Method for regulating multi-gene expression in microorganism and use thereof

Abstract

The present invention belongs to the field of genetic engineering. Specifically provided are a microorganism for regulating multi-gene expression, an expression vector, a promoter, and a method for regulating multi-gene expression in a microorganism, and the use thereof. The method for regulating multi-gene expression uses a plurality of inducible expression systems, and the plurality of inducible expression systems comprise a plurality of inducers, a plurality of regulatory proteins responsive to the inducers, a plurality of promoters that act with the regulatory proteins responsive to the inducers, and product synthesis genes controlled by the inducers. By changing the concentration of each inducer, the expression intensity of each gene can be changed. By means of the method, control of the expression intensity of each gene in a microorganism can be realized, and thus accurate control of the gene expression intensity and rapid construction of high-yield strains can be realized, which is conducive to balancing metabolic flux during the synthesis of metabolites in a microorganism and increasing the yield of biosynthesis. The method is of great significance for biosynthesis and "green manufacturing".

Description

A method for regulating multi-gene expression in microorganisms and its application Technical Field

[0001] The present invention relates to the field of genetic engineering, specifically to the field of microbial genetic engineering technology, and in particular to a method for regulating the expression of multiple genes in microorganisms using multiple inducible expression systems and its application. Background Art

[0002] Biosynthesis has brought a wide range of products to human production and life, and has broad prospects (Ko et al., 2020). Biosynthesis generally occurs within microorganisms and requires the participation of enzymes, which are encoded by genes related to the biosynthetic pathway. Generally speaking, biosynthesis requires multiple enzymatic reactions to convert substrates into target products. In other words, more than one enzyme catalyzes biosynthesis, and the product of the previous enzymatic reaction is the substrate for the next enzymatic reaction. However, due to the different catalytic efficiencies of the enzymes used in each enzymatic reaction, if the genes related to the biosynthetic pathway are expressed with the same intensity, that is, the same number of enzymes are provided for each enzymatic reaction, the products of the enzymes with high catalytic efficiency cannot be fully utilized by the enzymes with low catalytic efficiency, which in turn leads to the accumulation of intermediates in the cell and metabolic imbalance in the cell. Intermediates can inhibit the reaction rate, and toxic intermediates can also affect cell growth. In addition, unreasonable gene expression intensity can also cause overexpression of enzymes, resulting in waste of material and energy, and even affecting bacterial growth, ultimately reducing the yield of the target product (Farmer and Liao, 2000; Jung et al., 2021; Kent and Dixon, 2020; Li et al., 2022; Pitera et al., 2007).

[0003] If the expression intensity of each gene involved in biosynthesis could be individually regulated to achieve specific expression levels, a more optimized gene expression strategy could be achieved, allowing the product of each enzymatic reaction to be fully converted in the next enzymatic reaction, thereby reducing the accumulation of intermediates, balancing the metabolic flux of cells during biosynthesis, and increasing the yield of synthesized products (Meyer et al., 2019). To achieve this goal, it is necessary to develop methods that can individually regulate the expression intensity of multiple genes in the biosynthetic chassis cells.

[0004] Inducible expression systems can be used to regulate the expression intensity of genes. From the working principle, inducible expression systems can be divided into inhibitory inducible expression systems and activating inducible expression systems. Both rely on chemical signals (inducers), regulatory proteins and their corresponding inducible promoters to achieve gene expression regulation. Specifically, in the activating inducible expression system, the regulatory protein is an activator protein. In the absence of an inducer, the regulatory protein binds to the inducible promoter but cannot recruit RNA polymerase, and gene transcription is turned off. When an inducer is added, the inducer binds to the regulatory protein, and the regulatory protein undergoes a conformational change, allowing RNA polymerase to bind to the promoter, and gene transcription is turned on. In the inhibitory inducible expression system, the regulatory protein is a repressor protein. In the absence of an inducer, the regulatory protein binds to the inducible promoter, forming a steric hindrance to RNA polymerase, and gene transcription is turned off. When an inducer is added, the inducer binds to the regulatory protein, and the regulatory protein undergoes a conformational change, leaving the promoter region, allowing RNA polymerase to bind to the promoter, and gene transcription is turned on. In an inducible expression system, the expression intensity of a gene is controlled by the inducer concentration. Within a certain range, the higher the inducer concentration, the stronger the gene expression intensity (Li et al., 2022).

[0005] Therefore, a comprehensive multiple inducible expression system is needed to regulate the expression intensity of each gene involved in biosynthesis separately, so that it is expressed at a specific intensity and an optimized gene expression scheme is obtained.

[0006] Summary of the Invention

[0007] The present invention discloses a method for regulating the expression of multiple genes in microorganisms, using a variety of inducible expression systems, including multiple inducers, multiple regulatory proteins that respond to the inducers, and multiple promoters that interact with the regulatory proteins, as well as genes with biological functions controlled by them. By varying the concentration of each inducer, the expression intensity of each gene can be varied, achieving precise control of gene expression intensity and rapid construction of high-yield strains. Specifically,

[0008] In a first aspect, the present invention provides a microorganism for regulating the expression of multiple genes, wherein the microorganism comprises an expression vector, wherein the expression vector comprises a promoter that interacts with a regulatory protein, a nucleotide sequence encoding the regulatory protein, and a product synthesis gene.

[0009] Preferably, the nucleotide sequence encoding the regulatory protein and the product synthesis gene can be on one expression vector or two expression vectors, and the copy number of the expression vector can be one, two or more.

[0010] Preferably, the regulatory protein is selected from the group consisting of AraC, a regulatory protein that responds to arabinose, CinR, a regulatory protein that responds to OHC14 (N-(3-hydroxy-tetradecanoyl)-homoserine lactone), CymR, a regulatory protein that responds to cuminic acid, NahR, a regulatory protein that responds to sodium salicylate, VanR, a regulatory protein that responds to vanillic acid, TetR, a regulatory protein that responds to anhydrotetracycline, LuxR, a regulatory protein that responds to OC6 (N-(3-oxo-hexanoyl)-homoserine lactone), TtgR, a regulatory protein that responds to naringenin, PhlF, a regulatory protein that responds to DAPG (2,4-diacetylphloroglucinol), and one or more proteins with the same functions as the above regulatory proteins.

[0011] Further preferably, the regulatory protein AraC comprises SEQ ID No. 1 or comprises an amino acid sequence having more than 80% homology with SEQ ID No. 1 and having the same or similar activity.

[0012] The regulatory protein CinR comprises SEQ ID No. 2 or comprises an amino acid sequence having more than 80% homology with SEQ ID No. 2 and having the same or similar activity.

[0013] The regulatory protein CymR comprises SEQ ID No. 3 or an amino acid sequence having more than 80% homology with SEQ ID No. 3 and having the same or similar activity.

[0014] The regulatory protein NahR comprises SEQ ID No. 4 or comprises an amino acid sequence having more than 80% homology with SEQ ID No. 4 and having the same or similar activity.

[0015] The regulatory protein VanR comprises SEQ ID No. 5 or an amino acid sequence having more than 80% homology with SEQ ID No. 5 and having the same or similar activity.

