Engineering bacterium for adsorbing nitrate as well as construction method and application of engineering bacterium

Abstract

The invention belongs to the field of microorganisms, and particularly relates to an engineering bacterium for adsorbing nitrate as well as a construction method and application of the engineering bacterium. The engineering bacterium is used for expressing a fusion protein of a moringa oleifera seed MO2.1 protein and an escherichia coli prolipoprotein-outer membrane protein chimera. The nitrate adsorption rates exceed 56% and 52% respectively in 2 hours and 24 hours, the nitrate in the water body can be effectively adsorbed, and the electron donor utilization efficiency is improved by 9-13%. Engineering bacteria can generate a part of acetic acid and other substances through self metabolism in an anaerobic environment, or cells are decomposed when the engineering bacteria are apoptotic, and a carbon source can be provided for denitrifying bacteria in a nitrification tank.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and particularly relates to an engineered bacterium for adsorbing nitrate, a construction method thereof, and an application thereof. Background Art

[0002] The up-to-standard discharge of tail water is an important support for the green development of the aquaculture industry. Biological denitrification has the characteristics of low cost and high efficiency, and is the main denitrification method for aquaculture tail water. For example, CN112501099A discloses a denitrifying genetically engineered bacterium, which is optimized and synthesized according to the codons of the ammonia monooxygenase encoding gene and the hydroxylamine oxidase encoding gene of the nitrification pathway of the Bacillus subtilis host, and is assembled into the Bacillus subtilis free plasmid by DNA homologous recombination and tandem expressed under the P43 promoter to obtain the denitrifying genetically engineered bacterium.

[0003] However, aquaculture tail water has the characteristics of high dissolved oxygen concentration (usually 4-6 mg / L), low C / N, and the nitrogen concentration in the tail water fluctuates with factors such as feeding time. The low C / N of the tail water limits biological denitrification, and it is necessary to add electron donors to improve the denitrification efficiency of the tail water, including liquid (sodium acetate, glucose, methanol, sodium thiosulfate, etc.) and solid electron donors (polycaprolactone, agricultural waste, elemental sulfur particles, etc.); the biological denitrification process is ultimately realized through the denitrification pathway under anaerobic conditions, while the high dissolved oxygen concentration in the tail water limits the electron utilization efficiency, resulting in the meaningless consumption of the added electrons and increasing the denitrification cost of aquaculture tail water.

[0004] Based on the deficiencies of the existing denitrification technology caused by the existing electron efficiency, the present invention proposes a novel engineered bacterium modified by genetic engineering, which can adsorb nitrate in the water body, improve the electron donor utilization efficiency, and thus improve the denitrification efficiency. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention is modified by genetic engineering to construct a novel engineered bacterium that surface-displays the MO2.1 protein of Moringa oleifera seeds, which can adsorb nitrate in the water body, slowly migrate to the bottom anaerobic zone, improve the electron donor utilization efficiency, and the metabolites can be utilized by denitrifying bacteria to promote the denitrification process.

[0006] The first aspect of the present invention provides an engineered bacterium for adsorbing nitrate, the surface of the engineered bacterium displays the MO2.1 protein of Moringa oleifera seeds, and the amino acid sequence of the MO2.1 protein is as shown in SEQ ID NO: 1.

[0007] In some embodiments, the engineered bacterium expresses a fusion protein composed of the MO2.1 protein and an Escherichia coli prolipoprotein-outer membrane protein chimera, with the N-terminus of the MO2.1 protein connected to the C-terminus of the outer membrane protein. By fusing the N-terminus of the MO2.1 protein with the C-terminus of the outer membrane protein, it is surface-displayed on the outer membrane of the engineered bacterium, which is more conducive to adsorbing anions and thus adsorbing nitrate.

[0008] In some embodiments, the prolipoprotein-outer membrane protein chimera is an Lpp-OmpA chimera, and its amino acid sequence is as shown in SEQ ID NO: 2. The chimera consists of the first 9 N-terminal amino acid residues of the mature Escherichia coli lipoprotein and the amino acid residues 46 - 159 of the Escherichia coli outer membrane protein A (OmpA). The Lpp fragment helps the fusion protein correctly localize to the outer membrane; the OmpA fragment transports the C-terminally fused foreign protein across the outer membrane, enabling its expression on the surface of the engineered bacterium.

