Construction and application of xylan feruloyl esterase delivery plasmid

Abstract

The invention belongs to the technical field of genetic engineering and biology, and particularly discloses construction and application of a xylan feruloyl esterase delivery plasmid. The amino acid sequence of the xylan feruloyl esterase is shown as SEQ ID NO: 1, a xylan feruloyl esterase delivery plasmid pQ-XFE based on IncQ type plasmid and a recombinant genetic engineering bacterium are constructed, and thus the xylan feruloyl esterase is obtained. According to the delivery plasmid, efficient and stable delivery of xylan feruloyl esterase genes in various microorganisms is achieved, the enzyme expression quantity and activity can be remarkably improved, and the application range of the xylan feruloyl esterase genes is widened. The recombinant xylan feruloyl esterase has a remarkable degradation effect on ester compounds and can be applied to the fields of food, pharmacy, papermaking, feed and the like due to the specific activity and enzymatic characteristics of the recombinant xylan feruloyl esterase.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and biotechnology, and in particular relates to the construction and application of a xylan ferulic acid esterase delivery plasmid. Background Art

[0002] Xylan ferulate esterase (XFE), a unique bifunctional enzyme, demonstrates tremendous potential for application in numerous fields. In the feed industry, its xylanase activity effectively degrades anti-nutritional factors such as xylans in feed, significantly improving the nutritional value and utilization rate of feed. Furthermore, the ferulic acid released by ferulic acid activity possesses antioxidant and antimicrobial properties, effectively promoting animal growth and health and reducing disease incidence. The resulting xylooligosaccharides also optimize the balance of the animal's intestinal microbiome and improve intestinal function. In the food industry, when used in flour processing, this enzyme works synergistically with hemicellulases to reduce dough viscosity, improve rheological properties, and enhance baking quality. In fruit and vegetable processing, it aids in cell wall degradation, increasing juice yield and clarity. The released ferulic acid acts as a natural antioxidant, extending food shelf life. In the biomass energy sector, it synergizes with cellulases in lignocellulose pretreatment, enhancing degradation, improving biomass fermentability, and increasing bioethanol production. In the pulp and paper industry, it can help degrade xylan and ferulic acid ester bonds in plant fiber raw materials, making fiber separation and refining easier, reducing the use of chemical agents, reducing environmental pollution, and improving paper quality and performance.

[0003] However, the low expression levels and limited enzyme activity of xylan ferulic acid esterases produced by microorganisms in nature have greatly restricted their large-scale industrial application. For example, in actual feed production, insufficient enzyme quantity and poor activity make it difficult to fully degrade anti-nutritional factors, making it impossible to achieve the desired animal growth promotion effect and improve feed utilization. In food processing, low enzyme activity leads to low processing efficiency and limited product quality improvement. In the biomass energy production process, poor enzymatic hydrolysis directly affects the efficiency of bioenergy conversion. In the pulping and papermaking process, insufficient enzyme action leads to difficulties in fiber separation, high dependence on chemical agents, and prominent environmental pollution problems.

[0004] While a variety of plasmids are currently available for delivering functional genes in genetic engineering, specialized plasmids for delivering xylan feruloyl esterase have numerous drawbacks. Some plasmids have low copy numbers within their host bacteria, resulting in enzyme expression levels that are insufficient to meet industrial needs. Furthermore, many plasmids are highly selective for their host bacteria and have a narrow scope of application, making it difficult to effectively deliver the xylan feruloyl esterase gene in a variety of microorganisms. This limits their flexibility and universality in diverse environmental remediation and industrial production systems. Summary of the Invention

[0005] In response to the shortcomings of the current existing technology, the present invention aims to construct a xylan feruloyl esterase delivery plasmid based on the IncQ-type plasmid ubiquitous host, which not only achieves efficient and stable delivery of the xylan feruloyl esterase gene in a variety of microorganisms, but also significantly improves the expression level and activity of the enzyme, expands its application range, and provides strong biotechnology support for many industries such as feed, food, biomass energy, pulp and paper, and environmental remediation, thereby promoting industrial technology upgrading and sustainable development.

[0006] In a first aspect, the present invention provides a xylan feruloyl esterase, the amino acid sequence of the xylan feruloyl esterase is shown in SEQ ID NO: 1.

[0007] In a second aspect, the present invention provides a gene encoding the above-mentioned xylan feruloyl esterase, the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 2.

[0008] The expression cassette, recombinant vector, transgenic cell line and transgenic recombinant bacteria containing the above-mentioned xylan ferulic acid esterase gene are also within the protection scope of the present invention.