[0016] The regulatory protein TetR comprises SEQ ID No. 6 or an amino acid sequence having more than 80% homology with SEQ ID No. 6 and having the same or similar activity.

[0017] The regulatory protein LuxR comprises SEQ ID No. 7 or an amino acid sequence having more than 80% homology with SEQ ID No. 7 and having the same or similar activity.

[0018] The regulatory protein TtgR comprises SEQ ID No. 8 or an amino acid sequence having more than 80% homology with SEQ ID No. 8 and having the same or similar activity.

[0019] The regulatory protein PhlF comprises SEQ ID No. 9 or an amino acid sequence having more than 80% homology with SEQ ID No. 9 and having the same or similar activity.

[0020] Preferably, the promoter that interacts with the regulatory protein is an inducible promoter, and the inducible promoter contains a binding site for the regulatory protein.

[0021] Preferably, the inducible promoter can be selected from conventional inducible promoters in the prior art, as long as the inducible promoter contains a binding site for a regulatory protein and has inducible promoter activity. Further preferably, the inducible promoter is selected from a promoter P with a binding site for a regulatory protein AraC. BAD_NNNN , promoter P with regulatory protein CinR binding site Cin_NNNN , promoter P with regulatory protein CymR binding site Cym_NNNN , promoter P with a binding site for the regulatory protein NahR Sal_NNNN , promoter P with regulatory protein VanR binding site Van_NNNN , promoter P with regulatory protein TetR binding site Tet_NNNN , promoter P with a binding site for the regulatory protein LuxR Lux_NNNN , promoter P with regulatory protein TtgR binding site Ttg_NNNN , promoter P with a binding site for the regulatory protein PhlF PhlF_NNNN One or more of the following.

[0022] Further preferably, the promoter P BAD_NNNN The invention relates to a nucleotide sequence comprising SEQ ID No. 20 or comprising a nucleotide sequence having more than 80% homology with SEQ ID No. 20 and having the same or similar activity.

[0023] The promoter P BAD_NNNN The binding site of the regulatory protein comprises a nucleotide sequence shown in any one of SEQ ID NOs. 38-41.

[0024] SEQ ID NO.38

[0025] SEQ ID NO.39

[0026] SEQ ID NO.40

[0027] SEQ ID NO.41

[0028] The promoter P Cin_NNNN The invention relates to a nucleotide sequence comprising SEQ ID No. 21 or comprising a nucleotide sequence having 80% or more homology to SEQ ID No. 21 and having the same or similar activity.

[0029] The promoter P Cin_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.42.

[0030] SEQ ID NO.42

[0031] The promoter P Cym_NNNN The invention relates to a nucleotide sequence comprising SEQ ID No. 22 or comprising a nucleotide sequence having more than 80% homology with SEQ ID No. 22 and having the same or similar activity.

[0032] The promoter P Cym_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.43.

[0033] SEQ ID NO.43

[0034] The promoter P Sal_NNNN The invention relates to a nucleotide sequence comprising SEQ ID No. 23 or comprising a nucleotide sequence having more than 80% homology with SEQ ID No. 23 and having the same or similar activity.

[0035] The promoter P Sal_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.44.

[0036] SEQ ID NO.44

[0037] The promoter P Van_NNNN The invention relates to a nucleotide sequence comprising SEQ ID No. 24 or a nucleotide sequence having 80% or more homology to SEQ ID No. 24 and having the same or similar activity.

[0038] The promoter P Van_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.45.

[0039] SEQ ID NO.45

[0040] The promoter P Tet_NNNNThe invention relates to a nucleotide sequence comprising SEQ ID No. 25 or comprising a nucleotide sequence having more than 80% homology with SEQ ID No. 25 and having the same or similar activity.

[0041] The promoter P Tet_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.46.

[0042] SEQ ID NO.46

[0043] The promoter P Lux_NNNN The invention relates to a nucleotide sequence comprising SEQ ID No. 26 or comprising a nucleotide sequence having more than 80% homology with SEQ ID No. 26 and having the same or similar activity.

[0044] The promoter P Lux_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.47.

[0045] SEQ ID NO.47

[0046] The promoter P Ttg_NNNN The invention relates to a nucleotide sequence comprising SEQ ID No. 27 or comprising a nucleotide sequence having more than 80% homology with SEQ ID No. 27 and having the same or similar activity.

[0047] The promoter P Ttg_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.48.

[0048] SEQ ID NO.48

[0049] The promoter P PhlF_NNNN The invention relates to a nucleotide sequence comprising SEQ ID No. 28 or comprising a nucleotide sequence having more than 80% homology with SEQ ID No. 28 and having the same or similar activity.

[0050] The promoter P PhlF_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.49.

[0051] SEQ ID NO.49

[0052] Preferably, N in SEQ ID No. 20-28 can be any one of A, T, C, and G.

[0053] Preferably, the promoter P BAD_NNNN comprising SEQ ID No. 20, wherein NNNN is ACAC, TTGC, GGCG, AGAG, ACTC or GCTT; or, the promoter P Cin_NNNN Including SEQ ID No.21, wherein NNNN is GAAC, GCCC, GGGG, TGGG, TCAT or TTAA; or, the promoter P Cym_NNNN Including SEQ ID No.22, wherein NNNN is GCTG, AGGA, GAGA, TCTG, CCGG or CCAC; or, the promoter P Sal_NNNN comprising SEQ ID No.14; or, the promoter P Van_NNNN comprising SEQ ID No. 15; or, the promoter P Tet_NNNN comprising SEQ ID No.16; or, the promoter P Lux_NNNN comprising SEQ ID No. 26, wherein NNNN is CTGT, GGTG, GTGG, ATGT, TTGT or CATG; or, the promoter P Ttg_NNNN comprising SEQ ID No.18; or, the promoter P PhlF_NNNN Including SEQ ID No.19.

[0054] Preferably, the inducible promoter is selected from P BAD or its mutants, P Cin or its mutants, P Cym or its mutants, P Sal or its mutants, P Van or its mutants, P Tet or its mutants, P Lux or its mutants, P Ttg or its mutants, P PhlF or one or more of its mutants.

[0055] Further preferably, the mutant includes a mutation of at least one base, more preferably, the mutant includes a mutation of at least one base in the first four bases of the original promoter -10 region, and the four bases can be any combination of A, T, C, and G.

[0056] Further preferably, the promoter P BAD It comprises the nucleotide sequence shown in SEQ ID No.11.

[0057] The promoter P Cin It comprises the nucleotide sequence shown in SEQ ID No.12.

[0058] The promoter P Cym It comprises the nucleotide sequence shown in SEQ ID No.13.

[0059] The promoter P Sal It comprises the nucleotide sequence shown in SEQ ID No.14.

[0060] The promoter P Van It comprises the nucleotide sequence shown in SEQ ID No.15.

[0061] The promoter P Tet It comprises the nucleotide sequence shown in SEQ ID No.16.

[0062] The promoter P Lux It comprises the nucleotide sequence shown in SEQ ID No.17.

