CN114107359B - Method for improving cellulase expression capability of trichoderma reesei by regulating cell metabolism - Google Patents

Abstract

The invention relates to the field of genetic engineering, in particular to a method for improving the cellulase expression capability of trichoderma reesei by regulating cell metabolism. The present invention relates to a pentose phosphate pathway 6-phosphogluconate dehydrogenase gene of Trichoderma reesei72685And performing overexpression, and improving the protein expression quantity and the cellulase enzyme activity of the trichoderma reesei by constructing an overexpression plasmid of the gene and transforming the trichoderma reesei with the overexpression plasmid.

Description

A method for improving the ability of Trichoderma reesei to express cellulase by regulating cell metabolism

technical field

The invention relates to the field of genetic engineering, in particular to a method for improving the ability of Trichoderma reesei to express cellulase by regulating cell metabolism.

Background technique

As an important industrial production strain, the filamentous fungus Trichoderma reesei is widely used in the production of industrial products such as feed enzymes, organic acids, antibiotics, etc. It has the characteristics of high expression and strong secretion ability, and at the same time it has a variety of post-translational processing methods similar to eukaryotes, such as glycosylation, protease cleavage and disulfide bond formation and other modifications. In the mixed fermentation broth, the expression of cellobiohydrolase accounted for more than 50% of the total extracellular secreted proteins. However, the high cost of cellulase is still one of the main bottlenecks in cellulose biorefinery, so it is necessary to continue to develop new methods to improve the expression of cellulase and reduce the cost of application.

In T. reesei, cellulase expression is affected by multiple regulatory pathways. Among them, the main regulation occurs at the transcriptional level, including major activators, repressors, etc. Amino acid biosynthesis is a complex metabolic process that requires energy for both oxidative refolding of disulfide bonds and protein transport through secretory mechanisms. In Trichoderma reesei, protein production places a heavy metabolic burden on cells, potentially leading to limited supply of amino acids and redox energy species. An often overlooked stage that controls cellulase expression is cellular metabolism. In filamentous fungi, especially strains of Trichoderma reesei, there are few reports of increasing cellulase expression by altering the cell's own metabolic pathway.

Glucose-6-phosphate dehydrogenase in the pentose phosphate pathway or combined overexpression of different genes in the pathway have been reported in Pichia pastoris. The results showed that overexpression of glucose-6-phosphate dehydrogenase alone had no effect on the expression of superoxide dismutase, and the combination of glucose-6-phosphate dehydrogenase and 5-phosphate-ribulose genes or the combination of glucose-6-phosphate dehydrogenase Both catalase and 5-phosphate-ribulose gene overexpression inhibited the production of superoxide dismutase.

Therefore, the existing genetic engineering methods and the selection of regulatory genes have limitations, so it is crucial to continue to explore new regulatory genes.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for improving the ability of Trichoderma reesei to express cellulase by regulating cell metabolism.

The method for improving the ability of Trichoderma reesei to express cellulase by regulating cell metabolism according to the present invention comprises the step of overexpressing the Trichoderma reesei regulatory gene 72685 in Trichoderma reesei, wherein the Trichoderma reesei regulatory gene 72685 is a 6-phosphogluconate dehydrogenase gene of the pentose phosphate pathway , and its nucleotide sequence is shown in SEQ ID NO: 1.

The method for improving the ability of Trichoderma reesei to express cellulase by regulating cell metabolism according to the present invention includes the step of constructing an overexpression vector comprising the Trichoderma reesei regulatory gene 72685 .

According to the method for improving the ability of Trichoderma reesei to express cellulase by regulating cell metabolism of the present invention, the cDNA1 promoter, the 72685 gene, the cDNA1 terminator and the pyr4 expression cassette are spliced in series in a seamless splicing manner to obtain an overexpression plasmid.

In the present invention, the 6-phosphogluconate dehydrogenase gene 72685 of the pentose phosphate pathway of Trichoderma reesei is overexpressed, and the overexpression plasmid of the gene is constructed and used to transform Trichoderma reesei, thereby improving the efficiency of Trichoderma reesei. The protein expression and cellulase activity of mold.

Description of drawings

Fig. 1 is the 72685 overexpression plasmid map that improves cellulase expression;

Figure 2 shows the comparison of extracellular proteins of overexpressed 72685 strain and SUS4-2 strain;

Figure 3 shows the comparison of the cellulase activity of the overexpressed 72685 strain and the SUS4-2 strain;

Figure 4 shows the transcript abundance of the overexpressed 72685 strain-related cellulase self-genes.

Detailed ways

Example 1. Construction of overexpression regulatory gene 72685 plasmid

Construction of overexpression 72685 plasmid: including cDNA1 promoter, 72685 gene, cDNA1 terminator and pyr4 expression cassette plasmid. Taking the APA-GOD plasmid as a template, amplify the APA part ( AmpR-pyr4-AmpR ), and the APA sequence ( AmpR-pyr4-AmpR ) contains the expression cassette of the Trichoderma reesei auxotrophic selection marker pyr4 , which can be used with Transformation screening of Trichoderma reesei strains lacking the pyr4 gene. There are two tandemly repeated ampicillin genes at both ends of the pyr4 expression cassette. Homologous recombination of this direct tandem sequence can be used to knock out the pyr4 gene in the transformants of Trichoderma reesei more quickly, so that the pyr4 selection marker can be recycle. Using the Trichoderma reesei genome as a template, the CDNA1 promoter, 72685 gene (named CDNA) and CDNA1 terminator were amplified, and the four fragments recovered by electrophoresis were connected by homologous recombination. Transform E. coli Trans1-T1 competent cells, perform colony PCR on the colonies grown on the plate, and send the colonies identified as positive by PCR for sequencing, and the correctly sequenced plasmid is named pCDNA1p-72685 plasmid, as shown in Figure 1 .

