CN116589541A - FNR mutant and application thereof in gene expression regulation and control - Google Patents

Abstract

The application discloses an FNR mutant and application thereof in gene expression regulation. The mutant has R169G and/or G216D mutation on the amino acid sequence of the FNR, wherein the amino acid sequence of the FNR is shown as SEQ ID NO. 1 or has 99% identity with the amino acid sequence shown as SEQ ID NO. 1. The FNR mutant FNR (R169G) and FNR (G216D) can realize more strict regulation and control on the PfnrS promoter, solve the problem caused by leakage expression under the aerobic condition of the promoter, and improve the stability of strain growth and expression.

Description

FNR mutant and application thereof in gene expression regulation and control

The present application claims priority from China patent application 2022113380272 with the application date 2022/10/28. The present application incorporates the entirety of the above-mentioned chinese patent application.

Technical Field

The application belongs to the technical field of bioengineering, and particularly relates to an FNR mutant, a nucleic acid construct containing the FNR mutant, a genetically modified cell containing the FNR mutant and application of the FNR mutant in gene expression regulation and control, and a method for improving the regulation and control stringency of an FNR dependent promoter.

Background

In E.coli, the FNR proteins (fumarate and nitrate reduction regulator/agent) are the primary transcriptional activators controlling the switch from aerobic to anaerobic metabolism. In the anaerobic state, FNR dimerizes into active DNA binding proteins that activate hundreds of genes responsible for adaptation to anaerobic growth. In the aerobic state, FNR is prevented from dimerization by oxygen and is deactivated.

Under anaerobic and/or hypoxic conditions, FNR forms dimers and binds to specific sequences in the class I or class II FNR-dependent promoter (Class I or Class II FNR-dependent promoter), thereby activating the expression of the gene. Wherein the FNR-dependent promoter of class II has a consensus site (concentration site) located 41bp upstream of the transcription initiation site, and the FNR-dependent promoter of class I has a binding site located further upstream; however, under aerobic conditions, oxygen reacts with Iron-sulfur clusters (Iron-sulfur clusters) in FNR dimers and converts them to inactive form. In this way, the FNR-dependent promoter is suitable for regulating the expression of a protein or RNA.

Studies by Spiro S et al (Spiro S, guest J r.fnr and its role in oxygen-regulated gene expression in Escherichia coli [ J ]. FEMS microbiology reviews,1990,6 (4): 399-428.) showed that basal levels of active FNR were present in cells even under aerobic conditions, and that when FNR synthesis was amplified, active FNR increased. Therefore, when the FNR regulates gene expression, in an aerobic environment, the condition of leakage expression of the target gene exists, namely, the condition of non-strict FNR regulation exists.

In order to allow FNR to act as a transcriptional regulator, more precise regulation of the expression of subsequent genes, a variety of engineering approaches have been tried by those skilled in the art. Tim over et al (Transcription Activation at Escherichia coli FNR-Dependent Promoters by the Gonococcal FNR Protein: effects OF a Novel S18F Substitution and Comparisons with the Corresponding Substitution in E.coli FNR, JOURNAL OF BACTERIOLOGY, aug.2003, p.4734-4747) found that mutation OF S18F in N.gonorrhoeae FNR can enhance the stability OF iron-sulfur clusters, thereby enhancing activity under aerobic and anaerobic conditions.

CN102724988B promotes overexpression of meningococcal fhbp genes by mutating constitutively active FNR, which results in overexpression of FNR-activating genes such as fhbp even when oxygen levels are not restricted.

The above improvements to FNR have focused on maintaining or activating the activity of FNR in an aerobic environment to promote the expression of the gene of interest, but the demands for increasing the sensitivity of FNR to oxygen, decreasing the activity of FNR in an aerobic environment, and thus decreasing the leaky expression thereof, to increase the regulatory stringency of FNR-dependent promoters have not been satisfied.

The PfnrS promoter is a promoter which is endogenous to escherichia coli and is activated by FNR regulation, the expression of a target protein is inhibited under aerobic conditions, and the expression of the target protein is started under anaerobic or micro-aerobic conditions, so that the PfnrS promoter is often used for expressing a product which only needs to be expressed under anaerobic/micro-aerobic conditions in genetic engineering. It has been found through research that although the PfnrS promoter is theoretically inhibited from being expressed under aerobic conditions, in practice, when the PfnrS promoter is used in a genetically engineered strain expressing wild-type FNR, even if it leaks to express a target gene seriously under high-oxygen conditions, when the strain starts to express the target gene in a large amount when grown to the end of the log phase in a test tube or shake flask which does not control the dissolved oxygen amount, the growth of the strain is severely affected when the expression product is toxic to the strain, a higher strain concentration cannot be achieved, even the strain dies and cracks prematurely, affecting the stability of the strain.

Disclosure of Invention

In order to solve the problem of leakage of promoter expression in the prior art, the application provides an FNR mutant and application thereof in gene expression regulation. The application uses the mutant FNR, so that the regulation and expression of the type I or type II FNR dependent promoter on the downstream gene is more strict, the expression is inhibited in aerobic culture, namely, the FNR dependent promoter can only start the expression under the condition that dissolved oxygen is low enough, thereby realizing strict regulation and control on the target gene, and the growth condition and stability of the strain are improved.