[0063] The promoter P Ttg It comprises the nucleotide sequence shown in SEQ ID No.18.

[0064] The promoter P PhlF It comprises the nucleotide sequence shown in SEQ ID No.19.

[0065] In a specific embodiment of the present invention, the mutant comprises the nucleotide sequence shown in any one or more of SEQ ID No. 29-37.

[0066] Preferably, the microorganism is a eukaryotic microorganism and / or a prokaryotic microorganism, the prokaryotic microorganism includes any one of the genus Escherichia, Ralstonia, Bacillus Cohn, Corynebacterium, Alcaligenes, Pseudomonas, Aeromonas or Halomonas, and the eukaryotic microorganism includes any one of yeast, fungi or algae.

[0067] Preferably, the genus Escherichia includes Escherichia coli, the genus Ralstonia includes Ralstonia eutropha, the genus Bacillus includes Bacillus subtilis, the genus Corynebacterium includes Corynebacterium glutamicum, the genus Alcaligenes includes Alcaligenes faecalis, the genus Pseudomonas includes Pseudomonas entomophila, the genus Aeromonas includes Aeromonas hydrophila, and the genus Halomonas includes any one of Halomonas bluephagenesis, Halomonas campaniensis and Halomonas aydingkolgenesis.

[0068] In a specific embodiment of the present invention, the Halomonas includes but is not limited to Halomonas bluephagenesis TD01 CGMCC.No.4353, Halomonas campaniensis LS21 CGMCC No.6593 and Halomonas aydingkolgenesis M1 CGMCC No.19880.

[0069] Preferably, the product comprises an intracellular product, an enzyme secreted outside the cell, a protein related to cell morphology, or a protein or RNA related to gene regulation in the cell.

[0070] Preferably, the product comprises one or more of PHA, tetrahydropyrimidine, amino acids, inositol, 1,3-propanediol, 3-hydroxypropionic acid, 1,5-pentanediamine, 5-aminolevulinic acid, lipase, amylase, saccharifying enzyme, protease, cellulase, esterase, glucanase, pectinase, phospholipase, gelatinase, nuclease, microtubule protein FtsZ, cytoskeletal protein MreB, cell division regulatory factor MinCD, lactose operon repressor protein LacI, tetracycline operon repressor protein TetR, sgRNA used in CRISPRi / CRISPRa, or lycopene.

[0071] In one embodiment, the amino acid is arginine.

[0072] Preferably, the PHA is selected from 3-hydroxybutyric acid homopolymer PHB, 3-hydroxybutyric acid and 4-hydroxybutyric acid copolymer P3HB4HB, 3-hydroxybutyric acid, 4-hydroxybutyric acid and 3-hydroxyvaleric acid terpolymer PHBV4HB, 3-hydroxybutyric acid and 3-hydroxyhexanoic acid copolymer PHBHHx, a homopolymer or copolymer of 3-hydroxypropionic acid, the homopolymer of 3-hydroxypropionic acid is P3HP, and the copolymer of 3-hydroxypropionic acid is P(3HB-co-3HP) or PHBHP.

[0073] The product also contains various extracellular small molecule products and / or various extracellular hydrolases.

[0074] Preferably, the lycopene corresponding product synthesis genes include dxs, crtE, crtI, crtB, idi and dxs.

[0075] Preferably, the PHA corresponding product synthesis genes include phaA, phaB and phaC.

[0076] Preferably, the genes for synthesizing products corresponding to ectoine include ectA, ectB and ectC.

[0077] Preferably, the arginine corresponding product synthesis genes include gdh, argJ, argB, argC, argD, argF, argG, argH and pgl.

[0078] Preferably, an inducer is added to the microorganism during cultivation. The inducer binds to the regulatory protein, causing a conformational change in the regulatory protein, enabling RNA polymerase to bind to the promoter and initiating gene transcription. If no inducer is added, or if a substance that neutralizes or inhibits the inducer is added, the regulatory protein binds to the inducible promoter but is unable to recruit RNA polymerase, shutting down gene transcription. The intensity of gene expression is proportional to the concentration of the inducer. By varying the concentration of each inducer, the expression intensity of each gene can be altered.

[0079] Preferably, the inducer comprises one or more of arabinose, OHC14, cuminic acid, sodium salicylate, vanillic acid, anhydrotetracycline, OC6, naringenin and DAPG.

[0080] Preferably, the regulation of multi-gene expression uses a variety of inducible expression systems, which include inducers, regulatory proteins, and promoters that act with the regulatory proteins.

[0081] Preferably, the arabinose-inducible expression system comprises an inducer, arabinose, a regulatory protein AraC, and a promoter with a regulatory protein AraC binding site (e.g., P BAD_NNNN ).

[0082] Preferably, the OHC14 inducible expression system comprises an inducer OHC14, a regulatory protein CinR, and a promoter with a regulatory protein CinR binding site (e.g., P Cin_NNNN ).

[0083] Preferably, the cuminic acid inducible expression system comprises an inducer cuminic acid, a regulatory protein CymR, and a promoter with a regulatory protein CymR binding site (e.g., P Cym_NNNN ).

[0084] Preferably, the sodium salicylate inducible expression system comprises an inducer sodium salicylate, a regulatory protein NahR, and a promoter with a regulatory protein NahR binding site (e.g., P Sal_NNNN ).

[0085] Preferably, the vanillic acid inducible expression system comprises an inducer, vanillic acid, a regulatory protein VanR, and a promoter with a VanR binding site (e.g., P Van_NNNN ).

[0086] Preferably, the anhydrotetracycline inducible expression system comprises anhydrotetracycline as an inducer, a regulatory protein TetR, and a promoter with a regulatory protein TetR binding site (e.g., P Tet_NNNN ).

[0087] Preferably, the OC6 inducible expression system comprises an inducer OC6, a regulatory protein LuxR, and a promoter with a regulatory protein LuxR binding site (e.g., P Lux_NNNN ).

[0088] Preferably, the naringenin inducible expression system comprises an inducer naringenin, a regulatory protein TtgR, and a promoter with a regulatory protein TtgR binding site (e.g., P Ttg_NNNN ).

[0089] Preferably, the DAPG inducible expression system comprises an inducer DAPG, a regulatory protein PhlF and a promoter with a regulatory protein PhlF binding site (e.g., P PhlF_NNNN ).

[0090] Preferably, the inducible expression system comprises the following combination:

[0091] 1) Vanillic acid, OC6, OHC14, arabinose, and sodium salicylate inducible expression systems;

[0092] 2) Anhydrotetracycline, cuminic acid, and DAPG inducible expression systems;

[0093] 3) Naringenin, arabinose, and OHC14 inducible expression systems;

[0094] 4) Arabinose, OHC14, cuminic acid, sodium salicylate, vanillic acid, anhydrotetracycline, OC6, naringenin, and DAPG inducible expression systems; or,

[0095] 5) Sodium salicylate, OHC14 and arabinose inducible expression system.

[0096] Preferably, the expression vector is capable of replication, transcription, and translation in microorganisms. Therefore, it also contains other conventional expression elements, such as terminators, restriction enzyme cleavage sites, etc.

