Application of transcription activator and gene thereof in promoting cellulase production and growth of filamentous fungi

Abstract

The invention relates to a transcriptional activator and application of a gene of the transcriptional activator in improvement of cellulase produced by filamentous fungi and growth of the filamentous fungi, and belongs to the technical field of genes. In order to solve the problems that an existing filamentous fungus strain is still low in cellulase yield, high in cost and the like, the invention provides a transcriptional activator of cellulase and hemicellulase, and the nucleotide sequence is shown as SEQ ID NO.1; 1, and the amino acid sequence is as shown in SEQ ID NO. 2. The invention provides application of the transcriptional activator in improvement of growth of talaromyces filamentous fungi and production capacity of the talaromyces filamentous fungi on cellulase and hemicellulase after gene overexpression of the transcriptional activator.

Description

TECHNICAL FIELD

[0001] The present application relates to a transcriptional activator and its gene in improving the production of cellulase and growth of filamentous fungi, belonging to the field of genetic technology. BACKGROUND

[0002] Filamentous fungi are a kind of microorganisms with high economic value, which are widely used in fermentation industry, food, agriculture and environmental protection fields. Among filamentous fungi, transcriptional activators can enhance the synthesis ability of fungal cellulase by up-regulating the expression of genes related to cellulase production. By engineering these transcriptional activators, the production of cellulase and hemicellulase can be improved to meet the needs of industrial production. Although many studies have explored the improvement of enzyme production capacity of filamentous fungi through genetic engineering, existing technologies still face some challenges. For example, some transcription factors have limited effect and cannot significantly improve enzyme production; or the transcription factor may have a negative impact on the growth rate of fungi, resulting in a decrease in overall metabolic capacity. Therefore, the development of new transcriptional activators and their applications is of great significance for further improving the production of cellulase. SUMMARY

[0003] The purpose of the present application is to improve the production capacity of filamentous fungi for cellulase and hemicellulase, and to improve the growth rate and yield of filamentous fungi.

[0004] The present application provides a transcriptional activation gene of cellulase and hemicellulase, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0005] The present application provides a transcriptional activation factor of cellulase and hemicellulase, the amino acid sequence of which is shown in SEQ ID NO. 2.

[0006] The present application provides the use of the gene of the above-mentioned transcriptional activation factor in improving the growth of filamentous fungi and the production capacity of cellulase and hemicellulase.

[0007] Further, the method of the above-mentioned use is to overexpress the gene of the transcriptional activation factor in filamentous fungi.

[0008] Further, the filamentous fungi are Talaromyces endophyticus, and the strain preservation number is CCTCC NO: M 2022024.

[0009] Beneficial effects:

[0010] This invention provides transcription activator genes for cellulase and hemicellulase. Transforming a vector carrying these transcription activator genes into *Talaromyces endophyticus* CCTCC NO: M2022024 resulted in accelerated growth, elongated hyphae, and increased yield in the overexpression strain. Using microcrystalline cellulose as a carbon source, after 6 days of fermentation, the overexpression strain exhibited increased levels of endoglucanase (22.31 U / mL), exoglucanase (1.03 U / mL), β-glucosidase (3.22 U / mL), and xylanase (43.29 U / mL). Attached Figure Description

[0011] Figure 1 The results are PCR results for transcription activators; where M is the marker and Te18280 is the target gene.

[0012] Figure 2 PCR validation of the overexpression vector; where M is the marker; 1-3 are the overexpression vectors; 4 is the negative control; 5 is the blank control;

[0013] Figure 3 PCR validation of the overexpressing strains; where M is the marker; 1-6 are the overexpressing strains; 7 is the negative control; 8 is the blank control;

[0014] Figure 4 Colony size and hydrolysis zone of wild-type and overexpression strains; WT is the wild-type strain; OE-Te18280 is the overexpression strain;

[0015] Figure 5 Hyphae morphology of wild-type strain and overexpression strain; where A is the hyphae morphology of wild-type strain under 10x and 40x microscopes; B is the hyphae morphology of overexpression strain under 10x and 40x microscopes.

[0016] Figure 6 Dry weight analysis of wild-type and overexpression strains; where WT is the wild-type strain and OE-Te18280 is the overexpression strain.

[0017] Figure 7 Cellulase activities of wild-type strains and overexpression strains; where A is FPase and B is CMCase;

[0018] C is pNPCase; D is β-glucosidase; E is xylanase;

[0019] Figure 8Expression levels of transcription activator gene Te18280 and cellulase gene in the strain; where A is the expression level of Te18280 in the strain; and B is the expression level of cellulase gene in the strain.