In order to solve the technical problems, one of the schemes of the application provides a FNR mutant, which is characterized in that the FNR has R169G and/or G216D mutation, and the amino acid sequence of the FNR is shown as SEQ ID NO. 1 or has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence shown as SEQ ID NO. 1.

According to the application, the FNR mutant is found from the mutant strain, and experiments prove that the FNR (R169G) and FNR (G216D) mutants can realize more strict regulation and control on the PfnrS promoter, so that the problem caused by leakage expression of the promoter under the aerobic condition is solved, and the growth and expression stability of the strain are improved.

In some embodiments of the application, the FNR mutant has an amino acid sequence as shown in SEQ ID NO. 4, or SEQ ID NO. 8.

In some embodiments of the application, the nucleotide sequence encoding the FNR mutant is shown as SEQ ID NO. 3 or SEQ ID NO. 7.

To solve the above-mentioned problems, a second aspect of the present application provides an isolated nucleic acid encoding the FNR mutant according to one of the aspects of the present application.

In some embodiments of the application, the nucleotide sequence of the isolated nucleic acid is shown as SEQ ID NO. 3 or SEQ ID NO. 7.

In order to solve the above-mentioned problems, a third aspect of the present application provides a nucleic acid construct comprising the isolated nucleic acid of the second aspect of the present application and a gene expression cassette comprising an expression regulatory element and a gene of interest.

In some embodiments of the application, the expression regulatory element of the gene expression cassette is the PfnrS promoter.

In some embodiments of the application, the gene of interest is selected from the group consisting of IL-10, IL-22.

In order to solve the above-mentioned problems, a fourth aspect of the present application provides a genetically modified cell expressing an FNR mutant as described in one of the aspects of the present application or a nucleic acid construct as described in a third aspect of the present application, wherein the FNR mutant regulates the expression of a target gene.

In some embodiments of the application, the host cells of the cells include eukaryotic cells and prokaryotic cells.

In some embodiments of the application, the host cell is a bacterium, preferably E.coli, such as E.coli Nissle 1917.

To solve the above-described problems, a fifth aspect of the present application provides a method for improving the control stringency of an FNR-dependent promoter, which comprises controlling a target gene with the FNR mutant of one aspect of the present application or the isolated nucleic acid of the second aspect of the present application or the nucleic acid construct of the third aspect of the present application or the genetically modified cell of the fourth aspect of the present application, so that the target gene is expressed in a strictly anaerobic environment and the expression is inhibited in an aerobic environment.

In order to solve the above technical problems, a sixth aspect of the present application provides an FNR mutant according to one aspect of the present application, an isolated nucleic acid according to a second aspect of the present application, a nucleic acid construct according to a third aspect of the present application, or a genetically modified cell according to a fourth aspect of the present application, for use in preparing a reagent for regulating gene expression or for regulating gene expression.

The application has the positive progress effects that:

the FNR mutant in the application makes anaerobic regulation more strict. FNR with the R169G and G216D mutations is more sensitive to oxygen and normally has enough oxygen present to react with the iron-sulfur clusters in the FNR dimer, thereby destroying its activity. Whereas more sensitive FNRs are inactivated in the presence of trace amounts of oxygen and therefore are able to activate either class I or class II FNR dependent promoters only in more severe anaerobic environments. Strains with FNR (R169G) or FNR (G216D) mutation do not express or only express very little amount of a target gene such as IL-10 under aerobic conditions, do not cause toxicity to cells, are more stable under aerobic or anaerobic conditions than wild type FNR strains, and can reach higher OD ₆₀₀ And the bacterial cells can not die and crack under anaerobic conditions, and can secrete more target genes with activity.

Drawings

FIG. 1A shows growth of IL-10 expressing strains under aerobic culture.

FIG. 1B shows growth of IL-10 expressing strains under anaerobic culture conditions (beginning at the third hour).

FIG. 2A shows the total IL-10 production by IL-10 expressing strains under aerobic culture conditions.

FIG. 2B shows the overall IL-10 production by IL-10 expressing strains under anaerobic culture conditions (beginning at the third hour).

FIG. 3A shows growth of IL-22 expressing strains under aerobic culture.

FIG. 3B shows growth of IL-22 expressing strains under anaerobic culture conditions (beginning at third hour).

FIG. 4A shows the total IL-22 production by IL-22 expressing strains under aerobic culture conditions.

FIG. 4B shows the total IL-22 production by IL-22 expressing strains under anaerobic culture conditions (beginning at the third hour).

