Genetically engineered bacterium for efficiently expressing xylanase and use thereof in degradation of straw-type agricultural waste

Abstract

Provided are a genetically engineered bacterium for efficiently expressing xylanase and use thereof in the degradation of straw-type agricultural waste. By optimizing the expression cassette of a xylanase gene and increasing the expression level of an Hac1 gene, the expression level of xylanase is significantly improved, and the cost of industrial enzyme production is reduced. The successful construction of the engineered bacterium further reduces the cost of producing xylooligosaccharides by degrading the straw-type agricultural waste, so that the engineered bacterium is suitable for industrial production.

Description

High-efficiency xylanase-expressing genetically engineered bacteria and application thereof in degradation of straw agricultural waste TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioengineering, and particularly relates to high-efficiency xylanase-expressing genetically engineered bacteria and application thereof in degradation of straw agricultural waste. BACKGROUND

[0002] Agricultural waste, such as corn cob, sugarcane residue, cottonseed hulls and rapeseed straw, has been abandoned or burned for a long time, causing serious pollution to the environment. However, these agricultural wastes are rich in hemicellulose and have great application potential as biomass resources, and can be used to produce high-value bio-chemical products. In particular, xylan, as the main component of hemicellulose, is considered to be an ideal raw material for preparing xylooligosaccharide (XOS). Xylooligosaccharide has a wide application prospect in the fields of food, medicine, agriculture and the like as a prebiotic.

[0003] Xylanase (E.C.3.2.1.8) is a key enzyme for hydrolyzing xylan to produce xylooligosaccharide, and plays an important role in the high-value utilization of lignocellulose. Trichoderma reesei is an important strain in the field of biotechnology, and has a complete lignocellulose-degrading enzyme system. The xylanase Xyn2 (GenBank accession number: ETR98242.1) secreted by the strain has very high hydrolysis activity on xylan, and has been widely used in paper pulp biological bleaching and feed additives. The mutant Xyn2A obtained by introducing a pair of disulfide bonds (T2C / T28C) in Xyn2 significantly improves the enzyme activity and thermal stability of the enzyme at high temperature (65℃). Therefore, the large-scale production of Xyn2A and its degradation of hemicellulose in agricultural straw and saccharification process have important research significance and application value.

[0004] The lignocellulose in crop straw has a complex structure, wherein the cross-linking of lignin and the side chain modification of xylan limit the degradation efficiency of xylanase. Therefore, before the lignocellulose is hydrolyzed by using xylanase, the lignocellulose often needs to be pretreated. The pure physical method mainly adopts steam explosion method, which makes most of the xylan dissolved in the liquid, but a large amount of lignin hydrolysis products and furfural are also present in the solution, which is difficult to separate and purify and needs a large pressure vessel. The current pretreatment method is mainly the combination of physical method and chemical method. The material is often first crushed by a crusher and sieved to improve the efficiency of the subsequent chemical treatment. The chemical method mainly includes acid hydrolysis, alkali hydrolysis and hydrogen peroxide oxidation method. The acid hydrolysis makes the hemicellulose dissolved in the acid solution, which is difficult to separate from the lignin hydrolysis products, and the hydrogen peroxide method has a high cost in industrial production. Compared with the above methods, the alkali hydrolysis has a high efficiency in delignification and can deacetylate xylan, and is a more moderate and economical method, which provides an economically feasible way for the high-value utilization of straw agricultural waste. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a genetically engineered bacterium for efficiently expressing xylanase and its application in degrading straw agricultural waste.