[0020] Figure 9 Binding analysis of transcription activators to cellulase gene promoter regions; where A is the result of incubation of eg5B promoter DNA segment; B is the result of incubation of eg7A promoter DNA segment; C is the result of incubation of eg12A promoter DNA segment; D is the result of incubation of cbh6A promoter DNA segment; and E is the result of incubation of bgl3A promoter DNA segment. Detailed Implementation

[0021] The technical solution of the present invention will be further described in conjunction with the embodiments, but the scope of protection of the present invention is not limited thereto.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0023] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0024] The *Talaromyces endophyticus* used in the following examples was obtained from the China Center for Type Culture Collection (CCTCC), with accession number NO: M 2022024.

[0025] The pBARGPE1 vector (Amp and hygromycin resistance) and pET-32b vector (Amp resistance) used in the following examples were purchased from Wuhan Miaoling Biotechnology Co., Ltd.

[0026] The E. coli clone competent cells DH5α Chemically Competent Cell and E. coli expression competent cells Rosetta(DE3) Chemically Competent Cell in the following examples are products of Shanghai Weidi Biotechnology Co., Ltd.

[0027] PDA medium: 20g glucose, 200g clean potatoes cut into 1cm cubes, boiled to obtain filtrate, 15g agar, ddH2O to a final volume of 1L, dispensed into Erlenmeyer flasks, autoclaved at 121℃ for 20min. PDA liquid medium can be obtained by omitting agar from the PDA medium.

[0028] LB medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 12g agar, pH 7.2, add ddH2O to a final volume of 1L, then dispense into Erlenmeyer flasks and autoclave at 121℃ for 20min. LB liquid medium can be obtained by omitting agar from LB solid medium.

[0029] 50× Vogel's Inorganic Salt Medium: Ammonium nitrate 100g, potassium dihydrogen phosphate 250g, sodium citrate 125g, calcium chloride dihydrate 5g, magnesium sulfate heptahydrate 10g, 5ml Vog / Lel's Trace elements (zinc sulfate heptahydrate 50g, citric acid 50g, manganese sulfate monohydrate 0.5g, copper sulfate pentahydrate 2.5g, ferrous ammonium sulfate 10g, boric acid 0.5g, sodium aluminide dihydrate 0.5g)

[0030] Protoplast transformation upper medium: sorbitol 182g, glucose 20g, 50× Vogel's inorganic salt 20mL, 10g agarose 10g, add ddH2O to 1L mark, dispense into Erlenmeyer flasks, autoclave at 115℃ for 30min.

[0031] Protoplast transformation lower layer medium: 182g sorbitol, 20mL 50×Vogel's inorganic salt, 10g agarose, add ddH2O to the 1L mark, dispense into Erlenmeyer flasks, and autoclave at 115℃ for 30min.

[0032] CMC solid medium: 10g sodium carboxymethyl cellulose, 1.0g dipotassium hydrogen sulfate, 2.0g ammonium sulfate, 0.5g sodium chloride, 0.5g magnesium sulfate, 10g agar, ddH2O to a final volume of 1L, dispensed into Erlenmeyer flasks, and incubated at 121℃ for 20min under high temperature and high pressure. Spore germination solid medium: Based on CMC solid medium, add 1% wheat straw before use.

[0033] Mandels-Andreotti (MA) medium: Ammonium sulfate 1.4g, dipotassium hydrogen sulfate 2.0g, disodium hydrogen phosphate dodecahydrate 17.907g, magnesium sulfate 0.60g, calcium chloride 0.60g, urea 0.3g, Tween-80 0.5mL, peptone 2.0g, add 1mL of 1000× micronutrient solution (ferrous sulfate heptahydrate 5.0g, manganese sulfate monohydrate 1.6g, zinc sulfate heptahydrate 1.4g, cobalt chloride dihydrate 2.0g, ddH2O to bring the volume to 100mL, then filter sterilize using a sterile membrane). After bringing the volume to 1L with ddH2O, dispense into Erlenmeyer flasks and autoclave at 121℃ for 20min.

[0034] Liquid enzyme-producing medium: Add 1% (w / v) microcrystalline cellulose to MA liquid medium, and incubate at 121°C for 20 min under high temperature and high pressure.