Example 2. Introduction of Overexpression 72685 Plasmid

(1) Overexpression 72685 plasmid was transformed into Trichoderma reesei SUS4-2

Trichoderma reesei SUS4-2 was inoculated on a potato culture medium (PDA) plate, and cultured at 28 ºC for 7 days until the spores were produced. The spores were scraped and inoculated into 100 ml of PDB medium containing uracil at 28 ºC. , 160 rpm shaking culture overnight. The germinated mycelia were collected by filtration through a 200-mesh sieve, and 10 mg/ml of cellulase was added to digest at 30ºC for 2-3 hours. After the protoplasts were collected, the pCDNAP-72685 plasmid was digested with EcoR I and transformed into T. reesei host cells.

(2) PCR verification of the introduction of the pCDNAP-72685 plasmid into the Trichoderma reesei genome

Pick a single transformant, inoculate it in a 24-well plate containing MM-glucose medium, and culture at 28°C for 5-7 days. Genomic DNA was extracted to verify the introduction of the overexpression 72685 plasmid into the Trichoderma reesei genome. The transformants that could amplify PCR products with expected band size were inoculated on PDA medium to produce spores.

Example 3. The effect of overexpression of 72685 on the secretion and expression of Trichoderma reesei cellulase

(1) Shake flask induction of transformants overexpressing the 72685 gene

The transformants overexpressing the 72685 gene and the starting strain were respectively inoculated with 2×10 ⁷ spores in 50 ml MM-glucose medium, and cultured at 28°C and 160 rpm for 2 days. 10% of the inoculum was transferred to 50 ml MM+2% Avicel medium to induce cellulase expression. From the 3rd day, samples were taken every 24 h, and the samples were continuously sampled for 7 days.

(2) Determination of protein concentration and cellulase of transformants overexpressing 72685 gene

Coomassie brilliant blue method was used for protein quantification. After adding 250 L of 1 × dye reagent and 10 µl of protein standard, and reacting at room temperature for 10 minutes, the absorbance at 595 nm was measured. The results are shown in Figure 2. It can be seen that the transformants expressing the 72685 gene have a higher protein concentration than the original strain during the whole culture stage.

The determination of cellulase was carried out using sodium carboxymethylcellulose (1.5 % CMC-Na) as the substrate. Prepared in citric acid-disodium hydrogen phosphate buffer (0.05 M, pH 5.0). Take 100 µl of appropriately diluted enzyme solution, add it to 900 µl CMC-Na substrate, shake and mix evenly, incubate in a water bath at 50 °C for 30 min, when the reaction is terminated, add 1.5 ml of DNS reagent to each test tube, and boil in boiling water for 5 min. Cool quickly and measure the absorbance at 540 nm. 1 ml of liquid enzyme, at 50 ℃, pH 5.0, hydrolyzes sodium carboxymethylcellulose per hour to produce 1 μmol of reducing sugar (calculated as glucose) The amount of enzyme required is defined as one enzyme activity unit (U), The results are shown in Figure 3. It can be seen that the transformants expressing the 72685 gene have higher enzymatic activity of endocellulase than the original strain in the whole culture stage. After SUS4-2 was cultured in MM-Avicel for 6 days, the enzymatic activity of endo-cellulase was 40.85 U/mL, while that of the overexpressed strain 72685 was 57.56 U/mL, which was 0.40 times higher than that of the original strain. Therefore, overexpression of the 72685 gene can improve the enzymatic activity of cellulase.

Example 4. Overexpression 72685 strain-related cellulase and transcriptional abundance of its own genes

Extract the RNA of 72685 overexpressing strain and SUS4-2 strain after induction for 24 h on the medium with microcrystalline cellulose Avicel as the sole carbon source, and measure the transcriptional abundance of related cellulase and its own genes (see Figure 4). . It can be seen that after overexpression of 72685 , the transcription levels of cbh1 , cbh2 , egl1 , egl2 and 72685 genes were significantly higher than those of the control strain. In the detection experiment of the expression level of related genes, three samples were induced and cultured in parallel for each sample, and each sample was repeated three times by ^fluorescence quantitative technology. method.

The above embodiments are only used to explain the technical solutions of the present application, and do not limit the protection scope of the present application.

sequence listing

<110> Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences

<120> A method for improving the ability of Trichoderma reesei to express cellulase by regulating cell metabolism

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Claims (3)

1. a method that improves Trichoderma reesei expression cellulase ability by regulating and controlling cell metabolism, it is characterized in that, described method comprises the step of overexpressing Trichoderma reesei regulation gene 72685 in Trichoderma reesei, wherein, all The nucleotide sequence of the T. reesei regulatory gene is shown in SEQ ID NO: 1. 2. the method for improving the ability of Trichoderma reesei to express cellulase by regulating cell metabolism according to claim 1, is characterized in that, described method comprises the method of constructing the overexpression vector that comprises described Trichoderma reesei regulation gene 72685 . step. 3. the method for improving the ability of Trichoderma reesei to express cellulase by regulating cell metabolism according to claim 2, is characterized in that, by CDNA1 promoter, 72685 gene, CDNA1 terminator and pyr4 expression cassette seamless tandem splicing to obtain the overexpression vector.

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