PhiF* expression cassette, expression system and application of phiF* expression cassette in xanthomonas campestris

Abstract

The invention relates to the technical field of gene engineering, in particular to a phiF * expression cassette, an expression system and application of the phiF * expression cassette in xanthomonas campestris. The phiF * expression cassette is composed of a constitutive promoter Pj23119, an RBS sequence and a repressor protein gene phiFR *; the nucleotide sequence and the amino acid sequence of the repressor protein gene phiFR * are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2. According to the invention, a phiF * frame and a phiF * expression system which can be used in xanthomonas campestris are constructed, and a certain basis is provided for construction of xanthomonas campestris genetic engineering strains.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a phiF* expression frame, an expression system and application thereof in Xanthomonas campestris. Background Art

[0002] Xanthomonas campestris ( Xanthomonas campestris pv. campestris Xanthomonas campestris (Xcc) is a Gram-negative bacterium that uniquely produces the exopolysaccharide xanthan gum. Xanthan gum is currently the world's most abundant natural polysaccharide, boasting excellent stability, rheological properties, and pseudoplasticity, making it widely used in food, pharmaceuticals, and cosmetics. Furthermore, Xanthomonas campestris possesses a rich supply of polysaccharide substrates and holds great potential for the synthesis of polysaccharide products. Therefore, its application in metabolic engineering and synthetic biology is attracting increasing attention. However, there are currently few reports on related expression elements.

[0003] Chinese invention patent application CN 116875524 A discloses a method for regulating multi-gene expression in microorganisms, which involves a 2,4-diacetylphloroglucinol (DAPG) inducible expression system, including an inducer DAPG, a regulatory protein PhlF, and a promoter P that interacts with the regulatory protein. PhlF_NNNN , and an expression vector containing the above-mentioned 2,4-diacetylphloroglucinol inducible expression system, which can be a plasmid. The above-mentioned expression vector is introduced into a microorganism, and the expression vector can integrate the promoter into the microorganism's chromosomal genome or the expression vector is free in the microorganism. During the cultivation of the microorganism, an inducer is added, and the inducer binds to the regulatory protein, causing the regulatory protein to undergo conformational changes, allowing RNA polymerase to bind to the promoter and start gene transcription. The microorganisms applicable to this technical solution include Escherichia coli, Eutropha rhodochii ( Ralstonia eutropha ), Bacillus, Cereals, Alcaligenes megaterium, Pseudomonas spp. ( Pseudomonas entomophilia ), any of the genus Aeromonas or halophilic bacteria.

[0004] Research has shown that the inducible expression system provided by the above technical solution is not suitable for Xanthomonas campestris. Summary of the Invention

[0005] To address the current lack of expression elements for Xanthomonas campestris, the present invention provides a phiF* expression cassette, expression system, and their use in Xanthomonas campestris. This invention randomly mutates the gene sequence of the repressor protein phiFR to obtain a repressor protein mutant phiFR* with improved affinity and expression strength. This invention constructs a phiF* cassette and phiF* expression system that can be used in Xanthomonas campestris, providing a foundation for the construction of genetically engineered strains of Xanthomonas campestris.

[0006] The technical solutions of the present invention are as follows: In the first aspect, the present invention provides a phiF* expression cassette, which is composed of a repressor protein gene phiFR*, a constitutive promoter P j23119 and RBS sequence; The nucleotide sequence and amino acid sequence of the repressor protein gene phiFR* are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0007] Furthermore, the constitutive promoter P j23119 The nucleotide sequence is shown in SEQ ID NO. 3.

[0008] Furthermore, the nucleotide sequence of the RBS sequence is shown in SEQ ID NO. 4.

[0009] In a second aspect, the present invention provides a phiF* expression system, which is composed of an expression vector, the above-mentioned phiF* expression cassette and a target gene; The target gene contains P phiF promoter.

[0010] Furthermore, the expression vector is pBBRMCS-2 plasmid.

[0011] Furthermore, P phiF The nucleotide sequence of the promoter is shown in SEQ ID NO. 7.

[0012] In a third aspect, the present invention provides an application of the above-mentioned phiF* expression cassette in Xanthomonas campestris, specifically, the phiF* expression cassette is transformed into Xanthomonas campestris, and 2,4-diacetylphloroglucinol is used as an inducer to induce the strain to express the target gene, and the target gene contains P phiF promoter.

[0013] Furthermore, the target gene is superfolded green fluorescent protein (sfGFP), which is used as a marker gene to characterize the regulatory effect of the expression system.

