A process for returning straw fermentation to fields

Abstract

The present invention relates to the field of straw fermentation technology, and in particular to a process for returning sugarcane straw to the field by fermenting sugarcane straw, which can not only improve the sensory evaluation of sugarcane straw fermentation, but also improve the degradation efficiency of cellulose and hemicellulose in the straw and the fermentation efficiency of sugarcane straw by constructing CotA mutant engineering bacteria to ferment sugarcane straw, and has a good application prospect in returning sugarcane straw fermentation to the field.

Description

Technical Field

[0001] The invention relates to the technical field of straw fermentation, and in particular to a process for returning straw fermentation to fields. Background Art

[0002] Returning straw to the field is an effective measure to improve the comprehensive utilization rate of straw resources, balance the farmland ecosystem and achieve sustainable agricultural development. However, since most of the components of sugarcane straw are lignin and cellulose, the hydrogen bonds between and within molecules are the key issues that limit the hydrolysis of these components, making it difficult to degrade sugarcane straw after returning it to the field. At the same time, a large amount of residual unrotten straw will compete with crops for nitrogen nutrition during the decomposition process, seriously affecting the growth and development of the next crop. Therefore, screening sugarcane straw degrading bacteria is of great significance for the efficient utilization of sugarcane straw returning to the field in agricultural production. Studies have shown that adding microbial agents that are beneficial to straw decomposition can effectively promote straw decomposition. After adding exogenous microorganisms, the number of straw microorganisms can be increased under suitable conditions, which can accelerate the decomposition of straw to a certain extent and improve the utilization rate of straw returning to the field. Summary of the invention

[0003] The Lactococcus lactis NZ9000 competent cells used in the present invention were used for plasmid construction, and the lactic acid bacteria starter used in the experiment was purchased from Beijing Chuanxiu Technology Co., Ltd.

[0004] The medium for screening recombinant bacteria was LA solid medium: 1% peptone, 0.5% yeast extract, 1% sodium chloride, 1.5% agar powder, 37 °C for recombinant bacteria culture. Add 100 μg mL -1 of ampicillin.

[0005] The culture medium for the activated lactic acid bacteria preparation is liquid LB medium: 1 g of peptone, 0.5 g of yeast extract, 1 g of sodium chloride, and 100 mL of distilled water.

[0006] LB medium and sucrose medium were sterilized in an autoclave at 121°C for 30 min, and YPD medium was sterilized in an autoclave at 108°C for 20 min. They were all placed on a sterile operating table for ultraviolet sterilization for 30 min before inoculation.

[0007] The first object of the present invention is to provide an engineered bacterium for straw degradation, wherein the engineered bacterium is introduced with bacterial laccase CotA or a mutant thereof, and a recombinant Lactococcus lactis having the ability to secrete laccase is constructed.

[0008] The amino acid sequence of the CotA enzyme is shown in SEQ ID NO:1.

[0009] The codon-optimized nucleotide sequence of the CotA enzyme is shown in SEQ ID NO: 2, and the nucleotide sequence further comprises a usp45 signal peptide and a His tag.

[0010] Preferably, a mutant of bacterial laccase CotA is introduced into the engineered bacteria, which has one or more amino acid mutations, and can increase the decomposition rate of cellulose and hemicellulose to a certain extent.

[0011] The preferred percentage increase in the cellulose decomposition rate is 5% or more, or 10% or more, or 20% or more.

[0012] The preferred increase percentage of the hemicellulose decomposition rate is 5% or more, or 10% or more, or 20% or more.

[0013] Preferred CotA laccase mutation sites are one or more of H140Q, P241A, and V276D.

[0014] A more preferred mutation site is H140Q.

[0015] Preferably, the amino acid sequence of the mutated CotA enzyme is shown in SEQ ID NO: 3-5.

[0016] A more preferred amino acid sequence of the mutated CotA enzyme is shown in SEQ ID NO:3.

[0017] Another object of the present invention is to provide a method for constructing the above-mentioned engineered bacteria, which comprises the following steps:

[0018] (1) Design of the CotA laccase gene of Bacillus subtilis

[0019] The Bacillus subtilis CotA laccase gene sequence was obtained through GenBank, and the codons of the lactic acid bacteria expression system were optimized. The optimized CotA gene sequence was obtained by PCR, and the primers of the restriction site were designed using Primer 5.0 software. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0020] The preferred restriction sites are SnaB I and Eco81 I.