Detailed Description

The application is further illustrated by means of the following examples, which are not intended to limit the scope of the application. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

EXAMPLE 1 discovery of FNR mutant Gene and construction of engineering Strain containing FNR mutation

1.1 discovery of FNR mutant Gene

The applicant is constructing Nissle 1917 (EcN) engineering strain CBT4084FNR (WT) which uses PfnrS as promoter to express IL-10 (engineering strain construction process is shown in example 2), and carrying out the expression study of IL-10 subsequently, on the one hand, the very obvious leakage phenomenon of PfnrS promoter is found, even under the condition of aerobic culture, but the protein product IL-10 which drives expression has a certain influence on the growth survival of escherichia coli, when the strain grows to the end of the logarithm, IL-10 is expressed in a large amount, so that very large pressure is generated on the strain growth, the strain is cracked, the strain cannot reach higher strain concentration, and under the anaerobic condition, the strain can be rapidly and greatly cracked, and cannot normally grow, on the other hand, the constructed strain which uses PfnrS as promoter to express IL-10 has very many mutations, most of the mutant strains cannot normally express IL-10, and the expression quantity of target protein of a small amount is very poor although the protein is high enough, and the two mutant strains have good nucleotide sequences such as SEQ ID sequence No. 169F 3 and F ID No. 4 have the complete nucleotide sequence of the mutant strain as SEQ ID No. 3 and the nucleotide sequence No. 4 are found under the conditions of full-sequence No. 169; at the same time, other mutations in the FNR gene, such as L139Q (nucleotide sequence shown as SEQ ID NO:5 and protein sequence shown as SEQ ID NO: 6) were found in some strains which lost the IL-10 expression ability, so we speculate that the mutation of the FNR gene is most likely to be the cause of the phenotype differences of these mutants.

Wild type FNR nucleic acid sequence (SEQ ID NO: 1):

tcaggcaacgttacgcgtatgaccagcaagctgggccagcgcatcgttattttcgatagtgatgtatttacctttgactgccagcataccgcttttctggaagcgacccagcagacggctgatggtttcaaccgtcaggcccagatagttaccgatatcaccacgagtcatcgtcaggcggaattcacgaggggagaagccgcgttgggcaaaacgacgggacaggttgtagatgaatgcagccagacgttcctcggcatttttcttcgacaacagcaggatcatgtcctgatcgcctttgatttcaccgctcatcagacgcatcatctgctgacgcagattcggcattttaccagacaaatcgtccagcgtttcgaacgggatttcacataccatcgaggtttccagcgcctgcgcgaaactcgggtgatgaccgctgccgatggcatcaaatcccaccagatcgcctgctaaatggaaaccagtgatttgctcgtcgccttgctcagtgatggtataacttttaatcgtaccggagcggatggcataaagcgatttaagttcatctccagccttaaacagcgtctggcctttctgaataggcttcttccgctcaatgatattatcaagctgatcaagctcatgttcgttgagtgtgaacgggatgcaaagctggctgatgctgcaatcctggcaatggatagcacaaccgccagactgaatgcgccgtataattcgcttttccgggatcat

wild type FNR amino acid sequence (SEQ ID NO: 2):

MIPEKRIIRRIQSGGCAIHCQDCSISQLCIPFTLNEHELDQLDNIIERKKPIQKGQTLFKAGDELKSLYAIRSGTIKSYTITEQGDEQITGFHLAGDLVGFDAIGSGHHPSFAQALETSMVCEIPFETLDDLSGKMPNLRQQMMRLMSGEIKGDQDMILLLSKKNAEERLAAFIYNLSRRFAQRGFSPREFRLTMTRGDIGNYLGLTVETISRLLGRFQKSGMLAVKGKYITIENNDALAQLAGHTRNVA*

FNR (R169G) mutant nucleic acid sequence (SEQ ID NO: 3):

tcaggcaacgttacgcgtatgaccagcaagctgggccagcgcatcgttattttcgatagtgatgtatttacctttgactgccagcataccgcttttctggaagcgacccagcagacggctgatggtttcaaccgtcaggcccagatagttaccgatatcaccacgagtcatcgtcaggcggaattcacgaggggagaagccgcgttgggcaaaacgacgggacaggttgtagatgaatgcagccagaccttcctcggcatttttcttcgacaacagcaggatcatgtcctgatcgcctttgatttcaccgctcatcagacgcatcatctgctgacgcagattcggcattttaccagacaaatcgtccagcgtttcgaacgggatttcacataccatcgaggtttccagcgcctgcgcgaaactcgggtgatgaccgctgccgatggcatcaaatcccaccagatcgcctgctaaatggaaaccagtgatttgctcgtcgccttgctcagtgatggtataacttttaatcgtaccggagcggatggcataaagcgatttaagttcatctccagccttaaacagcgtctggcctttctgaataggcttcttccgctcaatgatattatcaagctgatcaagctcatgttcgttgagtgtgaacgggatgcaaagctggctgatgctgcaatcctggcaatggatagcacaaccgccagactgaatgcgccgtataattcgcttttccgggatcat

FNR (R169G) mutant amino acid sequence (SEQ ID NO: 4):

MIPEKRIIRRIQSGGCAIHCQDCSISQLCIPFTLNEHELDQLDNIIERKKPIQKGQTLFKAGDELKSLYAIRSGTIKSYTITEQGDEQITGFHLAGDLVGFDAIGSGHHPSFAQALETSMVCEIPFETLDDLSGKMPNLRQQMMRLMSGEIKGDQDMILLLSKKNAEEGLAAFIYNLSRRFAQRGFSPREFRLTMTRGDIGNYLGLTVETISRLLGRFQKSGMLAVKGKYITIENNDALAQLAGHTRNVA*