[0009] In a third aspect, the present invention provides a xylan feruloyl esterase delivery plasmid pQ-XFE based on an IncQ type plasmid, wherein the plasmid carries the nucleotide sequence encoding the xylan feruloyl esterase gene.

[0010] In a fourth aspect, the present invention provides a method for constructing a xylan ferulic acid esterase delivery plasmid based on an IncQ-type plasmid, the construction method specifically comprising the following steps:

[0011] S1. Obtain IncQ plasmid backbone: Use pQ-SacB-araC plasmid as template to design primers for PCR amplification to obtain the amplified product;

[0012] S2. Obtaining the gene encoding xylan feruloyl esterase: Extracting genomic DNA from the F5 strain, using it as a template, and performing PCR amplification with primers to obtain the target gene; the primers are

[0013] Primer F2 sequence: 5`-TCGCAACTCTCTACTGTTTCTCCATGAGGAGAAATACTAGCCCCC-3`,

[0014] Primer R2 sequence: 5`-CTCTTCGTTTTACTAAAGCAATGATCTACTTGAAGAGCATTGGAGCC-3`;

[0015] S3. Fragment recombination and transformation: The two amplification products obtained in step S1 and step S2 were purified and recovered by agarose gel electrophoresis, and the fragments were recombined using a homologous recombination kit and transformed into E. coli Dh5α competent cells. After recovery, the cells were spread on a medium containing ampicillin.

[0016] S4. Verification and screening: Pick a single colony from the culture medium in step S3, perform colony PCR, sequence verification, and screen positive bacteria to obtain the xylan ferulic acid esterase delivery vector pQ-XFE.

[0017] Preferably, the primers in step S1 are:

[0018] Primer F1 sequence: 5`-ATGGAGAAACAGTAGAGAGTTGC-3`;

[0019] Primer R1 sequence: 5`-ATCATTGCTTTAGTAAAACGAAGAGG-3`.

[0020] Preferably, the transformation step in step S3 includes: taking 300-500 ng of the recombinant product and adding it to 100 μL E.coli Dh5α competent cells, gently placing it on ice for 30 minutes, then transferring the centrifuge tube to a 42°C constant temperature water bath for precise heat shock for 60 seconds, and quickly moving it back to ice to cool for 5 minutes after the heat shock.

[0021] Preferably, the recovery culture method in step S3 is as follows: after transformation into competent cells, add 1 mL of LB liquid culture medium, culture at 37°C and 220 rpm for 1 h, then spread on an LB agar plate containing 100 μg / mL ampicillin, and culture at 37°C for 24 h.

[0022] Preferably, the nucleotide sequence of the delivery vector pQ-XFE is shown in SEQ ID NO: 3.

[0023] In a fifth aspect, the present invention provides a recombinant engineered bacterium producing xylan ferulic acid esterase, wherein the recombinant genetically engineered bacterium is obtained by transforming the recombinant plasmid pQ-XFE into Escherichia coli Dh5α.

[0024] In a sixth aspect, the present invention provides an application of the above-mentioned engineered bacteria in degrading ester compounds.

[0025] Preferably, the degradation application uses tributyrin as a substrate and is performed using Escherichia coli carrying the delivery plasmid pQ-XFE. Specifically, the degradation process includes the following steps: first, a basal medium containing 2% tributyrin, 1.5% agar, and a pH of 7.0-7.5 is prepared to prepare a tributyrin plate; then, Bacillus subtilis and Escherichia coli carrying the recombinant plasmid pQ-XFE are activated to the logarithmic growth phase; a bacterial solution from the positive control group is plated with an inoculation loop; and 100 μL of the diluted bacterial solution from the XFE clone group is plated with the plate; finally, the plate is inverted and incubated at 37°C for 18 hours. If a relatively large transparent zone appears around the E. coli colonies carrying the recombinant plasmid pQ-XFE, it is determined that the strain has excellent esterase production ability and enhanced esterase activity.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) Significantly improve the expression level of xylan feruloyl esterase: The pQ-XFE delivery plasmid constructed by the present invention can effectively enhance the expression level of xylan feruloyl esterase in microorganisms and improve the enzyme activity by optimizing elements such as promoter and gene sequence, and has a significant effect in the application of ester compound degradation.

[0028] (2) Broad host range: The vector of the present invention is based on an IncQ-type plasmid and has a broad host range, including not only different types of Gram-negative bacteria, but also some Gram-positive bacteria such as Actinobacteria and Firmicutes. The rep region-related genes on the vector plasmid can perform replication functions in the above-mentioned strains, and can achieve gene delivery to a variety of host bacteria from Gram-negative bacteria to Gram-positive bacteria, and is suitable for microorganisms in different environments. The pQ-XFE constructed by the present invention can be used for the delivery of xylan ferulic acid esterase, widely spread in the microbial community, realize microbial community editing, and achieve the purpose of expressing esterase in the intestinal microbial community. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the hydrolysis effect of xylan ferulic acid esterase expressed by the pQ-XFE plasmid of the present invention.