[0009] In some embodiments, a tag, such as a His tag, is connected to the C-terminus of the MO2.1 protein for protein expression verification.

[0010] In some embodiments, the coding sequence of the MO2.1 protein is as shown in SEQ ID NO: 3, and the coding sequence of the fusion protein is as shown in SEQ ID NO: 4. In some embodiments, the engineered bacterium is Escherichia coli.

[0011] The present invention also provides a method for constructing the engineered bacterium for adsorbing nitrate, including: constructing a plasmid vector containing the nucleotide sequences encoding the MO2.1 protein and the fusion protein, introducing the plasmid vector into a strain, and screening for positive strains to obtain the engineered bacterium.

[0012] In some embodiments, the construction method includes: constructing a plasmid vector pETduet-LppOmpA-MO2.1 containing the nucleotide sequence encoding the fusion protein composed of the MO2.1 protein and the Lpp-OmpA chimera, introducing the plasmid vector into a strain, and screening for positive strains to obtain the engineered bacterium.

[0013] The present invention also provides the use of the engineered bacterium for adsorbing nitrate for nitrate adsorption or biological denitrification, preferably for biological denitrification of aquaculture tail water.

[0014] Compared with the prior art, the beneficial effects of the present invention at least include:

[0015] 1. The engineered bacterium of the present invention can express a fusion protein of the Lpp-OmpA chimera and the MO2.1 protein, and can rapidly adsorb anions. For example, the nitrate adsorption rates exceed 56% and 52% at 2 h and 24 h, respectively, which are much higher than those of the control group. When used for biological denitrification, the electron donor utilization efficiency is effectively increased by 9 - 13%.

[0016] 2. When the engineered bacteria of the present invention are in an anaerobic environment, they will produce some substances such as acetic acid through their own metabolism, or when the engineered bacteria apoptose, the cells will be decomposed, which can provide a carbon source for denitrifying bacteria in the nitrification tank.

[0017] 3. The engineered bacteria of the present invention have low application cost, are easy to operate and will not cause secondary water pollution. Description of the Drawings

[0018] Figure 1 For SDS-PAGE to verify the expression of the target protein, where M: protein marker; 1 and 2: uninduced BL21 / pETduet-1; 3 and 4: uninduced engineered bacteria; 5 and 6: induced BL21 / pETduet-1; 7 and 8: induced engineered bacteria.

[0019] Figure 2 For the change in nitrate adsorption concentration of the engineered bacteria in 6 h.

[0020] Figure 3 For the change in nitrate adsorption concentration of the engineered bacteria in 96 h. Detailed Embodiments

[0021] In order to make the objectives, technical solutions and beneficial effects of the present invention clearer, the present invention will be further described in detail below in conjunction with the drawings and specific embodiments. Examples of the embodiments are shown in the drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only illustrative descriptions of the specific embodiments of the present invention, and are intended to explain the present invention, rather than limiting the present invention.

[0022] The endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values, and these ranges or values should be understood to include values approaching these ranges.

[0023] Example 1: Method for constructing engineered bacteria

[0024] (1) According to the codon preference of Escherichia coli, the nucleotide sequence of MO2.1 (GeneBank: AJ345072.1) was codon-optimized to ensure the correct folding and expression of the target protein in Escherichia coli. The amino acid sequence of MO2.1 is shown in SEQ ID NO: 1, and the nucleotide sequence encoding MO2.1 is shown in SEQ ID NO: 3. A His tag was added to the C-terminus of the protein for later verification.