[0006] The technical scheme adopted by the present application to solve its technical problems is:

[0007] The first purpose of the present application is to provide a recombinant plasmid pSc048, which is constructed by the following method:

[0008] 1) An Xyn2A expression frame is constructed by Golden Gate Cloning, which comprises an FMD promoter, an alphaMF* signal peptide, an Xyn2A gene and an AOX1 terminator, and the expression frame is loaded into a backbone plasmid pBK to construct a plasmid pSc026;

[0009] 2) An Hac1 expression frame is constructed by Golden Gate Cloning, which comprises an FBA2 promoter, an Hac1 gene and a DAS1 terminator, and the expression frame is loaded into a backbone plasmid pBK to construct a plasmid pSc035;

[0010] 3) The pSc026 plasmid is constructed into a recombinant plasmid pSc038 containing 2 copies of Xyn2A expression frame by enzyme digestion and ligation through the method of Biobrick;

[0011] 4) The pSc038 and pSc035 are constructed into a three-expression frame recombinant plasmid pSc048 containing 2 copies of Xyn2A expression frame and 1 copy of Hac1 expression frame by enzyme digestion and ligation through the method of Biobrick.

[0012] In specific embodiments, the nucleotide sequence of the FMD promoter is as set forth in SEQ ID NO: 3.

[0013] In specific embodiments, the nucleotide sequence of the aMF* signal peptide is as set forth in SEQ ID NO: 9.

[0014] In specific embodiments, the nucleotide sequence of the Xyn2A gene is as set forth in SEQ ID NO: 11.

[0015] In specific embodiments, the nucleotide sequence of the AOX1 terminator is as set forth in SEQ ID NO: 17.

[0016] In specific embodiments, the nucleotide sequence of the FBA2 promoter is as set forth in SEQ ID NO: 6.

[0017] In specific embodiments, the nucleotide sequence of the Hac1 gene is as set forth in SEQ ID NO: 14.

[0018] In specific embodiments, the nucleotide sequence of the DAS1 terminator is as set forth in SEQ ID NO: 19.

[0019] A second object of the present application is to provide a genetically engineered bacterium for efficiently expressing xylanase, which contains the recombinant plasmid pSc048 described above.

[0020] The genetically engineered bacterium is obtained by introducing the xylanase gene Xyn2A into Pichia pastoris, optimizing the promoter, signal peptide and copy number of the Xyn2A expression frame, and increasing the expression amount of the Hac1 gene.

[0021] A third object of the present application is to provide a method for constructing the genetically engineered bacterium described above, which comprises the following steps:

[0022] 1) Constructing the Xyn2A expression frame containing the FMD promoter, aMF* signal peptide, Xyn2A gene and AOX1 terminator by Golden Gate cloning, and loading the expression frame into the backbone plasmid pBK to construct the plasmid pSc026;

[0023] 2) Constructing the Hac1 expression frame containing the FBA2 promoter, Hac1 gene and DAS1 terminator by Golden Gate cloning, and loading the expression frame into the backbone plasmid pBK to construct the plasmid pSc035;

[0024] 3) Constructing the recombinant plasmid pSc038 containing 2 copies of the Xyn2A expression frame by biological brick method, enzyme digestion and ligation of the pSc026 plasmid;

[0025] 4) Construct the three-expression frame recombinant plasmid pSc048 containing two copies of Xyn2A expression frame and one copy of Hac1 expression frame by the biological brick method, enzyme digestion and ligation of pSc038 and pSc035;

[0026] 5) Transform pSc048 into Pichia pastoris X33, and screen positive transformants by means of the blasticidin plate.

[0027] The fourth object of the present application is a method for fermenting and producing xylanase by using the above-mentioned recombinant engineering bacteria, which comprises the following steps: taking BSM as the fermentation medium, inoculating at a rate of 10%, waiting for the glucose in the medium to be consumed, adding 70% glucose containing 12 mL / L PTM1 by means of exponential feeding, culturing the bacteria to OD 600 200, and then inducing the expression of xylanase by adding methanol containing 12 mL / L PTM1, and the concentration of methanol is maintained at 2.4 g / L, and the induction of expression is completed.

[0028] In a specific embodiment, the BSM medium is composed of the following: 85% H3PO4 26.7 mL / L, CaSO4 0.93 g / L, K2SO4 18.2 g / L, MgSO4·7H2O 14.9 g / L, KOH 4.13 g / L, glucose 20 g / L, and PTM1 4.4 mL / L.