FNR (L139Q) mutant nucleic acid sequence (SEQ ID NO: 5):

tcaggcaacgttacgcgtatgaccagcaagctgggccagcgcatcgttattttcgatagtgatgtatttacctttgactgccagcataccgcttttctggaagcgacccagcagacggctgatggtttcaaccgtcaggcccagatagttaccgatatcaccacgagtcatcgtcaggcggaattcacgaggggagaagccgcgttgggcaaaacgacgggacaggttgtagatgaatgcagccagacgttcctcggcatttttcttcgacaacagcaggatcatgtcctgatcgcctttgatttcaccgctcatcagacgcatcatctgctgacgctgattcggcattttaccagacaaatcgtccagcgtttcgaacgggatttcacataccatcgaggtttccagcgcctgcgcgaaactcgggtgatgaccgctgccgatggcatcaaatcccaccagatcgcctgctaaatggaaaccagtgatttgctcgtcgccttgctcagtgatggtataacttttaatcgtaccggagcggatggcataaagcgatttaagttcatctccagccttaaacagcgtctggcctttctgaataggcttcttccgctcaatgatattatcaagctgatcaagctcatgttcgttgagtgtgaacgggatgcaaagctggctgatgctgcaatcctggcaatggatagcacaaccgccagactgaatgcgccgtataattcgcttttccgggatcat

FNR (L139Q) mutant amino acid sequence (SEQ ID NO: 6):

MIPEKRIIRRIQSGGCAIHCQDCSISQLCIPFTLNEHELDQLDNIIERKKPIQKGQTLFKAGDELKSLYAIRSGTIKSYTITEQGDEQITGFHLAGDLVGFDAIGSGHHPSFAQALETSMVCEIPFETLDDLSGKMPNQRQQMMRLMSGEIKGDQDMILLLSKKNAEERLAAFIYNLSRRFAQRGFSPREFRLTMTRGDIGNYLGLTVETISRLLGRFQKSGMLAVKGKYITIENNDALAQLAGHTRNVA*

FNR (G216D) mutant nucleic acid sequence (SEQ ID NO: 7):

tcaggcaacgttacgcgtatgaccagcaagctgggccagcgcatcgttattttcgatagtgatgtatttacctttgactgccagcataccgcttttctggaagcgatccagcagacggctgatggtttcaaccgtcaggcccagatagttaccgatatcaccacgagtcatcgtcaggcggaattcacgaggggagaagccgcgttgggcaaaacgacgggacaggttgtagatgaatgcagccagacgttcctcggcatttttcttcgacaacagcaggatcatgtcctgatcgcctttgatttcaccgctcatcagacgcatcatctgctgacgcagattcggcattttaccagacaaatcgtccagcgtttcgaacgggatttcacataccatcgaggtttccagcgcctgcgcgaaactcgggtgatgaccgctgccgatggcatcaaatcccaccagatcgcctgctaaatggaaaccagtgatttgctcgtcgccttgctcagtgatggtataacttttaatcgtaccggagcggatggcataaagcgatttaagttcatctccagccttaaacagcgtctggcctttctgaataggcttcttccgctcaatgatattatcaagctgatcaagctcatgttcgttgagtgtgaacgggatgcaaagctggctgatgctgcaatcctggcaatggatagcacaaccgccagactgaatgcgccgtataattcgcttttccgggatcat

FNR (G216D) mutant amino acid sequence (SEQ ID NO: 8):

MIPEKRIIRRIQSGGCAIHCQDCSISQLCIPFTLNEHELDQLDNIIERKKPIQKGQTLFKAGDELKSLYAIRSGTIKSYTITEQGDEQITGFHLAGDLVGFDAIGSGHHPSFAQALETSMVCEIPFETLDDLSGKMPNLRQQMMRLMSGEIKGDQDMILLLSKKNAEERLAAFIYNLSRRFAQRGFSPREFRLTMTRGDIGNYLGLTVETISRLLDRFQKSGMLAVKGKYITIENNDALAQLAGHTRNVA*

construction of 1.2CBT4084FNR (R169G), CBT4084FNR (L139Q) and CBT4084FNR (G216D) mutant strains

To further confirm the effect of the found FNR mutant gene on the regulation of the PfnrS promoter in the genetically engineered strain, the applicant next constructed engineering strains CBT4084FNR (R169G), CBT4084FNR (L139Q) and CBT4084FNR (G216D) using the aforementioned FNR mutant gene in place of the wild-type FNR gene by homologous recombination based on CBT4084FNR (WT) carrying the wild-type FNR gene, as follows.

