Pichia pastoris engineering bacteria and preparation method thereof, and method for producing cellooligosaccharide / disaccharide by using corn straw or wheat straw

Abstract

The invention relates to pichia pastoris engineering bacteria and a preparation method thereof, and a method for producing cellooligosaccharide / disaccharide by using corn straws or wheat straws, and belongs to the technical field of genetic engineering. A cellulose incision enzyme gene and a cellulose excision enzyme gene are transferred into pichia pastoris engineering bacteria. The preparation method comprises the following steps: respectively inserting a cellulose incision enzyme gene and a cellulose excision enzyme gene into plasmids to construct expression vectors; transferring the expression vector into a competent cell of pichia pastoris GS115; and screening and identifying to obtain successfully transformed pichia pastoris engineering bacteria. According to the present invention, the pichia pastoris heterologous expression cellulose incision enzyme and cellulose excision enzyme are adopted to construct the pichia pastoris engineering strain capable of degrading the plant straw into the cellooligosaccharide / disaccharide, such that the reliable chassis strain is provided for the production of the starch from the cellulose;

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to an engineered Pichia pastoris strain and its preparation method, and a method for producing cellulosic oligosaccharides / disaccharides using corn stalks or wheat stalks. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] In the process of converting straw into starch, cellulose is partially degraded into cellulosic sugars by cellulase, then glucose-1-phosphate is generated by cellulosic sugar phosphorylase, and finally amylose is synthesized by α-glucan phosphorylase. This process requires no additional energy to generate glucose-1-phosphate, resulting in low overall conversion costs. Among the various types of cellulosic sugars, cellobiose phosphorylase is more readily expressed heterologously, thus facilitating the downstream pathway of cellobiose.

[0004] Natural cellulase-producing microorganisms, such as Trichoderma reesei and Aspergillus niger, have obvious limitations, such as: (1) These natural cellulase-degrading bacteria have complex regulatory networks. For example, Trichoderma reesei cells have multiple nuclei, which leads to uncontrollable enzyme system composition and non-specific hydrolysis; (2) The extracellular proteins secreted are diverse and difficult to serve as ideal hosts for the expression of single / double cellulase components; (3) If cellobiose is to be the target product, all β-glucosidase components in the enzyme system need to be removed. However, genetic engineering modification faces problems such as low transformation efficiency, long genetic operation cycle, and lack of mature gene editing tools. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an engineered Pichia pastoris strain and its preparation method, as well as a method for producing cellulosic oligosaccharides / disaccharides using corn or wheat straw. By heterologously expressing cellulase and cellulase in Pichia pastoris, a strain capable of decomposing straw into cellulosic oligosaccharides / disaccharides is constructed. The obtained strain can be used for the efficient production of cellulosic oligosaccharides or as a chassis strain for cellulose-to-starch synthesis.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: Firstly, a Pichia pastoris engineered strain was obtained by introducing cellulase endonuclease and cellulase exonuclease genes into Pichia pastoris GS115 as the starting strain. The cellulase gene includes A. nidulans A10, T. reesei cel5A, T. terrestris cel5A , T. terrestris cel5C , T. terrestris cel5D , T. terrestris cel7B and T. terrestris cel7C One or more of the following; The cellulase gene includes A. niger cbhB and T. emersonii cel7A fusion protein One or more of them.

[0007] Secondly, the preparation method of the above-mentioned engineered Pichia pastoris includes the following steps: S1. Insert the cellulose endonuclease gene and the cellulose exonuclease gene into the pPIC9K plasmid to construct the cellulose endonuclease gene expression vector and the cellulose exonuclease gene expression vector, respectively. S2. The expression vector was transferred into competent cells of Pichia pastoris GS115; S3. Screening and identification of successfully transformed Pichia pastoris engineered strains.

[0008] Thirdly, a method for producing fiber oligosaccharides / disaccharides using corn stalks or wheat stalks includes the following steps: S4. Treat corn stalks and / or wheat stalks by steam explosion or hydrothermal method to obtain natural cellulose substrates; S5. Mix the natural cellulose substrate, acetate-sodium acetate buffer, and the cellulase and cellulase obtained by fermentation of the above-mentioned Pichia pastoris engineered strain to obtain cellulosic oligosaccharide / disaccharide.

[0009] Fourthly, a method for obtaining cellulase and cellulase includes: fermenting the above-mentioned engineered Pichia pastoris.

[0010] The above one or more technical solutions have the following beneficial effects: This invention constructs an engineered Pichia pastoris strain capable of degrading plant straw into cellulosic oligosaccharides (especially cellobiose) through heterologous expression of cellulase and cellulase. This improves the purity and operability of cellulase expression and provides a reliable chassis strain for starch production from cellulose. The cellulase provided by this invention can effectively degrade cellulose to generate cellulosic oligosaccharides / disaccharides, improving the industrial production capacity of cellulosic oligosaccharides / disaccharides and possessing practical application value. Attached Figure Description

[0011] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0012] Figure 1 This is a schematic diagram of the overall technical route in Embodiment 2 of the present invention.

[0013] Figure 2 As in Embodiment 1 of the present invention p Schematic diagram of PIC9K plasmid.