[0029] In a more specific embodiment, PTM1 is composed of the following: CuSO4·5H2O 6 g / L, KI 0.08 g / L, MnSO4·H2O 3 g / L, MoNa2O4 0.2 g / L, H3BO3 0.02 g / L, CoCl2 0.5 g / L, ZnCl2 20 g / L, FeSO4·7H2O 65 g / L, Biotin 0.2 g / L, and H2SO4 5 mL / L.

[0030] The fifth object of the present application is to protect the xylanase prepared by the above-mentioned method.

[0031] The sixth object of the present application is the application of the above-mentioned xylanase in the degradation of straw agricultural waste to produce xylo-oligosaccharides, especially the degradation of corncob, sugarcane residue, cottonseed hulls and rape straw.

[0032] The specific steps are as follows: in a sodium acetate buffer solution with pH 5.0, add the pretreated straw, so that the concentration of straw powder in the reaction system is 100 g / L, and the amount of xylanase Xyn2A added is 1 mg / g of straw powder, and the reaction is carried out at 50°C and a stirring speed of 200 rpm for 12 h.

[0033] The seventh object of the present application is to promote the growth of plants, especially cucumber seedlings and tomato seedlings, by using the sugar solution produced by the degradation of the aforementioned straw agricultural waste. Advantages

[0034] The present application aims to provide a genetically engineered bacterium for efficiently expressing xylanase and its application in degrading straw agricultural waste, which has the following advantages compared with the prior art:

[0035] (1) The recombinant genetically engineered bacterium pSc048-X33 of the present application can efficiently express xylanase Xyn2A. In a 5L fermenter, the content of Xyn2A in the supernatant reaches 10.2g / L, and the enzyme activity is as high as 45100U / mL.

[0036] (2) The present application uses Xyn2A fermentation supernatant to treat agricultural waste. At a lower enzyme dosage (1g of material: 1mg of Xyn2A) and under the condition of enzyme hydrolysis solid-liquid ratio of 1:10, the reducing sugar content in the supernatant reaches 9.0-31.1mg / mL.

[0037] (3) The sugar solution obtained by degrading agricultural waste can significantly promote the growth of cucumber seedlings and tomato seedlings. The cucumber seedlings and tomato seedlings are sprayed with sugar solution at concentrations of 2400ppm and 100ppm, respectively, and the fresh weight of the aboveground parts of the plants increases by 34% and 49%, respectively. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is a schematic diagram of the construction of the Pichia pastoris expression toolbox in Example 1;

[0039] Figure 2 is the xylanase activity in the supernatant of the recombinant engineered bacterium in Example 2 after 72h of induced fermentation in a shake flask;

[0040] Figure 3 is the time curve of the pSc048-X33 recombinant bacterium in a 5L fermenter in Example 3;

[0041] Figure 4 is the protein SDS-PAGE result of the recombinant xylanase expressed in a 5L fermenter in Example 3; Lane 1 is the protein marker, and lanes 2-11 are the extracellular proteins induced and expressed for 0, 12, 24, 36, 48, 60, 72, 84, 96, and 108h, respectively;

[0042] Figure 5 is the result of searching the two obvious bands of SDS-PAGE by time-of-flight mass spectrometry combined with Mascot software in Example 3. (A) Result of band I; (B) Result of band II;

[0043] Figure 6 is the phenotype of cucumber seedlings in Example 8;

[0044] Figure 7 is the physiological index of cucumber seedlings in Example 8;

[0045] Figure 8 is a tomato seedling phenotype of Example 9;

[0046] Figure 9 is a physiological index of tomato seedlings of Example 9; DETAILED DESCRIPTION

[0047] The application will be further described in detail below in conjunction with examples. The reagents or instrument equipment used without specifying the manufacturer are all regarded as conventional products that can be purchased in the market.