[0030] Figure 2 This is an analysis of the results of Example 3 on the ability to decompose tributyrin. Figure A shows the blank control: containing only tributyrin; Figure B shows the empty group: composed of empty bacteria and tributyrin; Figure C shows the positive control: using Bacillus subtilis to decompose tributyrin; Figure D shows the XFE clone group sample: acting on tributyrin. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the examples of the present invention are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0033] Example 1

[0034] The construction of a xylan ferulic acid esterase delivery plasmid based on an IncQ-type plasmid ubiquitous host comprises the following steps:

[0035] (1) Obtaining IncQ plasmid backbone

[0036] Using the commercially available pQ-SacB-araC plasmid as a template, PCR amplification was performed using ingeniously designed primers in the mob and rep regions. After rigorous screening and optimization, primer sequences were identified as primers F1 and R1 to meet subsequent experimental requirements and ensure accurate and efficient amplification. The amplification system and conditions are shown in Tables 1 and 2. This precise process successfully yielded a 10,490 bp amplicon, with the mob and rep regions fully retaining their native functional properties, providing a robust framework supporting the plasmid's autonomous replication and transferability within the host bacteria.

[0037] The pQ-SacB-araC plasmid can be constructed according to conventional methods: using pQ-mini (the plasmid constructed earlier can refer to patent document CN 115109791 A) as the backbone, isolating the SacB gene from Bacillus subtilis as a negative selection marker, isolating the araC arabinose operon regulatory gene from Escherichia coli, and finally performing homologous recombination to form the pQ-SacB-araC plasmid.

[0038] The primers designed during the construction process are as follows:

[0039] Primer F1 sequence: 5`-ATGGAGAAACAGTAGAGAGTTGC-3`;

[0040] Primer R1 sequence: 5`-ATCATTGCTTTAGTAAAACGAAGAGG-3`;

[0041] The amplification system and conditions are as follows:

[0042] Table 1 High-fidelity PCR reaction system

[0043]

[0044] Table 2 Reaction conditions for high-fidelity enzyme PCR

[0045]

[0046]

[0047] (2) Obtaining the gene encoding xylan ferulic acid esterase

[0048] Genomic DNA was extracted from the F5 strain screened in the early stage for high production of xylan feruloyl esterase. Using this as a template, highly specific primers F2 and R2 were designed and synthesized for PCR amplification based on the unique sequence information of the xylan feruloyl esterase gene.

[0049] Primer F2 sequence:

[0050] 5`-TCGCAACTCTCTACTGTTTCTCCATGAGGAGAAATACTAGCCCCC-3`;

[0051] Primer R2 sequence:

[0052] 5`-CTCTTCGTTTTACTAAAGCAATGATCTACTTGAAGAGCATTGGAGCC-3`;

[0053] The reaction system remains the same as the previous amplification step, with strict control over the ratio and dosage of each component to ensure the stability and reproducibility of the amplification reaction. The amplified product is 1938 bp in size. The xylan feruloyl esterase (amino acid sequence shown in SEQ ID NO: 1) encoded by this gene (nucleotide sequence shown in SEQ ID NO: 2) has excellent catalytic activity and strict substrate specificity. It can accurately target and efficiently hydrolyze the glycosidic bonds and feruloyl ester bonds of the xylan backbone, laying a solid foundation for its core catalytic function for subsequent applications in various fields such as environmental remediation and industrial production.

[0054] (3) Fragment recombination to obtain pQ-XFE plasmid

[0055] Agarose gel electrophoresis was used to separate and purify the two amplified products. Specifically, a 1.0% TAE agarose gel was prepared, and the PCR amplified products were mixed with 10X loading buffer and spotted on the gel channels. The electrophoresis parameters were set to 100V and 88mA, and the electrophoresis was continued for 40 minutes to ensure that fragments of different sizes were effectively separated. Purification and recovery were performed using a gel recovery purification kit (Takara).

[0056] Subsequently, fragment recombination was performed using the One Step Cloning Kit for homologous recombination from Nanjing Novozymes Biotechnology Co., Ltd. The homologous recombination system configuration is shown in Table 3. The fragments were gently flicked to mix on ice, briefly centrifuged, and then incubated at 50°C for 30 minutes to ensure efficient and accurate recombination.