[0014] Figure 3 The graph shows the activities of cellulase CMC and cellulase pNPC in Example 1 of the present invention, where (a) represents the activity of cellulase CMC. Figure 1 (b) shows the activity of cellulase CMC. Figure 1 (c) is a graph showing the activity of cellulase CMC. Figure 4 The image shows the filter paper enzyme activity measured by the combination of cellulase and cellulase in Example 1 of this invention, wherein (a) shows the filter paper enzyme activity of different combination schemes. Figure 1 (b) shows the enzyme activity of filter paper with different combination schemes. Figure 2 .

[0015] Figure 5 The above are liquid chromatograms of the products of hydrolyzing microcrystalline cellulose by combining cellulose endonuclease and cellulose exonuclease in Example 1 of the present invention. (a) is a liquid chromatogram of a carbohydrate standard solution, and (b) is a liquid chromatogram of the products of hydrolyzing microcrystalline cellulose by different combinations. Figure 1 (c) shows the liquid chromatography of different combinations of hydrolyzed microcrystalline cellulose products. Figure 2 (d) shows the liquid chromatography of different combinations of hydrolyzed microcrystalline cellulose products. Figure 3 .

[0016] Figure 6 This refers to the protein concentration during the methanol-induced fermentation process of the double-transformation recombinant Pichia pastoris in Example 3 of this invention.

[0017] Figure 7 The enzyme activity of the fermentation broth on day 6 of methanol-induced fermentation of the double-transformation recombinant Pichia pastoris in Example 3 of this invention is shown in (a) and (b) is shown in (b). p NPC enzyme activity.

[0018] Figure 8 The filter paper enzyme activity of the double-transformation recombinant Pichia pastoris in Example 3 of the present invention.

[0019] Figure 9 This is a liquid chromatogram of the product of hydrolyzed microcrystalline cellulose by double-conversion recombinant Pichia pastoris in Example 3 of the present invention.

[0020] Figure 10 The images show liquid chromatograms of the products from the hydrolysis of different cellulose substrates by the dual-transformation recombinant Pichia pastoris in Example 5 of the present invention. Among them, (a) is the result of hydrolysis of corn straw pretreated by steam explosion, and (b) is the result of hydrolysis of wheat straw pretreated by hydrothermal treatment. Detailed Implementation

[0021] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0022] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0023] Cellulose endonuclease gene A. nidulans A10 The corresponding NCBI number is XM_653797.1.

[0024] Cellulose endonuclease gene T. reesei cel5A The corresponding NCBI number is KX255673.1.

[0025] Cellulose endonuclease gene T. terrestris cel5A The corresponding NCBI number is XM_003656198.1.

[0026] Cellulose endonuclease gene T. terrestris cel5C The corresponding NCBI number is XM_003656967.1.

[0027] Cellulose endonuclease gene T. terrestris cel5D The corresponding NCBI number is XM_003649228.1.

[0028] Cellulose endonuclease gene T. terrestris cel7B The corresponding NCBI number is XM_003649434.1.

[0029] Cellulose endonuclease gene T. terrestris cel7C The corresponding NCBI number is XM_003653428.1.

[0030] Cellulose exonuclease gene A. niger cbhB The corresponding NCBI number is HM769954.1.

[0031] Cellulose exonuclease gene T. emersonii cel7A fusion protein The corresponding NCBI number is AGU68246.1.

[0032] One or more embodiments of the present invention provide a Pichia pastoris engineered strain, which is obtained by transforming Pichia pastoris GS115 into cellulase endonuclease genes and cellulase exonuclease. The cellulase gene includes A. nidulans A10, T. reesei cel5A, T. terrestris cel5A , T. terrestris cel5C , T. terrestris cel5D , T. terrestris cel7B and T. terrestris cel7C One or more of the following; The cellulase gene includes A. niger cbhB and T. emersonii cel7A fusion protein One or more of them.

[0033] Optionally, the Pichia pastoris GS115 is a wild-type strain.

[0034] One or more embodiments of the present invention provide a method for preparing the above-mentioned engineered Pichia pastoris strain, comprising the following steps: S1. Insert the cellulose endonuclease gene and the cellulose exonuclease gene into the pPIC9K plasmid to construct the cellulose endonuclease gene expression vector and the cellulose exonuclease gene expression vector. S2. The expression vector was transferred into competent cells of Pichia pastoris GS115; S3. Screening and identification of successfully transformed Pichia pastoris engineered strains.

[0035] Optionally, in S1, after removing the signal peptide from the cellulase gene and cellulase gene, codon optimization is performed using Pichia pastoris as the host.

[0036] Optionally, in S1, when the cellulase gene is A. nidulans A10 At that time, the nucleotide sequence shown in SEQ ID NO.1 (primer A10-F) and the nucleotide sequence shown in SEQ ID NO.2 (primer A10-R) were used as primers for amplification to add upstream and downstream homologous arms.

[0037] Optionally, in S1, when the cellulase gene is T. reesei cel5A At that time, the nucleotide sequence shown in SEQ ID NO.3 (primer Tr5A-F) and the nucleotide sequence shown in SEQ ID NO.4 (primer Tr5A-R) were used as primers for amplification to add upstream and downstream homologous arms.