[0048] Example 1 Construction of Pichia expression toolbox

[0049] Based on the principle of Golden Gate Cloning seamless ligation, a toolbox for Pichia expression system was constructed using Type IIS enzyme. As shown in Figure 1, the toolbox consists of three levels of plasmids: Level 0, Level 1 and Level 2.

[0050] 1. Construction of Level 0 element plasmid

[0051] The Level 0 plasmid contains specific functional elements, including promoter, signal peptide, target gene and terminator, which are loaded into the backbone plasmid pYTK001 (Addgene product, product number #65108) by BsmBI-mediated Golden Gate Cloning. The promoters, signal peptides, target genes and terminators used are synthesized by Anhui General Company and loaded into pUC57 plasmid. The reaction system used for the construction of Level 0 plasmid is as follows: 10 fmol pYTK001 plasmid, 10 fmol pUC57 plasmid containing functional elements, 1 μL T4 DNA Ligase (NEB), 1 μL BsmBI (NEB), 2 μL T4 DNA ligase buffer, ddH2O to 20 μL. Mix the above system, use PCR instrument for assembly reaction, the program is as follows:

[0052] 8 uL of the assembly product was taken and transformed into E. coli, and the sequence-corrected level 0 element plasmid was obtained by sequencing verification.

[0053] 2. Construction of Level 1 single expression frame plasmid

[0054] Level 1 is a single expression cassette plasmid, which is assembled by Bsal mediated Golden Gate Assembly. Specific Level 0 plasmid is loaded into the backbone plasmid pBK. The reaction system is as follows: 10 fmol pBK plasmid, 10 fmol Level 0 element plasmid, 1 μL T4 DNA Ligase (NEB), 1 μL Bsal (NEB), 2 μL T4 DNA ligase buffer, ddH2O to 20 μL. Mix the above system, use PCR instrument for assembly reaction, the program is as follows:

[0055] 8 uL of the assembly product is taken to transform E. coli, and the level 1 element plasmid with correct sequence is obtained by sequencing verification.

[0056] 3. Construction of Level 2 multi-expression cassette plasmid

[0057] Level 2 plasmid is constructed by EcoRI, Xbal, Spel and Pstl mediated bio-brick method to concatenate expression cassettes. All the constructed plasmids are as follows:

[0058] Example 2 Construction and expression of Pichia pastoris recombinant strain of Xyn2A

[0059] 1. Construction of recombinant strain

[0060] The plasmids pSc020, pSc021, pSc022, pSc023, pSc024, pSc025, pSc026, pSc027, pSc038, pSc039, pSc040, pSc041, pSc042, pSc043, pSc044, pSc045, pSc046, pSc047, pSc048, pSc049, pSc050 in Example 1 were linearized by restriction enzyme Kpnl, respectively, and then electrotransformed into the expression host P. pastoris X33. The transformed bacterial suspension was spread on a plate containing bleomycin, and positive transformants were screened by resistance, and were named as pSc020-X33, pSc021-X33, pSc022-X33, pSc023-X33, pSc024-X33, pSc025-X33, pSc026-X33, pSc027-X33, pSc038-X33, pSc039-X33, pSc040-X33, pSc041-X33, pSc042-X33, pSc043-X33, pSc044-X33, pSc045-X33, pSc046-X33, pSc047-X33, pSc048-X33, pSc049-X33, pSc050-X33 recombinant bacteria, respectively.

[0061] 2. Shake flask fermentation

[0062] The above recombinant strains were subjected to shake flask fermentation. Single colonies were inoculated in 25 mL BMGY (yeast extract 10 g / L, peptone 20 g / L, K2HPO4 3 g / L, KH2PO4 12 g / L, YNB 1.34 g / L, glucose 10 g / L), and cultured at 30°C, 200 rpm for 20 h. The bacterial cells were collected by centrifugation, and an appropriate amount of bacterial cells was transferred to 100 mL BMMY (yeast extract 10 g / L, peptone 20 g / L, K2HPO4 3 g / L, KH2PO4 12 g / L, YNB 1.34 g / L) culture medium, and the initial OD600 in the shake flask was controlled at 2.0. 1 mL of methanol was added for induction. Thereafter, 1 mL of methanol was added every 24 h, and the expression was induced for 72 h. The supernatant xylanase activity and protein content were determined. The definition of xylanase activity unit: the amount of enzyme required to release 1 μmol of reducing sugar (xylose equivalent) from xylan per minute at pH 5.0 and 60°C is one enzyme activity unit. The protein content of the fermentation supernatant was determined by the Bradford method.