[0097] Preferably, the expression vector is a plasmid.

[0098] The second aspect of the present invention provides an expression vector comprising at least one inducible expression system.

[0099] Preferably, the inducible expression system includes an inducer, a regulatory protein, and a promoter that acts on the regulatory protein.

[0100] Preferably, the inducible expression system includes one or more selected from the group consisting of arabinose inducible expression system, N-(3-hydroxy-tetradecanoyl)-homoserine lactone (OHC14) inducible expression system, cuminic acid inducible expression system, sodium salicylate inducible expression system, vanillic acid inducible expression system, anhydrotetracycline inducible expression system, N-(3-oxy-hexanoyl)-homoserine lactone (OC6) inducible expression system, naringenin inducible expression system and 2,4-diacetylphloroglucinol (DAPG) inducible expression system.

[0101] Preferably, the arabinose-inducible expression system comprises an inducer, arabinose, a regulatory protein AraC, and a promoter with a regulatory protein AraC binding site (e.g., P BAD_NNNN ).

[0102] Preferably, the OHC14 inducible expression system comprises an inducer OHC14, a regulatory protein CinR, and a promoter with a regulatory protein CinR binding site (e.g., P Cin_NNNN ).

[0103] Preferably, the cuminic acid inducible expression system comprises an inducer cuminic acid, a regulatory protein CymR, and a promoter with a regulatory protein CymR binding site (e.g., P Cym_NNNN ).

[0104] Preferably, the sodium salicylate inducible expression system comprises an inducer sodium salicylate, a regulatory protein NahR, and a promoter with a regulatory protein NahR binding site (e.g., PSal_NNNN ).

[0105] Preferably, the vanillic acid inducible expression system comprises an inducer, vanillic acid, a regulatory protein VanR, and a promoter with a VanR binding site (e.g., P Van_NNNN ).

[0106] Preferably, the anhydrotetracycline inducible expression system comprises anhydrotetracycline as an inducer, a regulatory protein TetR, and a promoter with a regulatory protein TetR binding site (e.g., P Tet_NNNN ).

[0107] Preferably, the OC6 inducible expression system comprises an inducer OC6, a regulatory protein LuxR, and a promoter with a regulatory protein LuxR binding site (e.g., P Lux_NNNN ).

[0108] Preferably, the naringenin inducible expression system comprises an inducer naringenin, a regulatory protein TtgR, and a promoter with a regulatory protein TtgR binding site (e.g., P Ttg_NNNN ).

[0109] Preferably, the DAPG inducible expression system comprises an inducer DAPG, a regulatory protein PhlF and a promoter with a regulatory protein PhlF binding site (e.g., P PhlF_NNNN ).

[0110] Further preferably, the regulatory protein AraC comprises SEQ ID No. 1 or comprises an amino acid sequence having more than 80% homology with SEQ ID No. 1 and having the same or similar activity.

[0111] The regulatory protein CinR comprises SEQ ID No. 2 or comprises an amino acid sequence having more than 80% homology with SEQ ID No. 2 and having the same or similar activity.

[0112] The regulatory protein CymR comprises SEQ ID No. 3 or an amino acid sequence having more than 80% homology with SEQ ID No. 3 and having the same or similar activity.

[0113] The regulatory protein NahR comprises SEQ ID No. 4 or comprises an amino acid sequence having more than 80% homology with SEQ ID No. 4 and having the same or similar activity.

[0114] The regulatory protein VanR comprises SEQ ID No. 5 or an amino acid sequence having more than 80% homology with SEQ ID No. 5 and having the same or similar activity.

[0115] The regulatory protein TetR comprises SEQ ID No. 6 or an amino acid sequence having more than 80% homology with SEQ ID No. 6 and having the same or similar activity.

[0116] The regulatory protein LuxR comprises SEQ ID No. 7 or an amino acid sequence having more than 80% homology with SEQ ID No. 7 and having the same or similar activity.

[0117] The regulatory protein TtgR comprises SEQ ID No. 8 or an amino acid sequence having more than 80% homology with SEQ ID No. 8 and having the same or similar activity.

[0118] The regulatory protein PhlF comprises SEQ ID No. 9 or an amino acid sequence having more than 80% homology with SEQ ID No. 9 and having the same or similar activity.

[0119] Further preferably, the promoter P BAD_NNNN The invention relates to a nucleotide sequence comprising SEQ ID No. 20 or comprising a nucleotide sequence having more than 80% homology with SEQ ID No. 20 and having the same or similar activity.

[0120] The promoter P BAD_NNNN The binding site of the regulatory protein comprises a nucleotide sequence shown in any one of SEQ ID NOs. 38-41.

[0121] The promoter P Cin_NNNN The invention relates to a nucleotide sequence comprising SEQ ID No. 21 or comprising a nucleotide sequence having 80% or more homology to SEQ ID No. 21 and having the same or similar activity.

[0122] The promoter P Cin_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.42.

[0123] The promoter P Cym_NNNN The invention relates to a nucleotide sequence comprising SEQ ID No. 22 or comprising a nucleotide sequence having more than 80% homology with SEQ ID No. 22 and having the same or similar activity.

[0124] The promoter P Cym_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.43.

[0125] The promoter P Sal_NNNNThe invention relates to a nucleotide sequence comprising SEQ ID No. 23 or comprising a nucleotide sequence having more than 80% homology with SEQ ID No. 23 and having the same or similar activity.

[0126] The promoter P Sal_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.44.

[0127] The promoter P Van_NNNN The invention relates to a nucleotide sequence comprising SEQ ID No. 24 or a nucleotide sequence having 80% or more homology to SEQ ID No. 24 and having the same or similar activity.

[0128] The promoter P Van_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.45.

[0129] The promoter P Tet_NNNN The invention relates to a nucleotide sequence comprising SEQ ID No. 25 or comprising a nucleotide sequence having more than 80% homology with SEQ ID No. 25 and having the same or similar activity.

[0130] The promoter P Tet_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.46.

[0131] The promoter P Lux_NNNN The invention relates to a nucleotide sequence comprising SEQ ID No. 26 or comprising a nucleotide sequence having more than 80% homology with SEQ ID No. 26 and having the same or similar activity.

[0132] The promoter P Lux_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.47.

[0133] The promoter P Ttg_NNNN The invention relates to a nucleotide sequence comprising SEQ ID No. 27 or comprising a nucleotide sequence having more than 80% homology with SEQ ID No. 27 and having the same or similar activity.

[0134] The promoter P Ttg_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.48.

[0135] The promoter P PhlF_NNNN The invention relates to a nucleotide sequence comprising SEQ ID No. 28 or comprising a nucleotide sequence having more than 80% homology with SEQ ID No. 28 and having the same or similar activity.

[0136] The promoter P PhlF_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.49.

[0137] Among them, N in SEQ ID No. 20-28 can be any one of A, T, C, and G.

[0138] More preferably, the promoter P BAD_NNNN Contains P BAD or its mutants, further preferably, the promoter P BAD It comprises the nucleotide sequence shown in SEQ ID No.11.