[0038] Optionally, in S1, when the cellulase gene is T. terrestris cel5A At that time, the nucleotide sequence shown in SEQ ID NO.5 (primer Tt5A-F) and the nucleotide sequence shown in SEQ ID NO.6 (primer Tt5A-R) were used as primers for amplification to add upstream and downstream homologous arms.

[0039] Optionally, in S1, when the cellulase gene is T. terrestris cel5C At that time, the nucleotide sequence shown in SEQ ID NO.7 (primer Tt5C-F) and the nucleotide sequence shown in SEQ ID NO.8 (primer Tt5C-R) were used as primers for amplification to add upstream and downstream homologous arms.

[0040] Optionally, in S1, when the cellulase gene is T. terrestris cel5D At that time, the nucleotide sequence shown in SEQ ID NO.9 (primer Tt5D-F) and the nucleotide sequence shown in SEQ ID NO.10 (primer Tt5D-R) were used as primers for amplification to add upstream and downstream homologous arms.

[0041] Optionally, in S1, when the cellulase gene is T. terrestris cel7B At that time, the nucleotide sequence shown in SEQ ID NO.11 (primer Tt7B-F) and the nucleotide sequence shown in SEQ ID NO.12 (primer Tt7B-R) were used as primers for amplification to add upstream and downstream homologous arms.

[0042] Optionally, in S1, when the cellulase gene is T. terrestris cel7C At that time, the nucleotide sequence shown in SEQ ID NO.13 (primer Tt7C-F) and the nucleotide sequence shown in SEQ ID NO.14 (primer Tt7C-R) were used as primers for amplification to add upstream and downstream homologous arms.

[0043] Optionally, in S1, when the cellulase gene is A. niger cbhB At that time, the nucleotide sequence shown in SEQ ID NO.15 (primer AnB-F) and the nucleotide sequence shown in SEQ ID NO.16 (primer AnB-R) were used as primers for amplification to add upstream and downstream homologous arms.

[0044] Optionally, in S1, when the cellulase gene is T. emersonii cel7A fusion protein (abbreviated (Referred to as T. emersonii cel7A-FP) At that time, the nucleotide sequence shown in SEQ ID NO.17 (primer Te7A-F) and the nucleotide sequence shown in SEQ ID NO.18 (primer Te7A-R) were used as primers for amplification to add upstream and downstream homologous arms.

[0045] The nucleotide sequences of the primers mentioned above are shown in Table 1.

[0046] Table 1

[0047] Optionally, in S1, the combination of cellulase endonuclease and cellulase exonuclease genes is inserted into the pPIC9K plasmid using a one-step cloning method.

[0048] Optionally, in S2, the transition method includes the following steps: S21. Linearize the cellulose endonuclease gene expression vector and the cellulose exonuclease gene expression vector with restriction endonucleases, and dissolve them in TE buffer, respectively.

[0049] S22. Take Pichia pastoris GS115 electrotransformation competent cells, TE buffer containing a linearized cellulose endonuclease gene expression vector, and TE buffer containing a linearized cellulose exonuclease gene expression vector, mix them, and transfer them into an electrotransfer cuvette. S23. The substance in the electric rotating cup is electrically agitated with parameters of voltage 1400~1600V, capacitance 20~30μF and resistance 150~250Ω.

[0050] Optionally, in S22, equal volumes of TE buffer containing the linearized cellulose endonuclease gene expression vector and TE buffer containing the linearized cellulose exonuclease gene expression vector are mixed.

[0051] Optionally, in S3, screening is performed using an amino acid deficiency screening method, and the genome is identified using a PCR identification method.

[0052] One or more embodiments of the present invention provide a method for producing fiber oligosaccharides / disaccharides using corn stalks or wheat stalks, comprising the steps of: S4. Treat corn stalks or wheat stalks by steam explosion or hydrothermal method to obtain natural cellulose substrates; S5. Mix the natural cellulose substrate, acetate-sodium acetate buffer, and the cellulase and cellulase obtained by fermentation of the above-mentioned Pichia pastoris engineered strain to obtain cellulosic oligosaccharide / disaccharide.

[0053] Optionally, in S4, the natural cellulose substrate includes one or more of the following: corn stalks pretreated by steam explosion, corn stalks pretreated by hydrothermal method, wheat stalks pretreated by steam explosion, and wheat stalks pretreated by hydrothermal method.

[0054] "Producing fiber oligosaccharides / disaccharides" refers to producing fiber oligosaccharides, including cellotrisaccharides and cellotetrasaccharides, or producing only cellobiose.

[0055] When only cellobiose is produced, it can be separated and purified by methods such as column chromatography or membrane separation technology to obtain high-purity cellobiose.

[0056] One or more embodiments of the present invention provide a method for obtaining cellulase endonuclease and cellulase exonuclease, comprising: fermenting the above-mentioned Pichia pastoris engineered strain; the fermentation method comprising: methanol-induced fermentation for 6-7 days.