[0063] The results are shown in Figure 2. The supernatant xylanase activity of the pSc048-X33 strain reached the highest value of 1400 U / mL after 72 h of induction and expression.

[0064] Example 3: Induction of recombinant strain pSc048-X33 in a 5L fermenter

[0065] Four single clones were selected and inoculated into four 50 mL bottles of YPD medium (20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone) and cultured at 30 °C and 200 rpm for 18 h. The clones were then transferred at a 10% inoculum to a 5 L fermenter containing 1800 mL of BSM medium (85% H3PO4 26.7 mL / L, CaSO4 0.93 g / L, K2SO4 18.2 g / L, MgSO4·7H2O 14.9 g / L, KOH 4.13 g / L, glucose 20 g / L, PTM1 4.4 mL / L). The fermentation parameters were: temperature 30 °C, stirring speed 300 rpm, aeration rate 1 vvm, pH 5.0. After the fermenter was fully incubated with glucose, 70% glucose containing 12 mL / L PTM1 (CuSO4·5H2O 6 g / L, KI 0.08 g / L, MnSO4·H2O 3 g / L, MoNa2O4 0.2 g / L, H3BO3 0.02 g / L, CoCl2 0.5 g / L, ZnCl2 20 g / L, FeSO4·7H2O 65 g / L, Biotin 0.2 g / L, H2SO4 5 mL / L) was added to the fermenter in an exponential feeding manner. The feed volumes at different times are shown in the table below:

[0066] After feeding was completed and the bacterial OD600 reached 200, induction expression was initiated. Methanol containing 12 mL / L PTM1 was added, maintaining the methanol concentration in the fermenter at 2.4 g / L. The methanol concentration was added via an online methanol feed controller. Induction expression lasted 108 hours. OD600 was measured periodically during this period. 600 Protein concentration and xylanase activity. The results are shown in Figure 3. After 108 h of induction, OD... 600 The concentration of protein in the supernatant reached 10.2 g / L, and the enzyme activity in the supernatant reached 45100 U / mL. The fermentation broth was analyzed by SDS-PAGE protein electrophoresis, and the results are shown in Figure 4. Due to differences in glycosylation, Xyn2A showed two distinct bands on the SDS-PAGE image, at 22 kDa and 18 kDa, respectively. Time-of-flight mass spectrometry (TOF-MS) was performed on both bands and compared using Mascot, and the results are shown in Figure 5. Both bands were Xyn2A, and the molecular weight difference was due to partial glycosylation of Xyn2A.

[0067] Example 4: Direct hydrolysis of corn cob using fermentation supernatant

[0068] Corn cob (cellulose 39.5%, hemicellulose 33.9%, lignin 10.7%) was cut into 10 mm length, put into a high-speed grinder for grinding, and the powder was passed through a 20 mesh screen. 200 g of the above 20 mesh corn cob was weighed into a 5 L beaker, 2% KOH solution was added according to a solid-liquid ratio of 1:10, and soaked at 25°C for 24 h. An appropriate amount of 5% dilute sulfuric acid was added for neutralization, and the liquid was filtered through 8 layers of gauze. The pretreated corn cob was washed and collected, dried at 70°C, and weighed as 139.2 g. The contents of cellulose, hemicellulose and lignin in the pretreated corn cob were determined as 57.9%, 33.4% and 3.6%, respectively. 100 g of the pretreated corn cob was taken and put into an enzymatic hydrolysis tank, 10 mL of xylanase-containing fermentation supernatant was added according to a solid-liquid ratio of 1:10, and pH 5.0, 50 mM sodium acetate buffer was added at the same time. The stirring speed was set to 200 rpm, the temperature was 50°C, and the reaction was carried out for 12 h. After the reaction was completed, the reducing sugar content in the enzymatic hydrolysate was 20.2 mg / mL. The enzymatic hydrolysate was filtered, and the mass of the dried residue was 73.4 g, of which the contents of cellulose, hemicellulose and lignin were 77.2%, 8.1% and 4.3%, respectively. The degradation rate of hemicellulose in the pretreated corn cob after enzymatic hydrolysis was 82.2%.