[0035] Example 1: Construction of strains overexpressing transcription activator genes

[0036] (1) Cloning of the target gene

[0037] Using *T. endophyticus* CCTCC NO:M 2022024 cDNA as a template, PCR amplification was performed using the transcription factor gene primers Te18280-F and Te18280-R and a high-fidelity enzyme PrimeSTAR Max DNA Polymerase (TaKaRa) kit to obtain the PCR amplification products. The primers are shown below:

[0038] Te18280-F: 5'-GAAACTAAGCAATGCCGAAT-3';

[0039] Te18280-R: 5'-GGATATTGCTTCGGAGTG-3'.

[0040] PCR amplification system (50 μl): 2×PrimeSTARMaxPremix 25 μL, 10 μM Forward Primer 1 μL, 10 μM Reserve Primer 1 μL, cDNA template 1 μL, ddH2O 22 μL, Total volume 50 μL. PCR amplification conditions: 98℃ for 10 s, 57℃ for 5 s, 72℃ for 10 s, 35 cycles, terminated at 4℃. The PCR products were separated into target bands by electrophoresis using 1% agarose gel.

[0041] like Figure 1 As shown, the target band was obtained with the predicted size. The PCR amplification product was 1572 bp with a molecular weight of 58.5 kDa. Its nucleotide sequence is shown in SEQ ID NO.1, and its protein amino acid sequence is shown in SEQ ID NO.2.

[0042] (2) Construction of overexpression vector

[0043] A *T. endophyticus* NEAU-6 overexpression strain was constructed using homologous recombination. Before constructing the overexpression vector, the resistance selection marker gene needed to be determined. The overexpression vector contained hygromycin B. The strain was sent to a sequencing facility, and using the correct PCR product as a template, PCR amplification was performed using the PrimeSTARMax DNA Polymerase (TaKaRa) kit with primers pBARGPE1-Hygr-Te18280-BamHI-F and pBARGPE1-Hygr-Te18280-BamHI-R to amplify the Te18280 gene carrying the homologous arm of plasmid pBARGPE1. The primers are shown in the figure below: pBARGPE1-Hygr-Te18280-BamHI-F:

[0044] 5'-gtcgacctcgactctagaATGTACCCATACGATGTTCCAGATTACGCTggatccATGCCGAATATATC TGTC-3'

[0045] pBARGPE1-Hygr-Te18280-BamHI-R:

[0046] 5'-tcgcccttgctcaccatggatccGAGATTAGATGTAAGCCCCGGATATTGCTTCGGAGT-3'

[0047] The pBARGPE1 vector was digested with BamHI as the restriction site, and the Te18280 gene was inserted via homologous recombination. The resulting cells were transformed into *E. coli* DH5α competent cells. The transformation products were plated on LB agar plates containing Amp antibiotic. The plates were incubated overnight at 37°C, and positive clones were selected. PCR verification of the recombinant vector was performed as follows. Figure 2 As shown.

[0048] (3) Screening for overexpression strains

[0049] The strain was cultured in PDA medium, then inoculated into sorghum grains and cultured until sporulation. A spore suspension was prepared using physiological saline. A layer of sterile cellophane was placed on spore germination medium, and approximately 100 μL of the spore suspension was added, spread evenly, and incubated at 30°C for approximately 16 h. 1% (w / v) lysozyme (Sigma) and 1% snailase (Lambolid) were added to 30 mL of S1 solution and dissolved thoroughly. The solution was filtered to obtain a lysis buffer. The bacteria on the cellophane were lysed using the lysis buffer at 30°C for 6-7 h. The protoplast suspension was filtered into sterile centrifuge tubes. The tubes were centrifuged at 3000 × g, 4°C for 20 min, and the supernatant was discarded. The protoplasts were resuspended after washing once with S2 solution.

[0050] Add 3 μg of linearized recombinant vector and 25 μL of T1 solution to 100 μL of protoplasts, and incubate on ice for 20 min. Add 1 mL of T1 solution and incubate at room temperature for 5 min. Add 2 mL of S2 solution and mix well. Add the above transformation system to the upper selection medium (containing 70 μg / mL hygromycin), mix well, pour into a selection plate containing the lower medium, and incubate at 30°C in the dark for 5–10 days.

[0051] Conidia were picked and cultured on single-spore medium at 30°C for 5 days. The pure single-spore culture was then inoculated into 30 mL of liquid PDA medium (containing 70 μg / mL hygromycin) and cultured at 30°C with shaking at 200 rpm for 2 days. Recombinant genomic DNA was extracted using the Fungal DNA Extraction Kit (OMEGA) and verified by PCR. Figure 3 As shown. Te18280 overexpression strain was obtained.