[0139] The promoter P Cin_NNNN Contains P Cin or its mutants, further preferably, the promoter P Cin It comprises the nucleotide sequence shown in SEQ ID No.12.

[0140] The promoter P Cym_NNNN Contains P Cym or its mutants, further preferably, the promoter P Cym It comprises the nucleotide sequence shown in SEQ ID No.13.

[0141] The promoter P Sal_NNNN Contains P Sal or its mutants, further preferably, the promoter P Sal It comprises the nucleotide sequence shown in SEQ ID No.14.

[0142] The promoter P Van_NNNN Contains P Van or its mutants, further preferably, the promoter P Van It comprises the nucleotide sequence shown in SEQ ID No.15.

[0143] The promoter P Tet_NNNN Contains P Tet or its mutants, further preferably, the promoter P Tet It comprises the nucleotide sequence shown in SEQ ID No.16.

[0144] The promoter P Lux_NNNN Contains P Lux or its mutants, further preferably, the promoter P Lux It comprises the nucleotide sequence shown in SEQ ID No.17.

[0145] The promoter PTtg_NNNN Contains P Ttg or its mutants, further preferably, the promoter P Ttg It comprises the nucleotide sequence shown in SEQ ID No.18.

[0146] The promoter P PhlF_NNNN Contains P PhlF or its mutants, further preferably, the promoter P PhlF It comprises the nucleotide sequence shown in SEQ ID No.19.

[0147] Wherein, the mutant includes a mutation of at least one base. Preferably, the mutant includes a mutation of at least one base in the first four bases of the original promoter -10 region, and the four bases can be any combination of A, T, C, and G.

[0148] In a specific embodiment of the present invention, the mutant comprises the nucleotide sequence shown in any one or more of SEQ ID No. 29-37.

[0149] In a specific embodiment of the present invention, the inducible expression system comprises the following combination:

[0150] 1) Vanillic acid, OC6, OHC14, arabinose, and sodium salicylate inducible expression systems;

[0151] 2) Anhydrotetracycline, cuminic acid, and DAPG inducible expression systems;

[0152] 3) Naringenin, arabinose, and OHC14 inducible expression systems;

[0153] 4) Arabinose, OHC14, cuminic acid, sodium salicylate, vanillic acid, anhydrotetracycline, OC6, naringenin, and DAPG inducible expression systems; or,

[0154] 5) Sodium salicylate, OHC14 and arabinose inducible expression system.

[0155] Preferably, the expression vector also includes a product synthesis gene.

[0156] Preferably, the product comprises an intracellular product, an enzyme secreted outside the cell, a protein related to cell morphology, or a protein or RNA related to gene regulation in the cell.

[0157] Preferably, the product comprises one or more of PHA, tetrahydropyrimidine, amino acids, inositol, 1,3-propanediol, 3-hydroxypropionic acid, 1,5-pentanediamine, 5-aminolevulinic acid, lipase, amylase, saccharifying enzyme, protease, cellulase, esterase, glucanase, pectinase, phospholipase, gelatinase, nuclease, microtubule protein FtsZ, cytoskeletal protein MreB, cell division regulatory factor MinCD, lactose operon repressor protein LacI, tetracycline operon repressor protein TetR, sgRNA used in CRISPRi / CRISPRa, or lycopene.

[0158] In one embodiment, the amino acid is arginine.

[0159] Preferably, the PHA is selected from 3-hydroxybutyric acid homopolymer PHB, 3-hydroxybutyric acid and 4-hydroxybutyric acid copolymer P3HB4HB, 3-hydroxybutyric acid, 4-hydroxybutyric acid and 3-hydroxyvaleric acid terpolymer PHBV4HB, 3-hydroxybutyric acid and 3-hydroxyhexanoic acid copolymer PHBHHx, a homopolymer or copolymer of 3-hydroxypropionic acid, the homopolymer of 3-hydroxypropionic acid is P3HP, and the copolymer of 3-hydroxypropionic acid is P(3HB-co-3HP) or PHBHP.

[0160] The product also contains various extracellular small molecule products and / or various extracellular hydrolases.

[0161] Preferably, the product synthesis gene comprises one or more of dxs, crtE, crtI, crtB, idi, dxs, phaA, phaB, phaC, ectA, ectB, ectC, gdh, argJ, argB, argC, argD, argF, argG, argH and pgl.

[0162] The expression vector can replicate, transcribe and translate in the host cell. Therefore, it also contains other conventional expression elements, such as terminators, restriction enzyme cleavage sites, etc.

[0163] Preferably, the expression vector can be a prokaryotic expression vector or a eukaryotic expression vector, preferably a prokaryotic expression vector.

[0164] Preferably, the expression vector is a plasmid.

[0165] The third aspect of the present invention provides a method for regulating the expression of multiple genes in a microorganism, wherein the method comprises introducing the above-mentioned expression vector into the microorganism for culturing, or the method comprises culturing the above-mentioned microorganism.

[0166] Preferably, the method further comprises adding an inducer, and further preferably, the inducer comprises one or more of arabinose, N-(3-hydroxy-tetradecanoyl)-homoserine lactone (OHC14), cumic acid, sodium salicylate, vanillic acid, anhydrotetracycline, N-(3-oxy-hexanoyl)-homoserine lactone (OC6), naringenin and 2,4-diacetylphloroglucinol (DAPG).

[0167] Preferably, the gene expression intensity is proportional to the concentration of the added inducer.

[0168] Preferably, the regulation involves adding an inducer during the culture process. The inducer binds to the regulatory protein, causing a conformational change in the regulatory protein, enabling RNA polymerase to bind to the promoter and initiating gene transcription. If no inducer is added, or if a substance that neutralizes or inhibits the inducer is added, the regulatory protein binds to the inducible promoter but is unable to recruit RNA polymerase, shutting down gene transcription. The intensity of gene expression is proportional to the concentration of the inducer. By varying the concentration of each inducer, the expression intensity of each gene can be altered.

[0169] Preferably, the inducible expression system includes one or more of an arabinose inducible expression system, an OHC14 inducible expression system, a cuminic acid inducible expression system, a sodium salicylate inducible expression system, a vanillic acid inducible expression system, anhydrotetracycline inducible expression system, an OC6 inducible expression system, a naringenin inducible expression system, and a DAPG inducible expression system.

[0170] Preferably, the regulation of multi-gene expression uses a variety of inducible expression systems, which include inducers, regulatory proteins, and promoters that act with the regulatory proteins.

[0171] Preferably, the arabinose-inducible expression system comprises an inducer, arabinose, a regulatory protein AraC, and a promoter with a regulatory protein AraC binding site (e.g., P BAD_NNNN ).

[0172] Preferably, the OHC14 inducible expression system comprises an inducer OHC14, a regulatory protein CinR, and a promoter with a regulatory protein CinR binding site (e.g., P Cin_NNNN ).

[0173] Preferably, the cuminic acid inducible expression system comprises an inducer cuminic acid, a regulatory protein CymR, and a promoter with a regulatory protein CymR binding site (e.g., P Cym_NNNN ).