[0021] (2) PCR amplification of CotA laccase gene and construction of recombinant plasmid

[0022] Using pPIC9K-CotA as a template, PCR amplification was performed to obtain the target gene CotA. The original plasmid 368-PGK was digested with two restriction endonucleases, SnaBI and Eco81 I, to expose the sticky ends at both ends of the vector. The CotA amplified by PCR was also digested with SnaBI and Eco81 I and recovered.

[0023] The CotA recovered after restriction digestion and the vector pHBM-368-PGK fragment were mixed and reacted at 16°C overnight using T4 DNA ligase.

[0024] The recombinant plasmid can be identified by conventional methods, such as gel electrophoresis or sequencing after PCR amplification.

[0025] (3) Screening of positive transformants

[0026] The ligation product was transformed conventionally using Lactococcus lactis NZ9000 competent bacteria as the host, spread on LB solid medium containing ampicillin, cultured at 37°C overnight, and a single recombinant colony was picked.

[0027] The third object of the present invention is to provide a process for returning straw to the field by fermentation, which comprises returning straw to the field by fermentation of genetically engineered bacteria, wherein the genetically engineered bacteria are introduced with an expression vector of the CotA enzyme or its variant, and the CotA enzyme or its variant significantly improves the ability of the engineered bacteria to degrade cellulose and hemicellulose in the straw.

[0028] The straw returning process comprises the following steps:

[0029] 1) Dry the sugarcane stalks and crush them into small pieces of 5-10 cm;

[0030] 2) Put the straw into the reaction tank and add an appropriate amount of genetically engineered bacteria;

[0031] 3) Add water to cover the straw, and return it to the field after a period of reaction.

[0032] The concentration of the engineered bacteria was 1-5×10 9 / mL; preferably 2×10 9 Pieces / mL.

[0033] The added ratio of the engineered bacteria is 0.1-0.5% of the volume of the straw, preferably 0.2%.

[0034] The process of the invention can not only improve the sensory evaluation of sugarcane straw fermentation, but also improve the degradation efficiency of nitrogen, cellulose and hemicellulose in the straw, and has a good application prospect in returning straw fermentation to the field. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 : Schematic diagram of laccase expression vector pHBM-368-PGK-CotA;

[0036] Figure 2 : Enzyme activities of wild type and mutant CotA enzymes;

[0037] Figure 3: Ammonium nitrogen content after straw fermentation;

[0038] Figure 4 : Cellulose content of straw after fermentation;

[0039] Figure 5 : Hemicellulose content after straw fermentation. DETAILED DESCRIPTION

[0040] Example 1 Design of CotA laccase gene and construction of recombinant cloning vector

[0041] The mature peptide of CotA laccase from Bacillus subtilis was obtained through GenBank (accession number: FJ663050:1), and the codons of the lactic acid bacteria expression system were optimized. Primers with restriction sites were designed using Primer 5.0 software, and the restriction sites were SnaB I and Eco81 I. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the optimized CotA gene sequence was obtained by PCR. The specific sequence is shown in SEQ ID NO:2.

[0042] Using pPIC9K-CotA as a template, PCR amplification was performed to obtain the target gene CotA. The original plasmid 368-PGK was digested with two restriction endonucleases, SnaBI and Eco81 I, to expose the sticky ends at both ends of the vector. The CotA amplified by PCR was also digested with SnaBI and Eco81 I and recovered.

[0043] The CotA recovered after enzyme digestion and the vector pHBM-368-PGK fragment were mixed and reacted overnight at 16°C using T4 DNA ligase. Figure 1 shown.

[0044] Example 2 Construction of CotA enzyme mutants and recombinant strains

[0045] According to the amino acid map, a group of amino acids that may affect the spatial structure of the CotA enzyme were screened and mutated. The variants were H140Q, P241A, and V276D. The nucleotide sequences of muH140Q, muP241A, and muV276D were obtained by site-directed mutagenesis. The wt and its variants were successfully cloned into the plasmid pHBM-368-PGK, respectively. The successfully constructed recombinant plasmids were transformed into Lactococcus lactis NZ9000 competent cells, recorded as Lab-pHBM-368-PGK-CotA-WT and Lab-pHBM-368-PGK-CotA-muH140Q, Lab-pHBM-368-PGK-CotA-muP241A, and Lab-pHBM-368-PGK-CotA-muV276D. The recombinant single colony was picked, cultured in the culture medium, and the colony PCR was performed for identification. After restriction digestion identification, sequencing verification was performed, and the wild-type and mutant engineering bacteria were selected for preservation.