[0052] Example 2: Observation of colony size and hydrolysis zone of overexpression strain

[0053] Single colonies were selected from the recombinant strain and transferred to PDA solid medium for culture. After spore formation, fresh physiological saline was prepared and collected by filtration through a sterile filter membrane, resulting in a spore concentration of 1 × 10⁻⁶ per ml. 6 Spore suspensions were collected. 2 μL of fresh spore suspension was pipetted onto PDA, isolation, and CMC solid media sequentially. After incubation at 30°C for 3 days, samples were taken to examine the morphological characteristics of all media. On CMC solid media, colonies underwent Congo red staining followed by NaCl destaining. The diameter and hydrolysis zone of the recombinant colonies were measured. Compared with the wild type, the overexpression strain showed larger colony diameters and larger hydrolysis zones, indicating that the transcription activator Te18280 promoted strain growth and increased cellulase activity. The results are as follows: Figure 4 As shown

[0054] Example 3: Observation of hyphal branching status of overexpression strain

[0055] A sterile coverslip is inserted obliquely into a plate containing PDA medium. A suspension of bacterial spores is inoculated at the interface between the coverslip and the medium. The plate is incubated at 30°C for 3 days. The coverslip is then removed and observed under a microscope. Figure 5 As shown, the hyphae of the overexpressing strain were longer, more branched, and denser than those of the wild-type strain, indicating that the transcription activator Te18280 could promote the growth of the strain, and the strain grew faster after overexpression.

[0056] Example 4: Biomass Observation of Overexpression Strains

[0057] After 6 days of cultivation, the fermentation broth was centrifuged at 10,000 × g for 5 min. The resulting solid was washed with deionized water and dried at 40 °C to constant weight, and recorded as M1. M1 (mainly composed of cellulose and stem cells) was suspended in 3 mL of acetic acid-nitric acid reagent (80% acetic acid to nitric acid ratio of 10:1) and boiled in a water bath for 30 min. After cooling and centrifugation, the resulting solid was washed with distilled water until neutral, and then dried at 40 °C (mainly composed of cellulose) to constant weight, and recorded as M2. The mycelial dry weight was calculated based on the difference between M1 and M2. Figure 6 As shown, the overexpression strains have higher biomass and heavier hyphae.

[0058] Example 5: Ability of overexpression strains to produce cellulase and hemicellulase

[0059] Mycelial suspensions were prepared by shaking culture in 30 mL of LPDA liquid medium at 30 °C and 200 rpm for 3 days. 2 mL of the mycelial suspension was inoculated into an enzyme-producing medium containing microcrystalline cellulose and cultured at 30 °C and 150 rpm for 1–7 days. The medium was then centrifuged at 8000 rpm for 10 min at 4 °C, and the supernatant was used to determine cellulase activity. Figure 7 As shown in AE.

[0060] Assay for filter paper enzyme (FPase) activity: Cut Whatman NO.1 filter paper into 1×6cm (approximately 50mg) pieces, add 1.5mL of citrate buffer, incubate at 50℃ for 5min, add 0.5mL of diluted enzyme solution, and add inactivated enzyme solution to the control group. Incubate at 50℃ for 60min, add 1.5mL of DNS chromogenic reagent, and stop the reaction by boiling in a water bath for 5min. Measure the absorbance at 540nm.

[0061] Assay for endoglucanase activity: Take 1.5 mL of 1% (w / v) CMC-Na solution, add 0.5 mL of diluted enzyme solution, and add inactivated enzyme solution to the control group. Incubate at 50℃ for 30 min, then add 1.5 mL of DNS chromogenic reagent. Terminate the reaction by boiling in a water bath for 5 min, and measure the absorbance at 540 nm.

[0062] Determination of exoglucanase activity: Take 50 μL of 1 mg / mL pNPC solution and add 100 μL of diluted enzyme solution. The control group is treated with inactivated enzyme solution. Incubate the reaction at 50℃ for 30 min, then add 300 μL of 10% Na2CO3 solution to terminate the reaction. Measure the absorbance at 420 nm.

[0063] Determination of β-glucosidase activity: Take 1.5 mL of 0.5% (w / v) salicin solution, add 0.5 mL of diluted enzyme solution, add inactivated enzyme solution to the control group, incubate at 50℃ for 30 min, add 1.5 mL of DNS colorimetric reagent, stop the reaction by boiling water bath for 5 min, and measure the absorbance value at 540 nm wavelength.