[0174] Preferably, the sodium salicylate inducible expression system comprises an inducer sodium salicylate, a regulatory protein NahR, and a promoter with a regulatory protein NahR binding site (e.g., P Sal_NNNN ).

[0175] Preferably, the vanillic acid inducible expression system comprises an inducer, vanillic acid, a regulatory protein VanR, and a promoter with a VanR binding site (e.g., P Van_NNNN ).

[0176] Preferably, the anhydrotetracycline inducible expression system comprises anhydrotetracycline as an inducer, a regulatory protein TetR, and a promoter with a regulatory protein TetR binding site (e.g., P Tet_NNNN ).

[0177] Preferably, the OC6 inducible expression system comprises an inducer OC6, a regulatory protein LuxR, and a promoter with a regulatory protein LuxR binding site (e.g., P Lux_NNNN ).

[0178] Preferably, the naringenin inducible expression system comprises an inducer naringenin, a regulatory protein TtgR, and a promoter with a regulatory protein TtgR binding site (e.g., P Ttg_NNNN ).

[0179] Preferably, the DAPG inducible expression system comprises an inducer DAPG, a regulatory protein PhlF and a promoter with a regulatory protein PhlF binding site (e.g., P PhlF_NNNN ).

[0180] Preferably, the inducible expression system comprises the following combination:

[0181] 1) Vanillic acid, OC6, OHC14, arabinose, and sodium salicylate inducible expression systems;

[0182] 2) Anhydrotetracycline, cuminic acid, and DAPG inducible expression systems;

[0183] 3) Naringenin, arabinose, and OHC14 inducible expression systems;

[0184] 4) Arabinose, OHC14, cuminic acid, sodium salicylate, vanillic acid, anhydrotetracycline, OC6, naringenin, and DAPG inducible expression systems; or,

[0185] 5) Sodium salicylate, OHC14 and arabinose inducible expression system.

[0186] Preferably, the microorganism is a eukaryotic microorganism and / or a prokaryotic microorganism, the prokaryotic microorganism includes any one of the genus Escherichia, Ralstonia, Bacillus Cohn, Corynebacterium, Alcaligenes, Pseudomonas, Aeromonas or Halomonas, and the eukaryotic microorganism includes any one of yeast, fungi or algae.

[0187] Preferably, the genus Escherichia includes Escherichia coli, the genus Ralstonia includes Ralstonia eutropha, the genus Bacillus includes Bacillus subtilis, the genus Corynebacterium includes Corynebacterium glutamicum, the genus Alcaligenes includes Alcaligenes faecalis, the genus Pseudomonas includes Pseudomonas entomophila, the genus Aeromonas includes Aeromonas hydrophila, and the genus Halomonas includes any one of Halomonas bluephagenesis, Halomonas campaniensis and Halomonas aydingkolgenesis.

[0188] In a specific embodiment of the present invention, the Halomonas includes but is not limited to Halomonas bluephagenesis TD01 CGMCC.No.4353, Halomonas campaniensis LS21 CGMCC No.6593 and Halomonas aydingkolgenesis M1 CGMCC No.19880.

[0189] Preferably, the expression vector integrates the promoter into the chromosomal genome of the microorganism or the expression vector is free in the microorganism.

[0190] A fourth aspect of the present invention provides a method for producing a product, wherein the method comprises introducing the above-mentioned expression vector into a microorganism for culturing, or the method comprises culturing the above-mentioned microorganism.

[0191] Preferably, the method further comprises adding an inducer, and further preferably, the inducer comprises one or more of arabinose, N-(3-hydroxy-tetradecanoyl)-homoserine lactone (OHC14), cumic acid, sodium salicylate, vanillic acid, anhydrotetracycline, N-(3-oxy-hexanoyl)-homoserine lactone (OC6), naringenin and 2,4-diacetylphloroglucinol (DAPG).

[0192] Preferably, the gene expression intensity is proportional to the concentration of the added inducer.

[0193] Preferably, the regulation involves adding an inducer during the culture process. The inducer binds to the regulatory protein, causing a conformational change in the regulatory protein, enabling RNA polymerase to bind to the promoter and initiating gene transcription. If no inducer is added, or if a substance that neutralizes or inhibits the inducer is added, the regulatory protein binds to the inducible promoter but is unable to recruit RNA polymerase, shutting down gene transcription. The intensity of gene expression is proportional to the concentration of the inducer. By varying the concentration of each inducer, the expression intensity of each gene can be altered.

[0194] Preferably, the regulation of multi-gene expression uses a variety of inducible expression systems, which include inducers, regulatory proteins, and promoters that act with the regulatory proteins.

[0195] Preferably, the inducible expression system includes one or more of an arabinose inducible expression system, an OHC14 inducible expression system, a cuminic acid inducible expression system, a sodium salicylate inducible expression system, a vanillic acid inducible expression system, anhydrotetracycline inducible expression system, an OC6 inducible expression system, a naringenin inducible expression system, and a DAPG inducible expression system.

[0196] Preferably, the arabinose-inducible expression system comprises an inducer, arabinose, a regulatory protein AraC, and a promoter with a regulatory protein AraC binding site (e.g., P BAD_NNNN ).

[0197] Preferably, the OHC14 inducible expression system comprises an inducer OHC14, a regulatory protein CinR, and a promoter with a regulatory protein CinR binding site (e.g., P Cin_NNNN ).

[0198] Preferably, the cuminic acid inducible expression system comprises an inducer cuminic acid, a regulatory protein CymR, and a promoter with a regulatory protein CymR binding site (e.g., P Cym_NNNN ).

[0199] Preferably, the sodium salicylate inducible expression system comprises an inducer sodium salicylate, a regulatory protein NahR, and a promoter with a regulatory protein NahR binding site (e.g., P Sal_NNNN ).

[0200] Preferably, the vanillic acid inducible expression system comprises an inducer, vanillic acid, a regulatory protein VanR, and a promoter with a VanR binding site (e.g., P Van_NNNN ).

[0201] Preferably, the anhydrotetracycline inducible expression system comprises anhydrotetracycline as an inducer, a regulatory protein TetR, and a promoter with a regulatory protein TetR binding site (e.g., P Tet_NNNN ).

[0202] Preferably, the OC6 inducible expression system comprises an inducer OC6, a regulatory protein LuxR, and a promoter with a regulatory protein LuxR binding site (e.g., P Lux_NNNN ).

[0203] Preferably, the naringenin inducible expression system comprises an inducer naringenin, a regulatory protein TtgR, and a promoter with a regulatory protein TtgR binding site (e.g., P Ttg_NNNN ).

[0204] Preferably, the DAPG inducible expression system comprises an inducer DAPG, a regulatory protein PhlF and a promoter with a regulatory protein PhlF binding site (e.g., P PhlF_NNNN ).