[0014] In a fourth aspect, the present invention also provides an application of the above-mentioned phiF* expression system in Xanthomonas campestris, specifically, transforming the phiF* expression system into Xanthomonas campestris, and using 2,4-diacetylphloroglucinol as an inducer to induce the strain to express the target gene.

[0015] Furthermore, the target gene is superfolded green fluorescent protein (sfGFP), which is used as a marker gene to characterize the regulatory effect of the expression system.

[0016] The beneficial effects of the present invention are: The present invention constructs an expression system using the commercial plasmid pBBRMCS-2 as an expression vector and superfolded green fluorescent protein (sfGFP) as a characterization to efficiently screen repressor protein mutants and obtain a phiF* expression cassette that can be successfully induced for expression in Xanthomonas campestris. The repressor protein phiFR*, compared with the existing repressor protein phiFR, can exhibit sufficiently higher affinity and expression intensity in Xanthomonas campestris, with low leakage and rigorous regulation, thus solving the problem that existing repressor proteins are basically unable to be induced and increasing the induction amplification factor to 4.75 times.

[0017] The invention provides basic elements for the construction of genetic engineering strains of Xanthomonas campestris. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0019] Figure 1 Schematic diagram of the phiF* expression system constructed in Example 1. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0021] A phiF* expression cassette of Xanthomonas campestris, consisting of a repressor protein gene phiFR*, a constitutive promoter P j23119 and RBS sequence.

[0022] As a preferred embodiment of the present invention, the nucleotide sequence of the repressor protein gene phiFR* (SEQ ID NO. 1) is as follows: ATGGCACGTACCCCGAGCCGTAGCAGCATTGGTAGCCTGCGTAGTCCGCATACCCATAAAGCAATTCTGACCAGCACCATTGAAATCCTGAAAGAATGTGGTTATAGCGGTCTGAGCATTGAAAGCGTGGCACGTCGCGCCGGTGCATCTAAACCGACCATTTATCGTTGGTGGACCAACAAAGCAGCACTGATTGCCGAAGTGTATGAAAATGAATCTGAACAGGTACGTAAATTTCCGGATTT GGGTAGCTTTAAAGCCGATCTGGATTTTCTGCTGCGTAATCTGTGGAAAGTTTGGCGTGAAAACCATTTGTGGTGAAGCATTTCGTTGTGTTATTGCAGAAGCACAGTTGGACCCTGCCACCCTGACCCAACTGAAAGATCAGTTTATGGAACGTCGTCGTGAGATGCCGAAAAACTGGTGGAAAACGCGATCTCCAACGGTGAGCTGCCAAAAGACACTAACCGTGAACTGCTGCTGGACATGA; The amino acid sequence of the repressor protein gene phiFR* (SEQ ID NO. 2) is as follows: MARTPSRSSIGSLRSPHTHKAILTSTIEILKECGYSGLSIESVARRAGASKPTIYRWWTNKAALIAEVYENESEQVRKFPDLGSFKADLDFLLRNLWKVWRETICGEAFRCVIAEAQLDPATLTQLKDQFMERRREMPKNWWKTRSPTVSCQKTLTVNCCWT*; constitutive promoter P j23119 The nucleotide sequence (SEQ ID NO. 3) is as follows: TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGC; The nucleotide sequence of the RBS sequence (SEQ ID NO. 4) is as follows: CTATGGACTATGTTTGAAAGGGAGAAATACTAG.

[0023] As a preferred embodiment of the present invention, the repressor protein gene phiFR* is produced by QuickMutation TM The gene random mutagenesis kit randomly mutated the original repressor protein to generate a repressor protein mutant library. The repressor protein mutant library and the super folded green fluorescent protein (sfGFP) expression cassette were then connected to the plasmid vector. E. coli The plasmid library was obtained by electroporation in DH5α, and then the mutant library was obtained by transforming Xanthomonas campestris by electroporation. The repressor protein gene with the best induction amplification effect was then screened by fluorescence intensity.

[0024] As a preferred embodiment of the present invention, the Xanthomonas campestris used herein is preferably DEOSEN-NS320024, which has been disclosed in Chinese invention patent CN 115093994 B, with a deposit date of December 7, 2021, and a depository institution of the General Microbiological Center of the China Culture Collection Administration Committee. The depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 24042, and is classified as Xanthomonas campestris ( Xanthomonas campestris ).

[0025] As a preferred embodiment of the present invention, the nucleotide sequence of superfolded green fluorescent protein (sfGFP) (SEQ ID NO. 5) is as follows: ATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAACGGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGTGGCCAACACTTGTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGACCTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAACTCGAGTACAACTTTAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAATAA; The amino acid sequence of superfolder green fluorescent protein (sfGFP) (SEQ ID NO. 6) is as follows: MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLV NRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK*.

