherein the gene ID of the pyrG gene is XM _{_} 001395395.2。

S13: culturing the pyrG knockout transformant in a PDA culture medium without hph to obtain a strain with lost pFC332, namely the aspergillus niger uracil auxotroph strain.

4. The method of claim 3, wherein the strain auxotrophic for aspergillus niger is introduced with a pFC330 plasmid using a PEG-mediated transformation of aspergillus protoplasts;

wherein, the PEG-mediated Aspergillus protoplast transformation method comprises the following steps:

enzymatic hydrolysis of Aspergillus niger strains into protoplasts;

blending the protoplast and the pFC330 plasmid in a PEG solution, and inducing the pFC330 plasmid to enter the protoplast through PEG;

protoplasts transformed into pFC330 plasmid were mixed with medium and transformants were grown.

5. The method according to claim 4, wherein the temperature for the enzymolysis of the Aspergillus niger strain into protoplasts is 28 ℃, the rotation speed is 80rpm, and the enzymolysis time is 4 hours.

6. The construction method according to claim 2, wherein the Pxylp promoter sequence is amplified from the genome of penicillium by the primer Zero-Pxylp-F/Zero-Pxylp-R;

wherein the nucleotide sequence of the Zero-Pxylp-F is shown in SEQ ID NO. 9;

the nucleotide sequence of the Zero-Pxylp-R is shown as SEQ ID NO. 10.

7. The construction method according to claim 2, wherein the hph gene fragment is amplified from the vector pAN7-1 by the primer Pro-hph-F/Not 1-hph-R;

wherein the nucleotide sequence of Pro-hph-F is shown as SEQ ID NO. 11;

the nucleotide sequence of Not1-hph-R is shown as SEQ ID NO. 12.

8. The method of constructing of claim 7, wherein the method of preparing the transformation medium comprises:

mixing 10g glucose, 218.6g D-sorbitol, 1mL trace element and 50mL 20 Xsalt solution, adding water to 1L, adjusting pH to be=6.5, and sterilizing at 115 ℃ for 30min to obtain the final product;

wherein the salt solution comprises the following components: naNO ₃ 120g/L、KCl 10.4g/L、MgSO ₄ ·7H ₂ O10.4g/L and KH ₂ PO ₄ 30.4g/L; the microelements comprise the following components: znSO (ZnSO) ₄ ·7H ₂ O 2.20g/100mL、H ₃ BO ₃ 1.10g/100mL、MnCl ₂ ·4H ₂ O 0.50g/100mL、FeSO ₄ ·7H ₂ O 0.16g/100mL、CoCl ₂ ·5H ₂ O0.16g/100mL、CuSO ₄ ·5H ₂ O 0.16g/100mL、(NH ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O0.11g/100mL and Na ₄ EDTA5.00g/100mL。

9. Use of an engineered strain of aspergillus niger according to claim 1 for liquid sporulation or constructed and synthesized according to the method of any of claims 2-8 for asexual sporulation and fermentation in a xylose liquid medium.

10. The use according to claim 9, characterized in that the composition of the xylose liquid medium, in units of 100mL, is: 1g D + xylose, 0.1mL trace element, 5mL20 Xsalt solution;

wherein the salt solution comprises the following components: naNO ₃ 120g/L、KCl 10.4g/L、MgSO ₄ ·7H ₂ O10.4g/L and KH ₂ PO ₄ 30.4g/L; the microelements comprise the following components: znSO (ZnSO) ₄ ·7H ₂ O 2.20g/100mL、H ₃ BO ₃ 1.10g/100mL、MnCl ₂ ·4H ₂ O 0.50g/100mL、FeSO ₄ ·7H ₂ O 0.16g/100mL、CoCl ₂ ·5H ₂ O0.16g/100mL、CuSO ₄ ·5H ₂ O 0.16g/100mL、(NH ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O0.11g/100mL and Na ₄ EDTA5.00g/100mL。