[0057] Example 1 S11. Insert the cellulose endonuclease gene and the cellulose exonuclease gene into the pPIC9K plasmid, respectively, to construct the cellulose endonuclease gene expression vector and the cellulose exonuclease gene expression vector; including the following steps: After removing the signal peptide from each cellulase gene and cellulase gene, codon optimization was performed using Pichia pastoris as the host. The cellulase gene was then amplified using primers A10-F and A10-R. A. nidulans A10 And add a His tag to the end of the gene; Cellulase gene was amplified using primers Tr5A-F and Tr5A-R. T. reesei cel5A And add a His tag to the end of the gene; Cellulase gene was amplified using primers Tt5A-F and Tt5A-R. T. terrestris cel5A And add a His tag to the end of the gene; Cellulase gene was amplified using primers Tt5C-F and Tt5C-R. T. terrestris cel5C And add a His tag to the end of the gene; Cellulase gene was amplified using primers Tt5D-F and Tt5D-R. T. terrestris cel5D And add a His tag to the end of the gene; Cellulase gene was amplified using primers Tt7B-F and Tt7B-R. T. terrestris cel7B And add a His tag to the end of the gene; Cellulase gene was amplified using primers Tt7C-F and Tt7C-R. T. terrestris cel7C And add a His tag to the end of the gene; Cellulase gene was amplified using primers AnB-F and AnB-R. A. niger cbhB And add a His tag to the end of the gene; Cellulase gene was amplified using primers Te7A-F and Te7A-R. T. emersonii cel7A fusion protein (abbreviated as T. emersonii cel7A FP) And a His tag is added to the end of the gene.

[0058] Additionally, amplification was performed using appropriate primers. T. reeseicel7B (Gene number HM769954.1) 、T. terrestrisTtCel45A (Gene number: XM_062864956.1) T. reeseicel6A (Gene number XM_006962518.1) T. reeseicel7A (Gene number XM_006969162.1) T. emersoniicbh1 (Gene number AY081766.1) N. crassacbh1(Gene number XM_957405.2) was used for screening; the corresponding primers are shown in Table 2.

[0059] Table 2

[0060] Using the restriction endonuclease EcoR I to... p PIC9K plasmid linearization, where p PIC9K plasmid such as Figure 2 As shown, a one-step cloning kit (Novozymes) was used to ligate the linearized plasmid and the target gene (i.e., one of the cellulase exonuclease gene and cellulase endonuclease gene mentioned above) to construct an expression vector; and 8 μg of each expression vector was dissolved in 8 μL of TE buffer.

[0061] S12. Transform the expression vector into competent Pichia pastoris GS115 cells; including the following steps: S121. Preparation of Pichia pastoris GS115 electrocompetent cells, including the following steps: Inoculate Pichia pastoris GS115 cells into 5 mL YPD medium test tubes and culture overnight at 30°C and 200 rpm; inoculate 2% of the cells into 50 mL YPD medium and culture overnight at 30°C and 200 rpm until OD... 600 The concentration was 0.9 ± 0.3. All culture medium was transferred to 50 mL centrifuge tubes and centrifuged at 4°C, 5000 rpm for 5 min. The supernatant was discarded, and the cells were washed with 50 mL of pre-chilled sterile water, followed by centrifugation at 4°C, 5000 rpm for 5 min. The supernatant was discarded, and the cells were washed with 25 mL of pre-chilled sterile water, followed by centrifugation at 4°C, 5000 rpm for 5 min. The supernatant was discarded, and the cells were washed with 25 mL of pre-chilled 1 M sorbitol solution, followed by centrifugation at 4°C, 5000 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in 1 mL of pre-chilled 1 M sorbitol solution to obtain electrotransformed competent cells, which could be used immediately or stored at -80°C for later use.

[0062] S122. Take 80 μL of Pichia pastoris GS115 electrotransformed competent cells obtained in S21, 8 μL of TE buffer containing the linearized cellulose endonuclease gene expression vector obtained in S21, and 8 μL of TE buffer containing the linearized cellulose exonuclease gene expression vector obtained in S21, mix them, and transfer them into a pre-cooled 0.2 cm electrotransfer cuvette.

[0063] S123. The substance in the electric rotating cup is electrically shocked with parameters of 1500 V voltage, 25 μF capacitance and 200 Ω resistance.

[0064] S13. The obtained electroporated strain is placed on MD medium. After screening for amino acid defects and identifying the genome by PCR, the target transformant is obtained, which is the first intermediate engineered strain that has successfully transformed into one of the above genes.

[0065] Next, the CMC activity of cellulose endonucleases was measured, and the activity of cellulose exonucleases was measured. p NPC enzyme activity was used to screen for cellulases and cellulases with high enzyme activity. The method for determining CMC enzyme activity includes: mixing 60 μL of 1% CMC solution with 60 μL of enzyme solution (the supernatant obtained by centrifuging the first intermediate engineered strain in S13 after methanol induction on day 6 is the enzyme solution), reacting in a 50℃ water bath for 30 min, then immediately adding 120 μL of DNS reagent, mixing well, and boiling for 10 min to terminate the reaction; transferring 200 μL of the reaction solution to an ELISA plate, and measuring the OD using an ELISA reader. 550 The result is as follows Figure 3 As shown in (a) and (b) in the figure: the enzyme with the highest activity is T. reesei Cel5A's CMC enzyme activity reached approximately 16 U / mL on the sixth day of fermentation, followed by... T. terrestris Cel5A can reach a CMC enzyme activity of about 13.5 U / mL on the fifth day of fermentation. T. terrestris Cel5C and A. nidulans The highest enzyme activity of A10 is similar, both reaching 12 U / mL on the sixth day of fermentation. T. terrestris Cel7B and T. terrestris The highest enzyme activity of Cel7C reached 10 U / mL on the sixth day and 8.5 U / mL on the fifth day of fermentation. T. terrestris The highest enzyme activity of Cel5D can only reach about 6 U / mL on the fifth day of fermentation.