[0026] As a preferred embodiment of the present invention, the fluorescence intensity is measured using a multifunctional microplate reader Cytation 3 (Berteng Instruments, Inc., USA). The specific method is based on the relative fluorescence intensity measurement method described in the literature Yang S, Liu Q, Zhang Y, et al. Construction and characterization of broad-spectrum promoters for synthetic biology [J]. ACS Synthetic Biology, 2018, 7(1): 287-291.

[0027] A Xanthomonas campestris phiF* expression system is composed of an expression vector, the phiF* expression frame and a target gene.

[0028] As a preferred embodiment of the present invention, the expression vector is the commercial plasmid pBBRMCS-2.

[0029] As a preferred embodiment of the present invention, the target gene contains P phiF promoter.

[0030] As a preferred embodiment of the present invention, P phiF The nucleotide sequence of the promoter (SEQ ID NO. 7) is as follows: CGACGTACGGTGGAATCTGATTCGTTACCAATTGACATGATACGAAACGTACCGTATCGTTAAGGT.

[0031] As a preferred embodiment of the present invention, the target gene is superfolded green fluorescent protein (sfGFP), which is used as a marker gene to characterize the regulatory effect of the expression system.

[0032] The phiF* expression cassette and phiF* expression system can be used to express the target gene in Xanthomonas campestris, and the inducer is 2,4-diacetylphloroglucinol.

[0033] Example 1 Construction of phiF* expression system and engineered strain (1) Construction of the phiF* expression cassette The phiF* expression cassette consists of the repressor protein gene phiFR* and the constitutive promoter P j23119 and RBS sequence; The nucleotide sequence and amino acid sequence of the repressor protein gene phiFR* are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively; the constitutive promoter P j23119 The nucleotide sequence of is shown in SEQ ID NO. 3; the nucleotide sequence of the RBS sequence is shown in SEQ ID NO. 4.

[0034] The repressor protein phiFR* can bind to the promoter P in the absence of inducer. phiF Binding represses expression from the promoter, while disengagement from the promoter in the presence of an inducing agent allows expression from the promoter.

[0035] (2) Construction of superfolded green fluorescent protein (sfGFP) expression cassette In order to characterize the effect of the phiF* expression cassette, superfolded green fluorescent protein (sfGFP) was used as the target gene of the phiF* expression system, and the fluorescence intensity of superfolded green fluorescent protein (sfGFP) was used to characterize the performance of the expression system.

[0036] The superfolded green fluorescent protein (sfGFP) expression cassette is driven by the inducible promoter P phiF Constituted with superfolded green fluorescent protein (sfGFP); The nucleotide sequence and amino acid sequence of superfolded green fluorescent protein (sfGFP) are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively; phiF The nucleotide sequence of the promoter is shown in SEQ ID NO. 7.

[0037] (3) Construction of phiF* expression system The phiF* expression cassette of Xanthomonas campestris constructed in step (1) and the super-folded green fluorescent protein (sfGFP) expression cassette constructed in step (2) were ligated into the vector pBBRMCS-2 plasmid to obtain the phiF* expression system, named pBBR-phiF*-sfGFP, with the following structure: Figure 1 shown.

[0038] (4) Construction of engineered strains The phiF* expression system was introduced into Xanthomonas campestris DEOSEN-NS320024 by electroporation at a voltage of 1200 V / cm and a current of 25 mA to prepare the engineered strain Xcc-phiF*.

[0039] Comparative Example 1 Construction of phiF expression system and engineered strain (1) Construction of the phiF expression cassette The phiF expression cassette consists of the original repressor protein gene phiFR and the constitutive promoter P j23119 and RBS sequence; The nucleotide sequence of the original repressor protein gene phiFR (SEQ ID NO. 8) is as follows: ; The amino acid sequence of the original repressor protein gene phiFR (SEQ ID NO. 9) is as follows: MARTPSRSSIGSLRSPHTKAILTSTIEILKECGYSGLSIESVARRAGASKPTIYRWWTNKAALIAEVYENESEQVRKFPDLGSFKADLDFLLRNLWKVWRETICGEAFRCVIAEAQLDPATLTQLKDQFMERRREMPKKLVENAISNGELPKDTNRELLLDMIFFGFCWYRLLTEQLTVEQDIEEFTFLLINGVCPGTQR*.

[0040] constitutive promoter P j23119 The nucleotide sequence of is shown in SEQ ID NO. 3; the nucleotide sequence of the RBS sequence is shown in SEQ ID NO. 4.