Preparation of 1.2.1CBT4084FNR (WT)/pCBT 001 electrotransformation competent cells

100-200ng of pCBT001 plasmid (plasmid expressing lamda red homologous recombinase and Cas9, sequence SEQ ID NO: 10) was added to 100. Mu.L of CBT4084FNR (WT) electrotransfer competent cells, and the mixture was transferred to a 2mm electrotransfer cup, and electrotransfer conditions were set at 2.5kV, 25. Mu.F, 200Ω. After electrotransformation, the cells were suspended in 1mL of SOC and cultured with shaking (220 rpm) at 30℃for two hours. All cells were then plated on LB agar plates with 50. Mu.g/mL of spectinomycin and streptomycin added and incubated overnight at 30 ℃. The colonies grown on the plates were CBT4084FNR (WT)/pCBT 001 with pCBT001 plasmid. Single CBT4084FNR (WT)/pCBT 001 colonies on resistant LB agar plates were inoculated into 3mL of LB liquid medium supplemented with 50. Mu.g/mL of spectinomycin and streptomycin, cultured overnight with shaking (220 rpm) at 30℃after which 300. Mu.L of this overnight culture was inoculated into 30mL of LB liquid medium supplemented with 50. Mu.g/mL of spectinomycin and streptomycinThe medium was incubated at 30℃with shaking (220 rpm). IPTG was added to the culture at a concentration of 1mM after 1 hour. When OD is ₆₀₀ When about 0.6 was reached, the cells were washed 3 times with 20mL, 10mL and 5mL of 10% glycerol (4 ℃) and finally resuspended in 300. Mu.L of 10% glycerol (4 ℃) and aliquoted at 100. Mu.L per tube into 1.5mL centrifuge tubes.

1.2.2 transformation of donor Gene fragments

Approximately 2. Mu.g of the donor gene fragment PCR product containing the LHA-chloramphenicol resistance gene-the FNR mutant gene of interest was transformed into electrocompetent cells of CBT4084FNR (WT)/pCBT 001. After electrotransformation, the cells were suspended in 1mL of SOC and cultured with shaking (220 rpm) at 30℃for two hours. All cells were then plated on LB agar plates with 50. Mu.g/mL spectinomycin and streptomycin and 6. Mu.g/mL chloramphenicol and incubated overnight at 30 ℃. The target FNR mutant genes are respectively: FNR (R169G) (nucleotide sequence shown as SEQ ID NO: 3), FNR (L139Q) (nucleotide sequence shown as SEQ ID NO: 5) and FNR (G216D) (nucleotide sequence shown as SEQ ID NO: 7).

1.2.3 verification of integration of the target FNR mutant Gene

Primers for verification were designed upstream of the LHA and downstream of the FNR gene, respectively. And selecting a single colony growing on the transformation plate, performing colony PCR by using a verification primer, sequencing the obtained PCR product, and judging that the wild FNR in the genome is successfully replaced by the clone of the target FNR mutant gene through the sequence of the PCR product.

1.2.4 removal of chloramphenicol resistance Gene and excess plasmid

100-200ng of plasmid pCB003_Cm_sgRNA expressing a chloramphenicol resistance gene (SEQ ID NO: 23) and approximately 2. Mu.g of a donor gene fragment PCR product containing the LHA-target FNR mutant gene were transformed into selected cloned electrocompetent cells. After electrotransformation, the cells were suspended in 1mL of SOC and cultured with shaking (220 rpm) at 30℃for two hours. All cells were then plated on LB agar plates with 50. Mu.g/mL spectinomycin and streptomycin and 100. Mu.g/mL ampicillin. PCR and sequencing with the target FNR mutant gene verification primer used in the previous step, verifying elimination of the resistance gene and reconfirming the FNR mutant groupDue to successful substitution on the genome. The selected correct clone was inoculated into 3mL of LB liquid medium added with 50. Mu.g/mL of spectinomycin and streptomycin and 10mM of arabinose, cultured overnight with shaking (220 rpm) at 30℃and then the culture was diluted 10 ⁶ 100. Mu.L of the mixture was spread on LB agar plates to which 50. Mu.g/mL of spectinomycin and streptomycin were added, and incubated overnight at 30 ℃. Single colonies were then picked and spotted simultaneously on LB agar plates with 50. Mu.g/mL of spectinomycin and streptomycin, and 100. Mu.g/mL of ampicillin. The only colonies grown on LB agar plates supplemented with 50. Mu.g/mL of spectinomycin and streptomycin were those from which plasmid pCB003_Cm_sgRNA was eliminated. The selected correct clone was inoculated into 3mL of LB liquid medium, cultured overnight with shaking (220 rpm) at 42℃and then the culture was diluted 10 ⁶ 100. Mu.L of the mixture was spread on LB agar plates and incubated overnight at 37 ℃. Single colonies were then picked and spotted simultaneously on LB agar plates and LB agar plates with 50. Mu.g/mL of spectinomycin and streptomycin added. The only colonies grown on LB agar plates were those from which plasmid pCB001 was eliminated.