[0069] Example 5 Direct use of fermentation supernatant to hydrolyze rape straw

[0070] Rape straw (cellulose 40.2%, hemicellulose 22.3%, lignin 16.2%) was cut into 10 mm length, put into a high-speed grinder for grinding, and the powder was passed through a 60 mesh screen. 200 g of the above 60 mesh rape straw was weighed into a 5 L beaker, 1% KOH solution was added according to a solid-liquid ratio of 1:10, and soaked at 25°C for 36 h. An appropriate amount of 5% dilute sulfuric acid was added for neutralization, and the liquid was filtered through 8 layers of gauze. The pretreated rape straw was washed and collected, dried at 70°C, and weighed as 148.3 g. The contents of cellulose, hemicellulose and lignin in the pretreated rape straw were determined as 46.5%, 28.5% and 17.1%, respectively. 100 g of the pretreated rape straw was taken and put into an enzymatic hydrolysis tank, 10 mL of xylanase-containing fermentation supernatant was added according to a solid-liquid ratio of 1:10, and pH 5.0, 50 mM sodium acetate buffer was added at the same time. The stirring speed was set to 200 rpm, the temperature was 50°C, and the reaction was carried out for 12 h. After the reaction was completed, the reducing sugar content in the enzymatic hydrolysate was 16.2 mg / mL. The mass of the dried residue after the enzymatic hydrolysate was filtered was 78.7 g, of which the contents of cellulose, hemicellulose and lignin were 55.5%, 15.5% and 20.2%, respectively. The degradation rate of hemicellulose in the pretreated rape straw after enzymatic hydrolysis was 57.2%.

[0071] Example 6 Direct use of fermentation supernatant to hydrolyze bagasse

[0072] Bagsass (cellulose 53.5%, hemicellulose 19.5%, lignin 16.2%) was put into a high-speed pulverizer for pulverization, and the powder was passed through a 20-mesh screen. 200 g of the above 20-mesh bagsass was weighed into a 5-L beaker, 2% KOH solution was added at a solid-liquid ratio of 1:10, and the mixture was soaked at 25°C for 24 h. An appropriate amount of 5% dilute sulfuric acid was added for neutralization, the liquid was filtered through 8 layers of gauze, the pretreated bagsass was washed and collected, and was dried at 70°C to a weight of 153.6 g. The contents of cellulose, hemicellulose and lignin in the pretreated bagsass were determined to be 54.5%, 15.1% and 15.8%, respectively. 100 g of the pretreated bagsass was weighed into an enzymatic hydrolysis tank, 10 mL of fermentation supernatant containing xylanase was added at a solid-liquid ratio of 1:15, pH 5.0 and 50 mM sodium acetate buffer was added at the same time, the stirring speed was set to 200 rpm, the temperature was set to 50°C, and the reaction was carried out for 12 h. After the reaction was completed, the content of reducing sugar in the enzymatic hydrolysate was 9.0 mg / mL. The mass of the filter residue after filtration of the enzymatic hydrolysate and drying was 76.6 g, and the contents of cellulose, hemicellulose and lignin were 50.1%, 7.2% and 10.1%, respectively. The degradation rate of hemicellulose in the pretreated bagsass after enzymatic hydrolysis was 63.2%.