[0066] p The method for determining NPC enzyme activity includes adding 40 μL of 1 mg / mL solution to a 1.5 mL EP tube. p NPC solution, 40 μL of enzyme solution (the supernatant obtained by centrifuging the first intermediate engineered strain in S13 after methanol induction on day 6) and 120 μL of acetate-sodium acetate buffer were mixed and reacted in a 50°C water bath for 30 min. The reaction was then terminated by adding 40 μL of 10% Na₂CO₃ solution. 200 μL of the reaction solution was transferred to a microplate and the OD was measured using a microplate reader. 420 The result is as follows Figure 3 As shown in (c): Because exonucleases are difficult to express heterologously, and pThe enzyme activity measured by the NPC method was low; therefore, among all cellulases, only [the enzyme activity was low]. T. emersonii Cel7A-FP exhibited relatively high activity, and only... A. niger CbhB showed weak activity on the seventh day of methanol-induced fermentation, while other cellulases remained similar to the control group, and pNPC enzyme activity could not be detected.

[0067] Enzyme activity unit (U) is defined as: the amount of enzyme required to catalyze the transformation of 1 μmol of substrate (or generate 1 μmol of product) per minute under specific conditions. Highly active cellulases (endonucleases or exonucleases) that can be expressed by the first intermediate engineered bacteria are screened. Highly active cellulases include: A. nidulans A10, T. reesei cel5A, T. terrestris cel5A , T. terrestris cel5C , T. terrestris cel5D , T. terrestris cel7B and T. terrestris cel7C Highly active cellulases include: A. niger cbhB and T. emersonii cel7A fusion protein (abbreviation) T. emersonii cel7A-FP ).

[0068] The seven selected cellulase endonucleases and two cellulase exonucleases were combined to obtain 14 possible combinations, as follows: A. nidulans A10- A. niger CbhB、 T. reese i Cel5A- A. niger CbhB、 T. terrestris - A. niger CbhB、 T. terrestris Cel5A- A. niger CbhB、 T. terrestris Cel5C-A.niger CbhB, T. terrestris Cel5D-A.niger CbhB, T. terrestris Cel7B-A.nigerCbhB, T. terrestris Cel7C- A. niger CbhB、 A. nidulans A10- T. emersonii Cel7A-FP, T. reesei Cel5A- T. emersonii Cel7A-FP, T. terrestris Cel5A- T. emersonii Cel7A-FP, T. terrestris Cel5C- T. emersonii Cel7A-FP, T. terrestris Cel5D- T. emersoniiCel7A-FP, T. terrestris Cel7B- T. emersonii Cel7A-FP and T. terrestris Cel7C- T. emersonii Cel7A-FP.

[0069] The total enzyme activity of each combination scheme was determined using the filter paper enzyme activity assay (FPase activity) method, including the following steps: Seven Whatman No. 1 filter papers were stapled together, and circular filter paper discs with a diameter of 5 mm were punched out. Three discs were then added as substrate to a 25 mL enzyme activity assay tube. 1.5 mL of acetate-sodium acetate buffer and 0.5 mL of diluted enzyme solution (not added to the control group) were added to the enzyme activity assay tube. The fermentation broth of the first intermediate engineered strain obtained above was centrifuged at 10,000 rpm at 4℃, and the fermentation supernatant was collected. Following the above combination scheme, 500 μL of cellulase fermentation supernatant and cellulase fermentation supernatant were added to each of the 25 mL enzyme activity assay tubes. All reaction systems were thoroughly mixed and placed in a 50℃ water bath for 1 h. Immediately after the reaction, 3 mL of DNS reagent was added, and the mixture was boiled for 10 min. After the boiling water bath, the mixture was immediately placed in ice water to cool, and then distilled water was added to bring the volume to 25 mL. After inverting and mixing, 200 μL of the solution was collected. μL was added to an enzyme-labeled tube, and the absorbance at 540 nm was measured. The result was then substituted into the glucose standard curve obtained under the same conditions for calculation to obtain the total cellulase activity for the corresponding combination. The results are as follows: Figure 4 As shown, (a) represents the enzyme activity of filter paper with different combinations. Figure 1 (b) shows the enzyme activity of filter paper with different combination schemes. Figure 2 It can be seen that the filter paper enzyme activity of the combination of cellulase and *T. emersonii* Cel7A-FP is significantly higher than that of the combination of the same cellulase and *A. niger* CbhB. Among them, the cellulase with the highest filter paper enzyme activity when combined with an exonuclease is... T. terrestris Cel5A, followed by T. reesei Cel5A, which contradicts the level of CMC enzyme activity, reveals that the catalytic activity of individual cellulase and exonuclease components is not significantly correlated with the overall catalytic efficiency of their synergistic system.