[0041] (2) Construction of superfolded green fluorescent protein (sfGFP) expression cassette In order to characterize the effect of the phiF expression cassette, superfolded green fluorescent protein (sfGFP) was used as the target gene of the phiF expression system, and the fluorescence intensity of superfolded green fluorescent protein (sfGFP) was used to characterize the performance of the expression system.

[0042] The superfolded green fluorescent protein (sfGFP) expression cassette is driven by the inducible promoter P phiF Constituted with superfolded green fluorescent protein (sfGFP); The nucleotide sequence and amino acid sequence of superfolded green fluorescent protein (sfGFP) are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively; phiF The nucleotide sequence of the promoter is shown in SEQ ID NO. 7.

[0043] (3) Construction of phiF expression system The phiF expression cassette of Xanthomonas campestris constructed in step (1) and the super-folded green fluorescent protein (sfGFP) expression cassette constructed in step (2) were ligated into the vector pBBRMCS-2 plasmid to obtain the phiF expression system.

[0044] (4) Construction of engineered strains The phiF expression system was transformed into Xanthomonas campestris to prepare the engineered strain Xcc-phiF.

[0045] Example 2 Verification of induction of expression system in engineered strains The engineered strain Xcc-phiF* constructed in Example 1 and the engineered strain Xcc-phiF constructed in Comparative Example 1 were cultured using NBG medium containing 18 g nutrient broth, 10 g glucose, and 50 mg kanamycin sulfate per liter. The cultures were first cultured overnight at 30°C, 220 rpm, and shaken to obtain a seed solution. A 4% volume percentage inoculum was then transferred to a 96-well plate containing 100 mg / L kanamycin sulfate and cultured at 30°C, 750 rpm, and shaken for 72 h. Five treatments were set up: treatment group 1 received no inducer during the culture process. Treatment groups 2-5 received 25 μM of the inducer 2,4-diacetylphloroglucinol at 0, 6, 12, and 15 h of culture, respectively. Fluorescence expression intensity was measured for each treatment group after the incubation period.

[0046] The results are shown in Table 1 below. It can be seen that the phiF expression system of Comparative Example 1 was essentially unable to induce expression in Xanthomonas campestris. The phiF* expression system provided by the present invention exhibited very low leakage in Xanthomonas campestris and could achieve induced expression with 25 μM DAPG, with an induction magnification of 4.75 times.

[0047] Table 1 Expression intensity of fluorescent proteins by engineered strains in Examples and Comparative Examples

[0048] Although the present invention has been described in detail with reference to the accompanying drawings and in conjunction with preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, persons of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any changes or substitutions that can be easily conceived by persons skilled in the art within the technical scope disclosed in the present invention shall be within the scope of protection of the present invention.

Claims

1. A phiF* expression cassette, characterized in that: The constitutive promoter P j23119 , RBS sequence and repressor protein gene phiFR*; The nucleotide sequence and amino acid sequence of the repressor protein gene phiFR* are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

2. A phiF* expression cassette according to claim 1, characterized in that constitutive promoter P j23119 The nucleotide sequence is shown in SEQ ID NO.

3.

3. The Xanthomonas campestris phiF* expression cassette according to claim 1, wherein: The nucleotide sequence of the RBS sequence is shown in SEQ ID NO.

4.

4. A phiF* expression system, characterized in that: It consists of an expression vector, a phiF* expression cassette, and a target gene; The phiF* expression cassette is the phiF* expression cassette according to any one of claims 1 to 3; The target gene contains P phiF Promoter.

5. A phiF* expression system according to claim 4, characterized in that: The expression vector is pBBRMCS-2 plasmid.

6. The phiF* expression system according to claim 4, wherein: P phiF The nucleotide sequence of the promoter is shown in SEQ ID NO.

7.

7. Use of the phiF* expression cassette according to claim 1 in Xanthomonas campestris, characterized in that: The phiF* expression cassette was transformed into Xanthomonas campestris, and 2,4-diacetylphloroglucinol was used as an inducer to induce the strain to express the target gene. The target gene contained P phiF Promoter.

8. The use according to claim 7, characterized in that The target gene is superfolded green fluorescent protein.

9. Use of the phiF* expression system according to any one of claims 4 to 6 in Xanthomonas campestris, characterized in that: The phiF* expression system was transformed into Xanthomonas campestris, and 2,4-diacetylphloroglucinol was used as an inducer to induce the strain to express the target gene.

10. The use according to claim 9, characterized in that The target gene is superfolded green fluorescent protein.

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