[0057] Table 3 Homologous recombination system

[0058]

[0059]

[0060] (4) Transformation and screening of recombinant plasmid pQ-XFE:

[0061] The recombinant product is transformed into E. coli Dh5α competent cells. These competent cells are prepared by a special process and have the ability to efficiently accept foreign DNA.

[0062] The transformation process is as follows: take 300-500 ng of the ligation product and add it to 100 μL E.coli Dh5α competent cells, gently place it on ice for 30 minutes to allow the recombinant plasmid to fully approach and adsorb on the surface of the competent cells; then transfer the centrifuge tube to a 42°C constant temperature water bath for precise heat shock for 60 seconds. This process promotes the formation of short-term, reversible holes in the cell membrane, helping the recombinant plasmid to enter the cell smoothly; after the heat shock, quickly move it back to ice and cool for 5 minutes to promote the closure of the cell membrane holes and restore integrity, thereby reducing cell damage and mortality.

[0063] Finally, add 1 mL of LB liquid medium and culture at 37°C and 220 rpm for 1 h to provide the cells with suitable nutrients and environmental conditions, promote their recovery and expression of the ampicillin resistance gene, and then spread on an LB agar plate containing 100 μg / mL ampicillin. After constant temperature culture at 37°C for 24 h, colonies containing the target plasmid can be stably grown and formed.

[0064] (5) Verification and screening of recombinant plasmid pQ-XFE

[0065] Single colonies were picked from the LB agar plate obtained by culture and colony PCR was performed using the designed primers F3 and R3.

[0066] Primer F3 sequence: 5`-CGATTCATTAATGCAGCTGGC-3`;

[0067] Primer R3 sequence: 5`-GGATCATTTTGCGCTTCAGC-3`;

[0068] Through this validation process, positive clones whose amplified bands matched expectations were selected and sent to a professional sequencing company for high-precision sequencing analysis. The sequencing results were carefully compared with the target xylan feruloyl esterase gene sequence. Only when the sequences were completely consistent could the recombinant plasmid construction be determined to be accurate and the xylan feruloyl esterase delivery vector pQ-XFE (nucleotide sequence see SEQ ID NO: 3) successfully obtained, laying a solid and reliable material foundation for subsequent widespread application and in-depth research.

[0069] At this point, the xylan ferulic acid esterase delivery plasmid pQ-XFE based on the IncQ type plasmid ubiquitous host was successfully constructed.

[0070] Example 2 Hydrolysis experiment of xylan ferulic acid esterase

[0071] Prepare LB agar plates containing 100 μg / mL of tributyrin and ampicillin, then dilute the bacterial solution containing the pQ-XFE plasmid (shaken to the mid-logarithmic growth phase in advance) to about 10², spread on the plates, and then incubate at 37°C for 18-22 hours. After a single colony grows, incubate at 4°C overnight and measure the hydrolysis ring diameter the next day.

[0072] The negative control was the empty vector strain pQ-mini; the experimental group was pQ-XFE (xylan feruloyl esterase).

[0073] The experimental results are shown in Figure 1 The pQ-XFE plasmid constructed in the present invention can effectively hydrolyze tributyrin and has esterase activity. This shows that the pQ-XFE plasmid constructed in the present invention can be used for esterase delivery, achieving the purpose of expressing esterase in intestinal flora.

[0074] Example 3

[0075] Application of Escherichia coli carrying recombinant plasmid pQ-XFE in the degradation of ester compounds

[0076] Experimental procedures

[0077] (1) Prepare the culture medium: Weigh 10 g / L of peptone, 5 g / L of yeast extract, and 3 g / L of beef extract and add them to distilled water. Heat and stir until dissolved to prepare the basal culture medium. Add tributyrin to the basal culture medium to a final concentration of 2%. Add 1.5% agar and continue heating and stirring until the agar is completely dissolved. Adjust the pH of the culture medium to 7.0-7.5. Dispense the culture medium into culture dishes, pouring an appropriate amount of culture medium (approximately 15-20 mL) into each dish. After cooling and solidification, the tributyrin plate is obtained.

[0078] (2) Inoculation of strains: Bacillus subtilis (which has been reported to be able to decompose tributyrin) and Escherichia coli carrying the recombinant plasmid pQ-XFE were activated and inoculated into liquid culture medium respectively. The culture medium was placed in a shaker at 37°C and 180 rpm for 6 h to reach the logarithmic growth phase.

[0079] For the positive control group, an appropriate amount of bacterial solution (Bacillus subtilis) was taken with an inoculating loop and evenly spread on a tributyrin plate. For the XFE clone group sample (E. coli carrying the recombinant plasmid pQ-XFE), 100 μL of the bacterial solution was evenly spread on the plate after diluting to an appropriate gradient.