[0070] The method for screening high-yield combinations of cellulosic sugar / cellobiose includes: adding 1 g of microcrystalline cellulose and 5 mL of acetate-sodium acetate buffer to a 50 mL Erlenmeyer flask; calculating based on the measured protein concentration, adding 500 μg of cellulase and 500 μg of cellulase to the reaction system according to the above combination scheme; and making up to 20 mL with distilled water. After wrapping the bottle mouth with tin foil, the reaction was carried out at 50℃ and 150 rpm for 24 h. Then, 1 mL of sample was taken from each of the 14 conical flasks representing the 14 different combinations, centrifuged at 1000 rpm for 1 min, and the supernatant was collected. The supernatant was boiled for 10 min to denature the protein and then incubated at 4℃ overnight (to ensure complete precipitation of the denatured protein and prevent column clogging during subsequent liquid chromatography detection). After centrifugation at 1000 rpm for 1 min, the supernatant was filtered through a 0.22 μm aqueous filter membrane and analyzed using a 42A column to determine the yield of cellulosic acid and cellobiose, and high-yielding combinations were selected. The results are as follows: Figure 5 As shown, (a) is the liquid chromatogram of a carbohydrate standard solution, and (b) is the liquid chromatogram of different combinations of hydrolyzed microcrystalline cellulose products. Figure 1 (c) shows the liquid chromatography of different combinations of hydrolyzed microcrystalline cellulose products. Figure 2 (d) shows the liquid chromatography of different combinations of hydrolyzed microcrystalline cellulose products. Figure 3 It can be seen that cellobiose is produced after all combinations of cellulase and exonuclease hydrolyze microcrystalline cellulose for 24 h. T. emersonii The amount of cellobiose produced by the combined enzymatic hydrolysis of microcrystalline cellulose by Cel7A-FP and cellulase is significantly higher than that produced by other enzymes. A. niger The combination of CbhB and cellulase. Figure 5 In (a) of the standard, the concentration of the glucose standard solution is 10 mg / mL, the concentrations of the standard solutions of cellobiose, cellotriose, cellotetraose, and cellopentaose are 1 mg / mL, and the concentration of the standard solution of cellohexaose is 0.1 mg / mL due to its poor solubility. Based on the liquid chromatography results of the standards, the corresponding... Figure 5 (b) Figure 5 (c) and Figure 5 Based on calculations, the combination with the highest cellobiose concentration is (d) among the products in the given data. T. terrestris Cel5C- T. emersonii Cel7A-FP had the highest concentration at 1.68 g / L, followed by... T. terrestris Cel5A- T. emersonii Cel7A-FP, with a maximum concentration reaching 1.59 g / L; the combination with the highest concentration of cellotriose in the product is... T. terrestris Cel5C- A. nigerCbhB, followed by T. reesei Cel5A- A. niger The concentrations of cellotriose in the CbhB product reached 0.38 g / L and 0.26 g / L, respectively.

[0071] Example 2 To reduce the cost and reduce the number of cultivation steps required in the cellulase production process, this embodiment provides Pichia pastoris capable of co-expressing cellulase and cellulase and its preparation method, thereby avoiding the cumbersome multiple batch fermentations and producing cellulase and cellulase in a single fermentation.

[0072] The overall technical route is as follows Figure 1 As shown, the method includes: S1. Following the method in S11 of Example 1, obtain cellulose endonuclease gene expression vectors and cellulose exonuclease gene expression vectors, respectively; digest the cellulose endonuclease expression vectors with restriction endonuclease Sac I and the cellulose exonuclease expression vectors with restriction endonuclease Sal I to generate different sticky ends; after enzyme digestion and recovery, dissolve them in TE buffer to obtain cellulose endonuclease-modified vector solutions (or cellulose exonuclease-modified vector solutions), and then measure the OD. 260 Add an appropriate amount of Elution buffer to ensure that the concentrations of the cellulose endonuclease-modified carrier solution and the cellulose exonuclease-linearized carrier solution are consistent.

[0073] S2. Following the method in S121 of Example 1, prepare Pichia pastoris GS115 electrotransformation competent cells resuspended in sorbitol solution. Take 80 μL of electrotransformation competent cells into a sterile 1.5 mL EP tube, add 10 μL of cellulose endonuclease-modified vector solution and 10 μL of cellulose exonuclease-modified vector solution, incubate on ice for 30 min, and transform into competent cells by electroporation. Spread on MD solid medium for amino acid defect screening to obtain a second intermediate engineered strain successfully transformed with at least one target gene.