[0205] Preferably, the inducible expression system comprises the following combination:

[0206] 1) Vanillic acid, OC6, OHC14, arabinose, and sodium salicylate inducible expression systems;

[0207] 2) Anhydrotetracycline, cuminic acid, and DAPG inducible expression systems;

[0208] 3) Naringenin, arabinose, and OHC14 inducible expression systems;

[0209] 4) Arabinose, OHC14, cuminic acid, sodium salicylate, vanillic acid, anhydrotetracycline, OC6, naringenin, and DAPG inducible expression systems; or,

[0210] 5) Sodium salicylate, OHC14 and arabinose inducible expression system.

[0211] Preferably, the microorganism is a eukaryotic microorganism and / or a prokaryotic microorganism, the prokaryotic microorganism includes any one of the genus Escherichia, Ralstonia, Bacillus Cohn, Corynebacterium, Alcaligenes, Pseudomonas, Aeromonas or Halomonas, and the eukaryotic microorganism includes any one of yeast, fungi or algae.

[0212] Preferably, the genus Escherichia includes Escherichia coli, the genus Ralstonia includes Ralstonia eutropha, the genus Bacillus includes Bacillus subtilis, the genus Corynebacterium includes Corynebacterium glutamicum, the genus Alcaligenes includes Alcaligenes faecalis, the genus Pseudomonas includes Pseudomonas entomophila, the genus Aeromonas includes Aeromonas hydrophila, and the genus Halomonas includes any one of Halomonas bluephagenesis, Halomonas campaniensis and Halomonas aydingkolgenesis.

[0213] In a specific embodiment of the present invention, the Halomonas includes but is not limited to Halomonas bluephagenesis TD01 CGMCC.No.4353, Halomonas campaniensis LS21 CGMCC No.6593 and Halomonas aydingkolgenesis M1 CGMCC No.19880.

[0214] Preferably, the product comprises an intracellular product, an enzyme secreted outside the cell, a protein related to cell morphology, or a protein or RNA related to gene regulation in the cell.

[0215] Preferably, the product comprises one or more of PHA, tetrahydropyrimidine, amino acids, inositol, 1,3-propanediol, 3-hydroxypropionic acid, 1,5-pentanediamine, 5-aminolevulinic acid, lipase, amylase, saccharifying enzyme, protease, cellulase, esterase, glucanase, pectinase, phospholipase, gelatinase, nuclease, microtubule protein FtsZ, cytoskeletal protein MreB, cell division regulatory factor MinCD, lactose operon repressor protein LacI, tetracycline operon repressor protein TetR, sgRNA used in CRISPRi / CRISPRa, or lycopene.

[0216] Preferably, the product is PHA, and the microorganism is the expression vector containing phaA, phaB and phaC genes introduced into Pseudomonas entomophila LACR or Halomonas campaniensis LSR.

[0217] Preferably, the method comprises using a sodium salicylate, OHC14 and arabinose inducible expression system, or an anhydrotetracycline, cuminic acid and DAPG inducible expression system.

[0218] Preferably, the product is lycopene, and the microorganism is the expression vector containing crtE, crtI, crtB, idi and dxs genes introduced into Halomonas bluephagenesis TDR.

[0219] Preferably, the method comprises using vanillic acid, OC6, OHC14, arabinose and sodium salicylate to induce the expression system.

[0220] Preferably, the product is ectoine, and the microorganism is the expression vector containing ectA, ectB and ectC genes introduced into Halomonas Haydingkolgenesis MR.

[0221] Preferably, the method comprises using naringenin, arabinose and an OHC14 inducible expression system.

[0222] Preferably, the product is arginine, and the microorganism is the above-mentioned expression vector containing the gdh, argJ, argB, argC, argD, argF, argG, argH and pgl genes introduced into R. eutropha HR.

[0223] Preferably, the method comprises using arabinose, OHC14, cuminic acid, sodium salicylate, vanillic acid, anhydrotetracycline, OC6, naringenin, or a DAPG inducible expression system.

[0224] In a fifth aspect of the present invention, a promoter is provided, wherein the promoter interacts with a regulatory protein, and the promoter comprises P BAD The mutant, P Cin The mutant, P Cym The mutant, P Sal The mutant, P Van The mutant, P Tet The mutant, P Lux The mutant, P Ttg mutants or P PhlF Further preferably, the mutant comprises a mutation of at least one base, and more preferably, the mutant comprises a mutation of at least one base in the first four bases of the -10 region of the original promoter, wherein the four bases can be any combination of A, T, C, and G.

[0225] Preferably, the promoter is an inducible promoter, and the promoter contains a binding site for a regulatory protein.

[0226] Preferably, the regulatory protein is selected from one or more of AraC, CinR, CymR, NahR, VanR, TetR, LuxR, TtgR or PhlF.

[0227] Preferably, the inducible promoter is selected from promoters with regulatory protein AraC binding sites. BAD_NNNN , promoter P with regulatory protein CinR binding site Cin_NNNN , promoter P with regulatory protein CymR binding site Cym_NNNN , promoter P with a binding site for the regulatory protein NahR Sal_NNNN , promoter P with regulatory protein VanR binding site Van_NNNN , promoter P with regulatory protein TetR binding site Tet_NNNN , promoter P with a binding site for the regulatory protein LuxR Lux_NNNN , promoter P with regulatory protein TtgR binding site Ttg_NNNN , promoter P with a binding site for the regulatory protein PhlF PhlF_NNNN One or more of the following.

[0228] Preferably, the promoter P BAD_NNNN The binding site of the regulatory protein comprises a nucleotide sequence shown in any one of SEQ ID NOs. 38-41.

[0229] Preferably, the promoter P Cin_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.42.

[0230] Preferably, the promoter P Cym_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.43.

[0231] Preferably, the promoter P Sal_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.44.

[0232] Preferably, the promoter P Van_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.45.

[0233] Preferably, the promoter P Tet_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.46.

[0234] Preferably, the promoter P Lux_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.47.

[0235] Preferably, the promoter P Ttg_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.48.

[0236] Preferably, the promoter P PhlF_NNNN The binding site of the regulatory protein comprises the nucleotide sequence shown in SEQ ID NO.49.

[0237] Further preferably, the original promoter P BAD It comprises the nucleotide sequence shown in SEQ ID No.11.

[0238] The original promoter P Cin It comprises the nucleotide sequence shown in SEQ ID No.12.

[0239] The original promoter P Cym It comprises the nucleotide sequence shown in SEQ ID No.13.

[0240] The original promoter P Sal It comprises the nucleotide sequence shown in SEQ ID No.14.

[0241] The original promoter P Van It comprises the nucleotide sequence shown in SEQ ID No.15.

[0242] The original promoter P Tet It comprises the nucleotide sequence shown in SEQ ID No.16.

[0243] The original promoter P Lux It comprises the nucleotide sequence shown in SEQ ID No.17.

[0244] The original promoter P Ttg It comprises the nucleotide sequence shown in SEQ ID No.18.

[0245] The original promoter P PhlF It comprises the nucleotide sequence shown in SEQ ID No.19.