[0046] Example 3 CotA expression in recombinant engineered bacteria

[0047] Three strains of each type were selected and cultured in liquid LB medium (containing 5 μg mL -1 The culture was cultured at 30°C overnight, and the next day, 5% of the inoculum was inoculated into 100 mL of LB liquid medium (containing 5 μg mL -1 Erythromycin), 30°C, static culture to OD600 = 0.5, add 100 μL of 0.3M CuSO4 that has been sterilized by filtration in an ultra-clean workbench, 30°C, static culture for 4 hours, and then culture at 16°C for 24 hours.

[0048] Take 90 μL of the supernatant of the recombinant strain cell wall breaking solution, mix with 30 μL of 4×Protein Loading Buffer, boil at 100℃ for 10 min, centrifuge at 4℃, 12,000 rpm for 5 min, collect the sample processing solution, and detect the enzyme activity using the commonly used colorimetric method.

[0049] like Figure 2As shown, the average extracellular enzyme activities of Lab-pHBM-368-PGK-CotA-WT, Lab-pHBM-368-PGK-CotA-muH140Q, Lab-pHBM-368-PGK-CotA-muP241A, and Lab-pHBM-368-PGK-CotA-muV276D were 98.6U / L, 135.7U / L, 13.2U / L, and 90.5U / L, respectively. The results of the extracellular enzyme activity assay of laccase showed that the recombinant Lactococcus lactis successfully achieved the secretory expression of CotA laccase, and compared with the wild type, the enzyme activity of muH140Q was greatly improved, while muP241A almost caused the enzyme activity to be lost, and the enzyme activity of muV276D was basically equivalent to that of the wild type.

[0050] Example 4 Recombinant engineering bacteria straw fermentation

[0051] Pour the dried sugarcane straw into a 1000 mL beaker, mix the bacterial solution with water according to the amount in Table 1, pour into a spray pot, spray evenly on the straw, and stir continuously until the spraying is completed. After standing for 15 min, the straw can fully absorb the bacterial solution. Pour the treated sample into a vacuum packaging bag, seal it with a vacuum sealer, and ferment it in a constant temperature incubator at 28 ℃ for 14 days.

[0052] Table 1 Sugarcane straw fermentation treatment system

[0053] project A B C D Fermentation bacteria Engineering bacteria A Engineering bacteria B Engineering bacteria C Engineering bacteria D Sugarcane straw addition amount / g 50 50 50 50 Bacterial liquid addition amount / mL 50 50 50 50 Sterile water addition / mL 100 100 100 100

[0054] Note: Engineered bacteria A is Lab-pHBM-368-PGK-CotA-W, engineered bacteria B is Lab-pHBM-368-PGK-CotA-muH140Q, engineered bacteria C is Lab-pHBM-368-PGK-CotA-muP241A, and engineered bacteria D is Lab-pHBM-368-PGK-CotA-muV276D

[0055] Example 5 Evaluation of fermentation effect

[0056] Sensory evaluation: The four indicators of smell, color, texture and shape were used for comprehensive evaluation, with 1 point for the worst effect and 5 points for the best effect. The sensory evaluation standards for fermented sugarcane are shown in Table 2.

[0057] Ammonium nitrogen (NH + 4-N) Content: The liquid component was obtained by sulfuric acid digestion and determined by phenol-sodium hypochlorite colorimetry.

[0058] Cellulose content: The liquid component was obtained by hydrolysis using the National Renewable Energy Laboratory (NREL) method and measured using the anthrone-sulfuric acid colorimetric method.

[0059] Hemicellulose content: After calcium nitrate pretreatment, 2 mol·L -1 The liquid component was obtained by hydrochloric acid hydrolysis and the pH value was determined by DNS method.