The sgRNA sequence targeting the chloramphenicol resistance gene (SEQ ID NO: 23):

ccgttgatatatcccaatgg

EXAMPLE 2 construction of IL-10 expressing engineering Strain CBT4084FNR (WT)

IL-10 coding sequences, signal peptides, promoters, etc., were synthesized by gold Style (GeneScript) on a cloning plasmid (e.g., pUC 57). IL-10 coding sequence, signal peptide, promoter, etc. are amplified by using synthetic plasmid as template, LHA and RHA of insertion site are amplified by using EcN genome as template, and transcription terminator is amplified by using plasmid pCBT003 (nucleotide sequence shown as SEQ ID NO: 9) as template. The PCR primers used to amplify these fragments have homologous sequences of 15-20bp from each other, so that they can be joined by overlapping PCR to give expression cassettes of donor gene fragments flanked by LHA and RHA sequences, respectively, with the elements of the cassettes arranged in the following order from 5 'to 3': 5' -promoter-signal peptide-IL-10 coding sequence-terminator. The resulting PCR product of the donor gene fragment expression cassette with LHA and RHA sequences on both sides and the pCBT003_maeB_sgRNA plasmid expressing the insertion site sgRNA were simultaneously transformed into competent cells containing pCBT001 (nucleotide sequence shown in SEQ ID NO: 10), and the resulting single colony was amplified with the verification primer of the insertion site, and clone CBT4084FNR (WT) in which IL-10 was successfully inserted into the genome was selected by the size of the amplified band. IL-10 production was detected using ELISA KIT (KIT 10947A, yiqiao Shenzhou).

Wherein the IL-10 coding sequence is a sequence for encoding wild type human IL-10 (SEQ ID NO: 11); the inserted locus is the maeB of EcN genome, the sequence of sgRNA of the targeted maeB locus is shown as SEQ ID NO. 12, the sequences of homologous arms on two sides of the maeB locus are respectively shown as SEQ ID NO. 13 and 14, the nucleotide sequence of FNR is shown as SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5 and SEQ ID NO. 7, the promoter is a PrnrS promoter, the nucleotide sequence of the promoter is shown as SEQ ID NO. 15, the signal peptide is a USP45 signal peptide, the amino acid sequence of the signal peptide is shown as SEQ ID NO. 16, the terminator is a rrnB_T1_T7Te terminator, and the signal peptide is obtained by concatenating a rrnB_T1_terminator and a T7Te_terminator, wherein the sequence of the rrnB_T1_terminator is shown as SEQ ID NO. 17, and the sequence of the T7Te_terminator is shown as SEQ ID NO. 18.

Amino acid sequence of human IL-10 (SEQ ID NO: 11):

SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLED FKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLP CENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN*

sgRNA sequence targeting maeB site (SEQ ID NO: 12):

gaaggggaagaggcgcgcgt

left Homology Arm (LHA) sequence of maeB site (SEQ ID NO: 13):

gtggtggatgacggtaaacgtaccctcgatgatgtgattgaaggcgcggatattttcctcggctgttccggcccgaaagtgctgacccaggaaatggtgaagaaaatggcccgtgcgccaatgatcctggcgctggcgaacccggaaccggaaattctgccgccgctggcgaaagaagtgcgtccggatgccatcatctgtaccggccgttccgactacccgaaccaggtgaacaacgtcctttgcttcccgtttatcttccgtggcgcgctggacgttggcgcaaccgccatcaacgaagagatgaaactggcggcagtacgcgcgattgcagaactggcccatgcggaacagagtgaagtggtggcttcagcgtatggcgatcaggatctgagctttggtccggaatacatcattcc

the Right Homology Arm (RHA) sequence of the maeB site (SEQ ID NO: 14):

gcgaaaccgatccttattggtcgtccgaacgtgatcgaaatgcgcattcagaaactgggcttgcagatcaagtcgggcgttgattttgagatcgtcaataacgaatccgatccgcgctttaaagagtactggaccgaatacttccagatcatgaagcgtcgcggtgtcactcaggagcaggcgcagcgtgcgctgatcagtaacccgacagtgatcggcgcgatcatggttcagcgtggcgaagccgatgcaatgatttgcggtacggtgggcgattatcatgaacactttagcgtggtgaaaaatgtctttggttatcgcgatggcgttcacaccgcaggtgcaat

nucleotide sequence of PfnrS (SEQ ID NO: 15):

aaaaacgccgcaaagtttgagcgaagtcaataaactctctacccattcagggcaatatctctctt

the amino acid sequence of USP45 (SEQ ID NO: 16):

MKKKIISAILMSTVILSAAAPLSGVYA

sequence of rrnB_T1_terminator (SEQ ID NO: 17):

caaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctc

sequence of t7te_terminator (SEQ ID NO: 18):