[0074] S3. Cultivate the second intermediate engineered bacteria obtained in S2, extract the genome from the grown colonies, and determine by PCR whether they are double-transformed transformants. The primers required for determining double-transformed transformants are... A. nidulans A10 Gene primers such as SEQ ID NO.19 (primer) An10 -F) is shown, used for T. reesei cel5A Gene primers such as SEQ ID NO.20 (primer) Trcel5A -F) is shown, used for T. terrestris cel5A Gene primers such as SEQ ID NO.21 (primer) Ttcel5A-F As shown in the figure, it is used for T. terrestris cel5C Gene primers such as SEQ ID NO.22 (primer) Ttcel5C-F As shown in the figure, it is used for T. terrestris cel5D Gene primers such as SEQ ID NO.23 (primer) Ttcel5D-F As shown in the figure, it is used for T. terrestris cel7B Gene primers such as SEQ ID NO.24 (primer) Ttcel7B-F As shown in the figure, it is used for T. terrestris cel7C Gene primers such as SEQ ID NO.25 (primer) Ttcel7C-F As shown in the figure, it is used for A. niger cbhB Gene primers such as SEQ ID NO.26 (primer) AncbhB-F As shown in the figure, it is used for T. emersonii cel7A fusion protein Gene primers such as SEQ ID NO.27 (primer) Tecel7A-F As shown in SEQ ID NO. 28, the reverse primers used for all the genes described in this step are as follows: 9K-R As shown in the figure; the primer sequences are shown in Table 3.

[0075] Table 3

[0076] The engineered bacteria that simultaneously introduce the target cellulase gene and the target cellulase gene are the obtained Pichia pastoris engineered bacteria.

[0077] The method for evaluating the ability of the dual-transformation recombinant Pichia pastoris engineered strain to hydrolyze cellulose substrates provided in this embodiment includes: The Pichia pastoris engineered strain obtained in this example was analyzed using a Bradford assay kit, including the following steps: the enzyme solution was diluted 20-fold, 150 μL was transferred to an ELISA plate, 150 μL of Bradford reagent was added, the mixture was quickly mixed, and the plate was incubated at room temperature for 5 min. A blank control of 150 μL water and 150 μL Bradford reagent was used. The OD value was then measured using an ELISA reader. 595 The concentration of fermented protein can be determined by calculation, and the results are as follows: Figure 6 As shown, the protein concentration changes during the fermentation of the four types of double-transformed Pichia pastoris are not significantly different, and the highest values ​​are all around 150 μg / mL.

[0078] The CMC and pNPC enzyme activities of the Pichia pastoris engineered fermentation broth obtained in this example were measured respectively. Analysis revealed the approximate ratio of cellulase endonucleases and cellulase exonucleases in the fermentation broth. The results are as follows: Figure 7 As shown in (a) and (b), the CMC enzyme activity measured when expressed alone, pThe comparison of NPC enzyme activities showed that the expression level of cellulase endonucleases was downregulated in the double-transformed strains, while the proportion of exonucleases in the secreted cellulases was increased.

[0079] The enzyme activity of the double-transformed recombinant Pichia pastoris engineered strain in this example was determined using the filter paper enzyme activity assay (FPase activity). The activity was compared with the filter paper enzyme activity in Example 1 to analyze the difference in secretion between the double-transformed recombinant Pichia pastoris and the recombinant Pichia pastoris expressing cellulase alone. The results are as follows: Figure 8 As shown, the relative levels of filter paper enzyme activity of the four double-transformed strains are consistent with those of the single-expression combinations, ranging from highest to lowest as follows: T. terrestris Cel5A- T. emersonii Cel7A-FP, T. terrestris Cel5C- T. emersonii Cel7A-FP, T. reesei Cel5A-A.niger CbhB, T. terrestris Cel5C- A. niger CbhB.

[0080] Following the method for screening high-yield combinations of cellulosic acid / cellobiose in Example 1, the double-transformation recombinant Pichia pastoris engineered strain obtained in this example was screened. After hydrolyzing microcrystalline cellulose, the product was analyzed by liquid chromatography, and the results are as follows: Figure 9 As shown, the double-transformation strain with the highest cellobiose production is... T. terrestris Cel5A- T. emersonii Cel7A-FP, the strain with the highest cellotriose production is T. terrestris Cel5C- T. emersonii Cel7A-FP.

[0081] Example 4 A method for obtaining cellulase endonucleases and cellulase exonucleases, comprising the steps of: S4. The constructed cellulase- and exonuclease-double-transformed strain was inoculated into 5 mL of YPD liquid medium and cultured at 30℃ and 200 rpm for 16 h until OD was obtained. 600 When ≈1, inoculate 2% of the culture medium into BMGY liquid medium and incubate at 30°C and 200 rpm for 24 h. Then, centrifuge at 4°C and 6000 rpm to transfer all the cells into BMMY medium (containing methanol for methanol-induced fermentation) for fermentation. Incubate at 30°C and 200 rpm for 6 days. The supernatant obtained after centrifuging the fermentation broth is the crude enzyme solution containing cellulase and exonuclease.

[0082] If purification is required, the cellulase endonuclease and cellulase are purified using a nickel chromatography column.