[0064] Determination of xylanase activity: Take 1.5 mL of 1% (w / v) xylan solution, add 0.5 mL of diluted enzyme solution, add inactivated enzyme solution to the control group, incubate at 50℃ for 30 min, add 1.5 mL of DNS colorimetric reagent, stop the reaction by boiling water bath for 5 min, and measure the absorbance value at 540 nm wavelength.

[0065] The enzyme activity values ​​above are defined as the amount of enzyme required to hydrolyze the substrate to produce 1 μmol of reducing sugar or p-nitrophenol per minute under the above conditions, which is defined as 1 U.

[0066] Using microcrystalline cellulose as a carbon source, the wild-type strain Talaromyces endophyticus showed the following levels of enzymes after 6 days of fermentation: endoglucanase (17.76 U / mL), exoglucanase (0.66 U / mL), β-glucosidase (2.21 U / mL), and xylanase (34.08 U / mL).

[0067] Using microcrystalline cellulose as a carbon source, the overexpressing strain showed the following levels of enzymes after 6 days of fermentation: endoglucanase (22.31 U / mL), exoglucanase (1.03 U / mL), β-glucosidase (3.22 U / mL), and xylanase (43.29 U / mL).

[0068] Example 6: Expression levels of transcription activator gene and cellulase gene in overexpression strains

[0069] RNA was extracted from the overexpressing strain and wild-type strain using the Fungal RNA Kit (OMEGA, USA), reverse transcription was performed using the PrimeScript RT Reagent Kit with gDNAeraser (TaKaRa, Japan), and qPCR was performed using the Taq SYBR Green qPCR Premix (Lambolid).

[0070] PCR amplification system (20 μl): Template 1 μL, Forward Primer 0.4 μL, Reverse Primer 0.4 μL, 2×Taq SYBR Green qPCR Premix 10 μL, ddH2O 8.2 μL, Total volume 20 μL. The amplification reaction was performed according to the following program: 95℃ for 30 s; 95℃ for 10 s, 60℃ for 30 s, for a total of 40 cycles. Figure 8 As shown in Figure A, the expression level of the transcription factor Te18280 gene in the overexpressing strain was significantly increased, and it could effectively promote the expression of multiple cellulase genes eg5B, eg7A, eg12A, cbh6A, and bgl3A. Figure 8 B).

[0071] Example 7: Binding analysis of transcription repressor to cellulase gene promoter

[0072] EMSA analysis was performed using a chemiluminescence EMSA kit (Beyotime). The promoters of the cellulase genes eg5B, eg7A, eg12A, cbh6A, and bgl3A in *Bacillus basilica* were labeled with biotinylate probes before EMSA analysis. Figure 9 As shown in Figure AE, the diffusion band represents the recombinant protein His-Te18280. DBD The binding complexes with the promoter regions of various enzyme genes show a clearly visible free band of unbound DNA at the bottom. As the amount of recombinant protein loaded increases, the diffuse band becomes more abundant, while the free band becomes fainter, indicating increasingly stronger binding. As a negative control, TrxA-His-S protein does not bind to the target fluorescent probe DNA, showing no diffuse band. As a competitive control, as the amount of non-fluorescent DNA probe loaded increases, the diffuse band gradually weakens, while the free band becomes clearer. These results indicate that the recombinant protein His-Te18280... DBD It can directly and specifically bind to the promoter regions of the genes it regulates, thereby promoting the transcriptional expression of enzyme genes eg5B, eg7A, eg12A, cbh6A, and bgl3A. Therefore, overexpression of transcription activators can effectively enhance the transcriptional expression of cellulase and hemicellulase genes.

Claims

1. A transcription activator gene for cellulase and hemicellulase, wherein the nucleotide sequence encoding the transcription activator is shown in SEQ ID NO.

1.

2. A transcription activator of cellulase and hemicellulase, wherein the amino acid sequence of the transcription activator is shown in SEQ ID NO.

2.

3. The application of gene overexpression of the transcription activator of claim 1 in improving the production capacity of cellulase and hemicellulase in filamentous fungi.

4. The application of gene overexpression of the transcription activator of claim 1 in improving the growth of filamentous fungi.

5. The application according to claims 3 and 4, characterized in that, The method used was to overexpress genes of transcription activators in filamentous fungi.

6. The application according to claim 5, characterized in that, The filamentous fungus mentioned is *Talaromyces endophyticus*.

7. The application according to claim 6, characterized in that, The strain preservation number of the *Talaromyces endophyticus* is CCTCC NO:M 2022024.

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