ggctcaccttcgggtgggcctttctgcg

example 3 functional verification of the target FNR mutant Gene

Single colonies containing wild type FNR and CBT4084FNR (WT), CBT4084FNR (R169G), CBT4084FNR (L139Q), CBT4084FNR (G216D) were inoculated into 3mL of LB liquid medium, cultured overnight with shaking (220 rpm) at 37℃followed by inoculating 500. Mu.L of this overnight culture into 50mL of LB liquid medium, cultured with shaking (220 rpm) at 37℃and OD measurements per hour ₆₀₀ Values. After 3 hours of cultivation, 20mL of bacterial liquid is taken, the rest bacterial liquid is continuously cultivated aerobically, the bacterial liquid taken out is centrifuged and then resuspended in 20mL of LB (phosphate-citrate buffer system pH=8.0) +1%w/v glucose culture liquid and anaerobically cultivated in an anaerobic incubator at 37 ℃. From 4 hours, 1mL to 1.5mL of the bacterial liquid in aerobic culture and anaerobic culture is taken from each hour into a centrifuge tube, the supernatant is transferred into a new 1.5mL centrifuge tube after centrifugation, and the supernatant and the cell pellet are stored in a refrigerator at-20 ℃. Supernatant and finesThe yield of IL-10 in cell pellet was detected by ELISA KIT (KIT 10947A, yiqiao Shenzhou), cell pellet was resuspended in 150. Mu.L of 10mM Tris-HCl before detection, then 150. Mu.L of 2mg/mL lysozyme solution was added, after about half an hour of lysis at 4℃freeze thawing was performed at-20℃for one time, after complete lysis of cells, centrifuged for 5min, and cell lysate supernatant was taken for detection of IL-10. The sum of the IL-10 production of the supernatant and the precipitated IL-10 production was determined to be the total IL-10 production.

As shown in FIGS. 1A and 1B, CBT4084FNR (R169G), CBT4084FNR (L139Q), CBT4084FNR (G216D) containing FNR mutant gene reached the maximum OD under aerobic culture conditions ₆₀₀ Values were significantly higher than CBT4084FNR (WT), which showed only short growth in the first hour into anaerobic environment, followed by OD under anaerobic culture conditions ₆₀₀ The values began to drop rapidly, showing massive lysis of the cells, whereas CBT4084FNR (R169G), CBT4084FNR (L139K), CBT4084FNR (G216D) grew significantly continuously for the first two hours after entering anaerobic environment, and OD for the next two hours ₆₀₀ The values remain substantially stable.

As shown in FIGS. 2A and 2B, CBT4084FNR (WT) had begun to express IL-10 at 4 hours of culture under aerobic conditions, after which the IL-10 production increased rapidly, indicating that the dissolved oxygen content of the medium had not been able to inhibit the PfnrS promoter in CBT4084FNR (WT) and after 5 hours the IL-10 production began to decrease rapidly, indicating that the bacteria had been inactivated. In the whole aerobic culture process, CBT4084FNR (R169G), CBT4084FNR (L139Q) and CBT4084FNR (G216D) have only very small IL-10 expression, which indicates that the three FNR mutants can strictly inhibit the PfnrS promoter under the aerobic condition.

In anaerobic environments, the IL-10 production of CBT4084FNR (WT) begins to decline after a short rapid rise, indicating that the majority of IL-10 in the supernatant is released by cell lysis, rather than being secreted by living cells, and that IL-10 also begins to degrade after cell lysis. CBT4084FNR (L139Q) also had no expression of IL-10 under anaerobic conditions, indicating that this FNR mutant completely lost the ability to activate the PfnrS promoter, whereas both CBT4084FNR (R169G) and CBT4084FNR (G216D) showed a sustained increase in IL-10 production, indicating that both FNR mutants were able to normally activate PfnrS under anaerobic conditions while strictly inhibiting PfnrS under aerobic conditions, and that strains regulated by both FNR mutants maintained better stability and activity under anaerobic conditions.

In addition, it is notable that under anaerobic conditions, the overall IL-10 yield of CBT4084FNR (R169G), CBT4084FNR (G216D) is significantly higher than that of CBT4084FNR (WT), indicating that changes in FNR regulatory stringency by the R169G and G216D point mutations are beneficial not only for maintaining cell stability, but also for increasing the overall IL-10 yield.

EXAMPLE 4 modulation of the IL-22 Gene of interest by FNR muteins

In order to prove that FNR mutation has universality on regulation of a target gene, engineering bacteria expressing IL-22 are constructed, and the strain construction method is carried out according to example 2, so that the strains CBT4098FNR (WT) and CBT4098FNR (R169G) are obtained. The IL-22 coding sequences in this example are all sequences for coding wild type human IL-22 (SEQ ID NO: 19), the inserted locus is the kefB of EcN genome, the sequence of the sgRNA of the targeting kefB locus is shown as SEQ ID NO:20, the two homologous arm sequences of the kefB locus are shown as SEQ ID NO:21 and 22 respectively, the nucleotide sequences of the FNR are shown as SEQ ID NO:1 and SEQ ID NO:3, the nucleotide sequence of the promoter PfnrS is shown as SEQ ID NO:15, the amino acid sequence of the signal peptide USP45 is shown as SEQ ID NO:16, the terminator rrnB_T1_T7Te_is obtained by concatenating rrnB_T1_terminator and T7Te_terminator, the sequence of rrnB_T1_terminator is shown as SEQ ID NO:17, and the sequence of the T7Te_terminator is shown as SEQ ID NO: 18.