[0083] Example 5 The production of cellulosic oligosaccharides (including disaccharides and trisaccharides) using different cellulose substrates includes the following steps: S5. To obtain a natural cellulose substrate by treating plant straw by steam explosion or hydrothermal method, including: one of the following: corn straw pretreated by steam explosion, corn straw pretreated by hydrothermal method, wheat straw pretreated by steam explosion and wheat straw pretreated by hydrothermal method; S6. Add 1 g of dry cellulose substrate and 5 mL of acetate-sodium acetate buffer to a 50 mL Erlenmeyer flask. Based on the measured protein concentration, add 500 μg of cellulose endonuclease and 500 μg of cellulose exonuclease obtained in Example 4 to the reaction system, respectively. Add distilled water to a final volume of 20 mL. After wrapping the flask opening with aluminum foil, react at 50°C and 150 rpm for 72 h. Measure the cellulose substrate processing capacity and the yield of cellobiose in the product. The results are as follows: Figure 10 As shown, (a) is the result of hydrolysis and steam explosion pretreatment of corn straw, and (b) is the result of hydrolysis and hydrothermal pretreatment of wheat straw. T. terrestrial Cel5C- T. emersonii Cel7A-FP performed poorly in the hydrolysis of hydrothermally pretreated wheat straw, producing only a small amount of cellobiose. This may be due to an altered ratio of endo- and exo-cellulases, leading to a decreased adsorption or catalytic effect on lignin-attached natural lignocellulose substrates. However, it showed better hydrolysis performance on steam-explosion pretreated corn straw. Conversely, T. terrestris Cel5A- T. emersonii Cel7A-FP showed poor hydrolysis performance on corn straw pretreated by steam explosion, but good hydrolysis performance on wheat straw pretreated by hydrothermal treatment. T. terrestris Cel5C- T. Emerson The cellobiose and trisaccharides produced during the pretreatment of corn stalks by Cel7A-FP hydrolysis and steam explosion were at their highest levels.

[0084] When only cellobiose is produced T. terrestris Cel5C- T. emersonii The Cel7A-FP hydrolysis and vapor explosion pretreatment of corn stalks yields the best results, producing a large amount of cellobiose. This cellobiose is then separated and purified by column chromatography to obtain high-purity cellobiose.

[0085] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A Pichia pastoris engineered strain, characterized in that, Using Pichia pastoris GS115 as the starting strain, the strain was obtained by introducing cellulase endonuclease and cellulase genes; the cellulase genes include A. nidulans A10, T. reesei cel5A, T. terrestris cel5A , T. terrestris cel5C , T. terrestris cel5D , T. terrestris cel7B and T. terrestris cel7C One or more of the following; the cellulase gene includes A. niger cbhB and T. emersonii cel7A fusion protein One or more of them.

2. The engineered Pichia pastoris strain as described in claim 1, characterized in that, The Pichia pastoris GS115 is a wild strain.

3. A method for preparing Pichia pastoris engineered strain as described in any one of claims 1-2, comprising the steps of: S1. Insert the cellulose endonuclease gene and the cellulose exonuclease gene into the pPIC9K plasmid to construct the cellulose endonuclease gene expression vector and the cellulose exonuclease gene expression vector. S2. The expression vector was transferred into competent cells of Pichia pastoris GS115; S3. Screening and identification of successfully transformed Pichia pastoris engineered strains.

4. The method for preparing Pichia pastoris engineered strains as described in claim 3, characterized in that, In S1, after removing the signal peptide from the cellulase gene and cellulase gene, codon optimization was performed using Pichia pastoris as the host.

5. The method for preparing Pichia pastoris engineered strains as described in claim 3, characterized in that, In S1, the combination of cellulose endonuclease gene and cellulose exonuclease gene was inserted into the pPIC9K plasmid using a one-step cloning method.

6. The method for preparing Pichia pastoris engineered strains as described in claim 3, characterized in that, In S2, the transition method includes the following steps: S21. Linearize the cellulose endonuclease gene expression vector and the cellulose exonuclease gene expression vector with restriction endonucleases, and dissolve them in TE buffer, respectively. S22. Take Pichia pastoris GS115 electrotransformation competent cells, TE buffer containing linearized cellulose endonuclease gene expression vector and TE buffer containing linearized cellulose exonuclease gene expression vector, mix them and transfer them into an electrotransfer cuvette. S23. The substance in the electric rotating cup is electrically agitated with parameters of voltage 1400~1600V, capacitance 20~30μF and resistance 150~250Ω.

7. The method for preparing Pichia pastoris engineered strains as described in claim 6, characterized in that, In S22, equal volumes of TE buffer containing a linearized cellulose endonuclease gene expression vector and TE buffer containing a linearized cellulose exonuclease gene expression vector are mixed. Alternatively, in S3, screening is performed using an amino acid deficiency screening method, and the genome is identified using a PCR identification method.

8. A method for producing fiber oligosaccharides / disaccharides using corn stalks or wheat stalks, characterized in that, Including the following steps: S4. Treat corn stalks and / or wheat stalks by steam explosion or hydrothermal method to obtain natural cellulose substrates; S5. Mix natural cellulose substrate, acetate-sodium acetate buffer, and cellulase and cellulase obtained by fermentation of Pichia pastoris engineered strain as described in any one of claims 1-2 to obtain cellulosic oligosaccharide / disaccharide.

9. The method for producing fiber oligosaccharides / disaccharides using corn stalks or wheat stalks as described in claim 8, characterized in that, In S4, the natural cellulose substrate includes one or more of the following: corn stalks pretreated by steam explosion, corn stalks pretreated by hydrothermal method, wheat stalks pretreated by steam explosion, and wheat stalks pretreated by hydrothermal method.

10. A method for obtaining cellulase endonuclease and cellulase exonuclease, characterized in that, include: Fermentation of the engineered Pichia pastoris strain as described in any one of claims 1-2.

Images