[0025] QGPGRQPDFQRCCQQLRNISPPCRCPSLRQAVQLTHQQQGQVGPQQVRQMYRVA SNIPST (SEQ IDNO: 1)

[0026] CAAGGTCCGGGTCGTCAGCCGGATTTTCAGCGTTGTTGTCAGCAACTGCGTAATATTTCTCCGCCGTGTCGTTGTCCGAGCCTGCGTCAGGCTGTTCAGCTGACTCATCAACAACAGGGTCAGGTTGGTCCGCAACAGGTTCGTCAGATGTATCGTGTTGCTTCTAATATTCCGTCTACTTAA(SEQ ID NO:3)

[0027] (2) Connect using the Escherichia coli prolipoprotein-outer membrane protein chimera and the optimized MO2.1 protein to construct the LPPOmpA-MO2.1 fragment. The amino acid sequence of the chimera is shown in SEQ ID NO: 2, and the nucleotide sequence of the LPPOmpA-MO2.1 fragment is shown in SEQ ID NO: 4.

[0028] MKATKLVLGNNNGPTHENQLGAGAFGGYQVNPYVGFEMGYDWLGRMPYKGSV ENGAYKAQGVQLTAKLGYPITDDLDIYTRLGGMVWRADTKSNVYGKNHDTGVSPVF AGGVEYAITPEIATR(SEQ ID NO:2)

[0029] ATGAAAGCTACTAAACTGGTACTGGGCAACAACAATGGCCCGACCCATGAAAACCAACTGGGCGCTGGTGCTTTTGGTGGTTACCAGGTTAACCCGTATGTTGGCTTTGAAATGGGTTACGACTGGTTAGGTCGTATGCCGTACAAAGGCAGCGTTGAAAACGGTGCATACAAAGCTCAGGGCGTTCAACTGACCGCTAAACTGGGTTACCCAATCACTGACGACCTGGACATCTACACTCGTCTGGGTGGCATGGTATGGCGTGCAGACACTAAATCCAACGTTTATGGTAAAAACCACGACACCGGCGTTTCTCCGGTCTTCGCTGGCGGTGTTGAGTACGCGATCACTCCTGAAATCGCTACCCGTCAAGGTCCGGGTCGTCAGCCGGATTTTCAGCGTTGTTGTCAGCAACTGCGTAATATTTCTCCGCCGTGTCGTTGTCCGAGCCTGCGTCAGGCTGTTCAGCTGACTCATCAACAACAGGGTCAGGTTGGTCCGCAACAGGTTCGTCAGATGTATCGTGTTGCTTCTAATATTCCGTCTACTCATCATCATCACCATCATTAAAAGCTT(SEQ ID NO:4)

[0030] (3) pETduet-1 is a plasmid stored in this laboratory and can also be commercially purchased. Using the NcoⅠ and HindⅢ restriction enzyme sites in the pETduet-1 plasmid, the LPPOmpA-MO2.1 nucleotide sequence was subcloned into the MCS-1 region of pETduet-1. The PCR target fragment was verified with the pET-MCS-F / pET-MCS-R primers to ensure the correct insertion of the target fragment. pET-MCS-F: CGGCGTAGAGGATCGAGATC(SEQ ID NO: 5); pET-MCS-R: GATTATGCGGCCGTGTACAATAC(SEQ ID NO: 6).

[0031] Example 2: Screening of positive engineering bacteria and expression conditions of target protein

[0032] The constructed pETduet-LPPOmpA-MO2.1 plasmid was transferred into the expression-competent BL21(DE3) by chemical transformation to construct the engineered strain BL21 / pETduet-LPPOmpA-MO2.1. The engineered strain was spread on LB solid medium with a final concentration of 100 μg / mL ampicillin and cultured in a constant temperature incubator at 37 °C for 14 - 16 h. Single colonies were picked and positive recombinants were identified using the primers pET-MCS-F / pET-MCS-R. The single colonies were transferred to 1 mL of LB liquid medium containing ampicillin resistance and cultured at 37 °C and 220 rpm / min on a shaker for 4 h. Then, 1 mL of the bacterial solution was transferred to 20 mL of LB liquid medium containing ampicillin resistance and cultured at 37 °C and 220 rpm / min on a shaker for 4 h. 8 mL was taken and transferred to 300 mL of LB liquid medium containing ampicillin resistance. When OD = 0.9 - 1.0, IPTG with a final concentration of 0.5 mM was added and cultured at 20 °C and 90 rpm / min for 12 h to induce the expression of the target protein. 1 mL of the bacterial solution was centrifuged at 12000 rpm / min for 1 min, the supernatant was removed, and the bacterial pellet was resuspended in 1 mL of lysis buffer and sonicated using a sonicator. 15 μL of the lysed bacterial solution was added to 5 μL of 4× protein loading buffer and heated at 100 °C for 10 min, and SDS-PAGE was used to verify the expression of the target protein.