IL-22 amino acid sequence (SEQ ID NO: 19)

APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERC YLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQK LKDTVKKLGESGEIKAIGELDLLFMSLRNACI*

The sgRNA sequence targeting the kefB site (SEQ ID NO: 20):

gccggaagacactatgaagc

the Left Homology Arm (LHA) sequence of the kefB site (SEQ ID NO: 21):

ttgtttatggatgcgctggggttgtcgatggcgctcggtacgtttattgcgggtgtgctactggcggaaagtgaatatcgccatgaactggaaacggctatcgatcccttcaaaggcttgctgctcggtttgttctttatctctgtcggcatgtcgctcaacctcggggtgctttatacccatctgttgtgggtagtgataagtgtggttgtgctggtggcggtgaaaattctcgtgctgtatctgctggcgcgattgtatggcgtgcgcagttctgagcggatgcagtttgctggcgtgttgagtcagggcggtgagtttgcctttgtcctcttttctaccgcttcttcacaacgcttattccagggcgaccagat

the Right Homology Arm (RHA) sequence of the kefB site (SEQ ID NO: 22):

ttgcatattcttgcgcgagcgcgcggacgtgtggaagcgcatgagttattacaggcaggggtgacgcagttttcccgtgaaacattctccagtgcgttagagctggggcgcaagacgctggtcacgcttggcatgcatccgcatcaggcgcagcgcgcgcaactgcattttcgccgcctggatatgcgaatgctgcgggagttaatcccgatgcatgctgataccgtacaaatttctcgcgccagggaagcccgacgtgaactggaagagattttccagcgtgaaatgcaacaagaacgacgccagctggacggctgggatgaatttgagt

the method for detecting the regulation of the FNR mutein on the target gene IL-22 was the same as in example 2. The yields of IL-22 in the supernatant and cell pellet were measured using ELISA KIT (KIT 13059, yinqiao), and the sum of the yields of IL-22 in supernatant and the yields of precipitated IL-22 was the total yield of IL-22.

As shown in fig. 3A and 3B, CBT4098FNR (R169G) containing the FNR mutant gene grew at a significantly higher rate than CBT4098FNR (WT) under aerobic culture conditions; under anaerobic culture conditions, CBT4098FNR (WT) grew briefly only the first hour into the anaerobic environment, OD two hours after entering the anaerobic environment ₆₀₀ The values began to drop rapidly, showing massive lysis of the cells, while CBT4098FNR (R169G) grew significantly continuously for the first three hours after entering anaerobic environment, and OD for the next hour ₆₀₀ The values remain substantially stable.

As shown in FIGS. 4A and 4B, under aerobic conditions, CBT4098FNR (WT) had begun to express IL-22 after 4 hours of culture and the yield of IL-22 had rapidly increased after 4 hours of culture, while CBT4098FNR (R169G) had only minimal IL-22 expression throughout the aerobic culture, indicating that the strict inhibition of the PFnrS promoter by this FNR mutant was equally applicable to IL-22 expression.

In anaerobic environment, the IL-22 production of CBT4098FNR (WT) stopped increasing after culturing for 6 hours, while the IL-22 production of CBT4098FNR (R169G) remained continuously increasing, and the IL-22 production of CBT4098FNR (WT) cultured for 7 hours was significantly higher than the highest production of CBT4098FNR (WT). This shows that for the IL-22 expressing strain, the R169G point mutation also increases IL-22 production while maintaining cell stability.

Claims (10)

1. A FNR mutant is characterized by having R169G and/or G216D mutation on the amino acid sequence of the FNR, wherein the amino acid sequence of the FNR is shown as SEQ ID NO. 1 or has 99% identity with the amino acid sequence shown as SEQ ID NO. 1.

2. The FNR mutant of claim 1, wherein the amino acid sequence is set forth in SEQ ID No. 4 or SEQ ID No. 8.

3. The FNR mutant of claim 1 or 2, wherein the nucleotide sequence encoding the FNR mutant is as set forth in SEQ ID No. 3 or SEQ ID No. 7.

4. An isolated nucleic acid encoding the FNR mutant of any one of claims 1 to 3.

5. The isolated nucleic acid of claim 4, wherein the nucleotide sequence of the isolated nucleic acid is set forth in SEQ ID NO. 3 or SEQ ID NO. 7.

6. A nucleic acid construct comprising the isolated nucleic acid of claim 4 or 5 and a gene expression cassette comprising an expression regulatory element and a gene of interest;

preferably, the expression regulatory element is the PfnrS promoter;

more preferably, the gene of interest is selected from the group consisting of IL-10, IL-22.

7. A genetically modified cell expressing the FNR mutant of any one of claims 1 to 3 or the nucleic acid construct of claim 6.

8. The genetically modified cell of claim 7, wherein the host cell of the cell comprises a eukaryotic cell and a prokaryotic cell;

preferably, the host cell is a bacterium, preferably E.coli, such as E.coli Nissle 1917.

9. A method of increasing the stringency of regulation of an FNR-dependent promoter, comprising regulating a gene of interest with an FNR mutant of any one of claims 1 to 3 or an isolated nucleic acid of claim 4 or 5 or a nucleic acid construct of claim 6 or a genetically modified cell of claim 7 or 8 such that expression of the gene of interest is inhibited in a strictly anaerobic environment and expression is inhibited in an aerobic environment.

10. Use of the FNR mutant of any one of claims 1 to 3 or the isolated nucleic acid of claim 4 or 5 or the nucleic acid construct of claim 6 or the genetically modified cell of claim 7 or 8 for the preparation of an agent or for the regulation of gene expression.