[0073] Example 7 Direct use of fermentation supernatant to hydrolyze cotton seed hulls

[0074] Cotton seed hulls (cellulose 36.6%, hemicellulose 27.2%, lignin 19.5%) were put into a high-speed pulverizer for pulverization, and the powder was passed through a 20-mesh screen. 200 g of the above 20-mesh cotton seed hulls was weighed into a 5-L beaker, 1% KOH solution was added at a solid-liquid ratio of 1:10, and the mixture was soaked at 25°C for 24 h. An appropriate amount of 5% dilute sulfuric acid was added for neutralization, the liquid was filtered through 8 layers of gauze, the pretreated cotton seed hulls was washed and collected, and was dried at 70°C to a weight of 163.2 g. The contents of cellulose, hemicellulose and lignin in the pretreated cotton seed hulls were determined to be 43.5%, 29.8% and 17.0%, respectively. 100 g of the pretreated cotton seed hulls was weighed into an enzymatic hydrolysis tank, 10 mL of fermentation supernatant containing xylanase was added at a solid-liquid ratio of 1:10, pH 5.0 and 50 mM sodium acetate buffer was added at the same time, the stirring speed was set to 200 rpm, the temperature was set to 50°C, and the reaction was carried out for 12 h. After the reaction was completed, the content of reducing sugar in the enzymatic hydrolysate was 31.1 mg / mL. The mass of the filter residue after filtration of the enzymatic hydrolysate and drying was 64.5 g, and the contents of cellulose, hemicellulose and lignin were 56.3%, 7.5% and 26.2%, respectively. The degradation rate of hemicellulose in the pretreated cotton seed hulls after enzymatic hydrolysis was 83.8%.

[0075] Example 8 Foliar spraying of cucumber seedlings with hydrolyzed sugar solution

[0076] The hydrolysis obtained sugar solution is diluted appropriately to make the reducing sugar concentration reach 2.4 mg / mL, as a spraying sugar solution. The cucumber seedlings (variety: Lu Feng) in the three-leaf stage are transplanted into 300 g of substrate soil, and the cucumber seedlings are sprayed with the sugar solution. Each seedling is sprayed with 5 mL, and spraying is performed once every 4 days, for a total of three times, with deionized water as a control. After three times of spraying, the phenotype is shown in Figure 1, and compared with the control group, the plant height, root length, aboveground fresh weight, and root fresh weight of the treatment group are increased by 32%, 28%, 34%, and 60%, respectively. The physiological indexes are shown in Figure 2, and compared with the control group, the content of root growth hormone IAA of the treatment group is increased by 45%, the leaf photosynthesis rate is increased by 51%, and the leaf intercellular carbon dioxide concentration is decreased by 7%.

[0077] Example 9: Leaf spraying of tomato seedlings with hydrolysis sugar solution

[0078] The hydrolysis obtained sugar solution is diluted appropriately to make the reducing sugar concentration reach 0.1 mg / mL, as a spraying sugar solution. The tomato seedlings (variety: Micro Tom) in the three-leaf stage are transplanted into 300 g of substrate soil, and the cucumber seedlings are sprayed with the sugar solution. Each seedling is sprayed with 5 mL, and spraying is performed once every 4 days, for a total of three times, with deionized water as a control. After three times of spraying, the phenotype is shown in Figure 3, and compared with the control group, the plant height, root length, aboveground fresh weight, and root fresh weight of the treatment group are increased by 34%, 52%, 49%, and 120%, respectively. The physiological indexes are shown in Figure 4, and compared with the control group, the content of root growth hormone IAA of the treatment group is increased by 40%, the leaf photosynthesis rate is increased by 80%, and the leaf intercellular carbon dioxide concentration is decreased by 11%. The protection content of the present application is not limited to the above examples. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and the appended claims are protected.