[0033] The results are as Figure 1 shown, from which it can be seen that the engineered strain (7,8) successfully expressed the fusion protein containing MO2.1.

[0034] Example 3: Nitrate adsorption experiment of the engineered strain

[0035] A blank control group (10 mg / L nitrate-nitrogen), control group 1 (BL21 / pETduet-1 + 10 mg / L nitrate-nitrogen), and experimental group (engineered strain + 10 mg / L nitrate-nitrogen) were set up in the experiment. Each group had three parallels, and samples were taken at 0 h, 0.5 h, 2 h, 6 h, 24 h, 48 h, 72 h, and 96 h under static conditions at 20 °C. 5 mL of the liquid was aspirated, centrifuged at 4000 rpm and 10 °C for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane to measure the remaining nitrate content in the liquid. The average value was taken to calculate the adsorption efficiency of the engineered strain for nitrate. The changes in the nitrate adsorption concentration of the engineered strain are respectively as Figure 2 and Figure 3 shown.

[0036] The results showed that the nitrate adsorption rates of the engineered strain at 2 h and 24 h exceeded 56% and 52% respectively, while those of the control group were only 41% and 19%. The engineered strain can effectively improve the electron donor utilization efficiency by 9 - 13%.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation to the content of the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. An engineered bacterium for adsorbing nitrate, characterized in that, The engineered bacterium surface-displayed the Moringa oleifera seed MO2.1 protein, and the amino acid sequence of the MO2.1 protein was as shown in SEQ ID NO:

1.

2. The engineered bacteria for adsorbing nitrate according to claim 1, characterized in that, The engineered bacterium expressed a fusion protein composed of the MO2.1 protein and the Escherichia coli prolipoprotein-outer membrane protein chimera, and the N-terminus of the MO2.1 protein was connected to the C-terminus of the outer membrane protein.

3. The engineered bacteria for adsorbing nitrate according to claim 2, characterized in that, The outer membrane protein was selected from partial amino acid sequences of the OmpA protein, OmpC protein or OmpF protein of Escherichia coli, and this partial amino acid transported the functional protein fused to the C-terminus across the outer membrane.

4. The engineered bacteria for adsorbing nitrate according to claim 2, characterized in that, The prolipoprotein-outer membrane protein chimera was the Lpp-OmpA chimera, and its amino acid sequence was as shown in SEQ ID NO:

2.

5. The engineered bacteria for adsorbing nitrate according to claim 4, wherein The coding sequence of the MO2.1 protein was as shown in SEQ ID NO: 3, and the coding sequence of the fusion protein was as shown in SEQ ID NO:

4.

6. The engineered bacteria for adsorbing nitrate according to claim 1, characterized in that, The engineered bacterium was Escherichia coli.

7. A method for constructing an engineered bacterium for adsorbing nitrate according to any one of claims 1-6, characterized in that, Including: Constructing a plasmid vector containing the nucleotide sequences encoding MO2.1 and the fusion protein, introducing the plasmid vector into the strain, and screening the positive strain to obtain it.

8. The construction method according to claim 7, wherein Including: Constructing a plasmid vector containing the nucleotide sequence encoding the fusion protein composed of the MO2.1 protein and the Lpp-OmpA chimera, introducing the plasmid vector into the strain, and screening the positive strain to obtain it; the plasmid vector was the pETduet-1 plasmid.

9. Use of an engineered bacterium for adsorbing nitrate according to any one of claims 1-6, characterized in that, For adsorbing nitrate or biological denitrification.

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