[0080] (3) Cultivation and observation

[0081] Place the inoculated plate upside down in a constant-temperature incubator at 37°C for 18 hours. Observe the area surrounding the colonies. If the strain produces esterase, it will break down tributyrin, forming a clear zone around the colonies. The size of the clear zone can, to a certain extent, reflect the strain's esterase-producing capacity; larger clear zones indicate higher esterase activity.

[0082] Result Analysis

[0083] like Figure 2 As shown, Figure A shows the blank control: containing only tributyrin, which serves as a basic reference to exclude the influence of environmental factors on tributyrin. Figure B shows the empty group: composed of empty bacteria and tributyrin, used to determine that the empty bacteria themselves have no effect on tributyrin. Figure C shows the positive control: tributyrin is decomposed by esterase, and a transparent edge is formed around the colony indicated by the arrow, indicating that tributyrin is decomposed. Figure D shows the XFE clone group sample: after it acts on tributyrin, the recombinant plasmid pQ-XFE constructed by the present invention is introduced into Escherichia coli, and then the xylan ferulic acid esterase produced by the bacteria growing on the plate degrades tributyrin to form a transparent edge. Compared with the other groups, a larger transparent edge is formed around the colony, reflecting that the strain's ability to produce esterase is improved and the esterase activity is enhanced. This shows that the XFE clone group has better lipid decomposition ability and has potential application value.

[0084] Obviously, the specific implementation scheme described above is only a further detailed description of the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above is only a specific example of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A xylan ferulic acid esterase, characterized in that The amino acid sequence of the xylan feruloyl esterase is shown in SEQ ID NO:

1.

2. The gene encoding the xylan feruloyl esterase according to claim 1, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO:

2.

3. An expression cassette, a recombinant vector, a transgenic cell line and a transgenic recombinant bacterium containing the gene encoding xylan feruloyl esterase according to claim 2.

4. A xylan ferulic acid esterase delivery plasmid pQ-XFE based on IncQ type plasmid, characterized in that The plasmid carries the nucleotide sequence of the gene encoding xylan ferulic acid esterase according to claim 2.

5. The method for constructing the xylan ferulic acid esterase delivery plasmid pQ-XFE based on IncQ type plasmid according to claim 4, characterized in that: The steps include: S1. Using the pQ-SacB-araC plasmid as a template, primers were designed and PCR was performed to obtain an amplified product; the primer was F: 5'-ATGGAGAAACAGTAGAGAGTTGC-3'; R: 5`-ATCATTGCTTTAGTAAAACGAAGAGG-3`; S2. Genomic DNA was extracted from the F5 strain and used as a template to perform PCR amplification using primers to obtain the target gene; the primers were F: 5'-TCGCAACTCTCTACTGTTTCTCCATGAGGAGAAATACTAGCCCCC-3', R: 5`-CTCTTCGTTTTACTAAAGCAATGATCTACTTGAAGAGCATTGGAGCC-3`; S3, purifying and recovering the amplified products obtained in step S1 and step S2 by electrophoresis, recombining the fragments, transforming them into competent cells, and coating them on a culture medium containing ampicillin after recovery; S4. Pick a single colony from the culture medium in step S3, perform colony PCR, sequence verification, screen positive bacteria, and obtain the xylan ferulic acid esterase delivery plasmid pQ-XFE.

6. The method for constructing a xylan ferulic acid esterase delivery plasmid pQ-XFE based on an IncQ type plasmid according to claim 5, characterized in that: The transformation step in step S3 includes: taking 300-500 ng of the recombinant product and adding it to 100 μL E.coli Dh5α competent cells, gently placing it on ice for 30 minutes, then transferring the centrifuge tube to a 42°C constant temperature water bath for precise heat shock for 60 seconds, and then quickly transferring it back to ice to cool for 5 minutes after the heat shock.

7. The method for constructing a xylan ferulic acid esterase delivery plasmid pQ-XFE based on an IncQ type plasmid according to claim 5, characterized in that: The nucleotide sequence of the delivery plasmid pQ-XFE is shown in SEQ ID NO:

3.

8. A recombinant engineered bacterium producing xylan ferulic acid esterase, characterized in that: The recombinant genetically engineered bacteria is obtained by transforming the plasmid pQ-XFE according to claim 4 into Escherichia coli E. coli Dh5α.

9. Use of the engineered bacteria according to claim 8 in degrading ester compounds.

10. The use according to claim 9, characterized in that The degradation application is as follows: using tributyrin as a substrate, degradation is performed using Escherichia coli carrying the delivery plasmid pQ-XFE.

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