Claims

1. A recombinant plasmid pSc048, wherein the recombinant plasmid Sc048 is constructed by the following method: 1) The Xyn2A expression cassette, which contains the FMD promoter, αMF* signal peptide, Xyn2A gene and AOX1 terminator, was constructed by cloning in Kinmen. The expression cassette was loaded into the backbone plasmid pBK to construct plasmid pSc026. 2) Construct the Hac1 expression cassette, which contains the FBA2 promoter, the Hac1 gene and the DAS1 terminator, by cloning in Kinmen. Load the expression cassette into the backbone plasmid pBK to construct plasmid pSc035. 3) The pSc026 plasmid was digested and ligated using the biobrick method to construct the recombinant plasmid pSc038 containing 2 copies of the Xyn2A expression cassette; 4) pSc038 and pSc035 were digested and ligated using the biobrick method to construct a three-expression-frame recombinant plasmid pSc048 containing 2 copies of the Xyn2A expression cassette and 1 copy of the Hac1 expression cassette.

2. The recombinant plasmid pSc048 according to claim 1, characterized in that, The nucleotide sequence of the Xyn2A gene is shown in SEQ ID NO:11; Preferably, the nucleotide sequence of the FMD promoter is shown in SEQ ID NO:3; Preferably, the nucleotide sequence of the αMF* signal peptide is shown in SEQ ID NO:9; Preferably, the nucleotide sequence of the AOX1 terminator is shown in SEQ ID NO:

17.

3. The recombinant plasmid pSc048 according to claim 1, characterized in that, The nucleotide sequence of the Hac1 gene is shown in SEQ ID NO:14; Preferably, the nucleotide sequence of the FBA2 promoter is shown in SEQ ID NO:6; Preferably, the nucleotide sequence of the DAS1 terminator is shown in SEQ ID NO:

19.

4. A genetically engineered bacterium that efficiently expresses xylanase, wherein the genetically engineered bacterium contains the recombinant plasmid pSc048 as described in any one of claims 1-3.

5. The method for constructing genetically engineered bacteria according to claim 4, characterized in that, The construction method includes the following steps: Step 1: Construct the recombinant plasmid pSc048 as described in any one of claims 1-3; Step 2: Transform the pSc048 obtained in Step 1 into Pichia pastoris X33, and screen positive transformants using bleomycin plates.

6. A method for producing xylanase by fermentation using the recombinant engineered bacteria of claim 4, the method comprising the following steps: using BSM as the fermentation medium, inoculating the recombinant engineered bacteria of claim 4 at an inoculum size of 10%; after the glucose in the medium is consumed, adding 70% glucose containing 12 mL / L PTM1 via exponential feeding; and culturing the bacteria to OD200. 600 Once the concentration reaches 200, xylanase expression is induced by adding methanol containing 12 mL / L PTM1, maintaining the methanol concentration at 2.4 g / L. Preferably, the BSM culture medium has the following composition: 85% H3PO4 26.7 mL / L, CaSO4 0.93 g / L, K2SO4 18.2 g / L, MgSO4·7H2O 14.9 g / L, KOH 4.13 g / L, glucose 20 g / L, PTM1 4.4 mL / L; Preferably, the PTM1 composition is as follows: CuSO4·5H2O 6g / L, KI 0.08g / L, MnSO4·H2O 3g / L, MoNa2O4 0.2g / L, H3BO3 0.02g / L, CoCl2 0.5g / L, ZnCl2 20g / L, FeSO4·7H2O 65g / L, Biotin 0.2g / L, H2SO4 5mL / L.

7. The xylanase prepared by the method of claim 6.

8. The application of the recombinant plasmid pSc048 according to any one of claims 1-3, the genetically engineered bacteria according to claim 4, the construction method according to claim 5, and the xylanase according to claim 7 in the degradation of straw-type agricultural waste.

9. The application according to claim 8, characterized in that, The agricultural waste consisting of straw includes corn cobs, sugarcane bagasse, cottonseed hulls, or rapeseed straw.

10. The application according to claim 8, characterized in that, The application includes the following specific steps: adding lignocellulose to sodium acetate buffer at pH 5.0, then adding the xylanase described in claim 7, and reacting at 50°C.

11. The application of the sugar solution produced according to any one of claims 8-10 in promoting plant growth, preferably, the plant is cucumber or tomato.

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