[0060] SAS software was used to perform variance analysis on the data (LSD method, α=0.05)

[0061] Table 2 Sensory evaluation criteria for fermented sugarcane straw

[0062] Evaluation indicators 1.0 points 2.0 points 3.0 points 4.0 points 5.0 points odor Corruption smell Rancidity sour Sour aroma Sweet and sour flavor Color Other colors brown tan yellow Bright yellow texture Agglomeration Sticky lumps Slightly sticky Loose Loose and soft Stem and leaf morphology Severe decay rot Slight decay Structural damage Well preserved

[0063] Table 3 Sensory evaluation of straw fermentation

[0064] Experimental Group odor Color texture form Average score Sensory level A 3.0 3.0 3.0 3.0 3.0 medium B 5.0 4.0 4.0 5.0 4.5 high quality C 2.0 2.0 4.0 1.0 2.25 Difference D 3.0 4.0 3.0 3.0 3.25 medium

[0065] Through evaluation, it was found that the sugarcane straw fermented by mutant engineered bacteria D had significant improvements in odor and morphology compared with those fermented by other strains. In comparison, the sour aroma was moderate and had a light wine aroma. The degree of erosion in morphology was reduced and the agglomeration phenomenon was reduced. The comprehensive evaluation results are shown in Table 3. The sugarcane straw fermented by engineered bacteria B was generally better than other treatment combinations, and the fermentation sensory properties of engineered bacteria D were close to those of wild-type engineered bacteria A.

[0066] NH + 4-N content is an important indicator for measuring the effect of straw fermentation. It is produced by the decomposition of protein in straw by spoilage bacteria. + The 4-N content can reflect the effect of different fermentation additives on the fermentation treatment of sugarcane straw. Figure 3 As shown, the fermentation of engineered bacteria B caused NH + The 4-N content was significantly increased compared with other groups (P<0.01), and its NH + The 4-N content was 45.69 mg / g. That is, the fermentation degree of engineered bacteria B on sugarcane straw was better than that of other groups, and it could effectively increase the protein nitrogen content of straw.

[0067] At the same time, the effect of the engineered bacteria group D is basically equivalent to that of the wild-type engineered bacteria A.

[0068] The ratio of hemicellulose to cellulose is an important indicator for measuring the degree of straw fermentation. Figure 4 , Figure 5 As shown in the figure, the contents of cellulose and hemicellulose in the straw fermented by engineered bacteria B were significantly lower than those in other groups (P<0.01), indicating that the straw fermented by it can effectively improve the degradation efficiency of cellulose and hemicellulose in the straw and improve the nutrient utilization value of the straw returned to the field.

[0069] At the same time, the effect of the engineered bacteria group D is basically equivalent to that of the wild-type engineered bacteria A.

[0070] Therefore, the engineered bacteria B and D constructed with CotA mutants have good application prospects in returning straw to the fields.

[0071] Example 6 Returning straw to the field

[0072] After drying the sugarcane stalks, crush them into small pieces of 5-10 cm, put them into a reaction tank, and add an appropriate amount of the genetically engineered bacteria activated in Example 4, the added ratio of the engineered bacteria being 0.1-0.5% of the volume of the stalks. Add water to submerge the stalks, and return them to the field after a period of reaction.

[0073] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A method for returning straw to the field by fermentation, wherein the method comprises returning the straw to the field by fermentation with genetically engineered bacteria, wherein the genetically engineered bacteria comprises an expression vector of a CotA enzyme mutant, wherein the CotA enzyme mutant significantly improves the ability of the engineered bacteria to degrade cellulose and hemicellulose in the straw, and the amino acid sequence of the CotA enzyme variant is shown in SEQ NO ID: 3; The method can not only improve the sensory evaluation of sugarcane straw fermentation, but also improve the degradation efficiency of nitrogen, cellulose and hemicellulose in the straw.

2. The method according to claim 1, characterized in that The straw returning to the field comprises the following steps: 1) Dry the sugarcane stalks and crush them into small pieces of 5-10 cm; 2) Put the straw into the reaction tank and add an appropriate amount of genetically engineered bacteria; 3) Add water to cover the straw, and return it to the field after a period of reaction.

3. The method according to claim 2, wherein in step 2), the amount of engineered bacteria added is 0.1-0.5% of the volume of the straw.

4. The method according to claim 2, wherein the concentration of the engineered bacteria in step 2) is 1-5×10 9 Pieces / mL.

5. The method according to claim 2, wherein the reaction time in step 3) is 10-15 days.

6. A bacterial agent for returning straw fermentation to fields, comprising the genetically engineered bacteria described in any one of claims 1 to 5.

7. Use of the microbial agent according to claim 6 in returning straw fermentation to fields.

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