[0246] Preferably, the promoter P BAD_NNNN The nucleotide sequence is shown in SEQ ID No. 20, wherein NNNN is ACAC, TTGC, GGCG, AGAG or GCTT; the promoter P Cin_NNNN The nucleotide sequence is shown in SEQ ID No. 21, wherein NNNN is GCCC, GGGG, TGGG, TCAT or TTAA; the promoter P Cym_NNNN The nucleotide sequence is shown in SEQ ID No. 22, wherein NNNN is GCTG, AGGA, GAGA, CCGG or CCAC; the promoter P Lux_NNNN The nucleotide sequence is shown in SEQ ID No. 26, wherein NNNN is CTGT, GGTG, GTGG, ATGT or CATG.

[0247] In a specific embodiment of the present invention, the mutant comprises the nucleotide sequence shown in any one or more of SEQ ID No. 29-37.

[0248] In a sixth aspect, the present invention provides a method for preparing the above-mentioned microorganism, which comprises introducing the above-mentioned expression vector or the above-mentioned promoter into the microorganism.

[0249] Preferably, the microorganism is a eukaryotic microorganism and / or a prokaryotic microorganism, the prokaryotic microorganism includes any one of the genus Escherichia, Ralstonia, Bacillus Cohn, Corynebacterium, Alcaligenes, Pseudomonas, Aeromonas or Halomonas, and the eukaryotic microorganism includes any one of yeast, fungi or algae.

[0250] Preferably, the genus Escherichia includes Escherichia coli, the genus Ralstonia includes Ralstonia eutropha, the genus Bacillus includes Bacillus subtilis, the genus Corynebacterium includes Corynebacterium glutamicum, the genus Alcaligenes includes Alcaligenes faecalis, the genus Pseudomonas includes Pseudomonas entomophila, the genus Aeromonas includes Aeromonas hydrophila, and the genus Halomonas includes any one of Halomonas bluephagenesis, Halomonas campaniensis and Halomonas aydingkolgenesis.

[0251] In a specific embodiment of the present invention, the Halomonas includes but is not limited to Halomonas bluephagenesis TD01 CGMCC.No.4353, Halomonas campaniensis LS21 CGMCC No.6593 and Halomonas aydingkolgenesis M1 CGMCC No.19880.

[0252] Preferably, the expression vector integrates the promoter into the chromosomal genome of the microorganism or the expression vector is free in the microorganism.

[0253] The seventh aspect of the present invention provides an expression vector or cell, wherein the expression vector or cell comprises the above-mentioned promoter.

[0254] In an eighth aspect, the present invention provides the use of the above-mentioned expression vector, the above-mentioned promoter, the above-mentioned cell, the above-mentioned microorganism, the above-mentioned method for regulating multi-gene expression in a microorganism and / or the above-mentioned method for producing a product in gene editing or product synthesis.

[0255] Preferably, the product comprises an intracellular product, an enzyme secreted outside the cell, a protein related to cell morphology, or a protein or RNA related to gene regulation in the cell.

[0256] Preferably, the product comprises one or more of PHA, ectoine, amino acids, inositol, 1,3-propanediol, 3-hydroxypropionic acid, 1,5-pentanediamine, 5-aminolevulinic acid, lipase, amylase, saccharifying enzyme, protease, cellulase, esterase, glucanase, pectinase, phospholipase, gelatinase, nuclease, tubulin FtsZ, cytoskeletal protein MreB, cell division regulator MinCD, lactose operon repressor protein LacI, tetracycline operon repressor protein TetR, sgRNA used in CRISPRi / CRISPRa, or lycopene. In a specific embodiment, the amino acid is arginine.

[0257] Preferably, the PHA is selected from 3-hydroxybutyric acid homopolymer PHB, 3-hydroxybutyric acid and 4-hydroxybutyric acid copolymer P3HB4HB, 3-hydroxybutyric acid, 4-hydroxybutyric acid and 3-hydroxyvaleric acid terpolymer PHBV4HB, 3-hydroxybutyric acid and 3-hydroxyhexanoic acid copolymer PHBHHx, a homopolymer or copolymer of 3-hydroxypropionic acid, the homopolymer of 3-hydroxypropionic acid is P3HP, and the copolymer of 3-hydroxypropionic acid is P(3HB-co-3HP) or PHBHP.

[0258] The product also contains various extracellular small molecule products and / or various extracellular hydrolases.

[0259] In a ninth aspect, the present invention provides a method for producing PHA, which comprises culturing the aforementioned microorganism, or introducing the aforementioned expression vector into the microorganism for culturing.

[0260] Preferably, the microorganism is the above-mentioned expression vector containing phaA, phaB and phaC genes introduced into Pseudomonas entomophila LACR or Halomonas campaniensis LSR.

[0261] Preferably, the method comprises adding an inducer, and further preferably, the inducer comprises one or more of arabinose, N-(3-hydroxy-tetradecanoyl)-homoserine lactone (OHC14), cumic acid, sodium salicylate, vanillic acid, anhydrotetracycline, N-(3-oxy-hexanoyl)-homoserine lactone (OC6), naringenin and 2,4-diacetylphloroglucinol (DAPG).

[0262] Preferably, the method comprises using a sodium salicylate, OHC14 and arabinose inducible expression system, or an anhydrotetracycline, cuminic acid and DAPG inducible expression system.

[0263] Preferably, the method comprises adjusting the concentration of the inducing agent.

[0264] The tenth aspect of the present invention provides a method for producing lycopene, which comprises culturing the aforementioned microorganism, or introducing the aforementioned expression vector into the microorganism for culturing.

[0265] Preferably, the microorganism is the above-mentioned expression vector containing crtE, crtI, crtB, idi and dxs genes introduced into Halomonas bluephagenesis TDR.

[0266] Preferably, the method comprises adding an inducer, and further preferably, the inducer comprises one or more of arabinose, N-(3-hydroxy-tetradecanoyl)-homoserine lactone (OHC14), cumic acid, sodium salicylate, vanillic acid, anhydrotetracycline, N-(3-oxy-hexanoyl)-homoserine lactone (OC6), naringenin and 2,4-diacetylphloroglucinol (DAPG).

[0267] Preferably, the method comprises using vanillic acid, OC6, OHC14, arabinose and sodium salicylate to induce the expression system.

[0268] Preferably, the method comprises adjusting the concentration of the inducing agent.

[0269] In an eleventh aspect of the present invention, a method for producing ectoine is provided, which comprises culturing the aforementioned microorganism, or introducing the aforementioned expression vector into the microorganism for culturing.

[0270] Preferably, the microorganism is the above-mentioned expression vector containing ectA, ectB and ectC genes introduced into Halomonas Haydingkolgenesis MR.

[0271] Preferably, the method comprises adding an inducer, and further preferably, the inducer comprises one or more of arabinose, N-(3-hydroxy-tetradecanoyl)-homoserine lactone (OHC14), cumic acid, sodium salicylate, vanillic acid, anhydrotetracycline, N-(3-oxy-hexanoyl)-homoserine lactone (OC6), naringenin and 2,4-diacetylphloroglucinol (DAPG).

[0272] Preferably, the method comprises using naringenin, arabinose and an OHC14 inducible expression system.

[0273] Preferably, the method comprises adjusting the concentration of the inducing agent.

[0274] A twelfth aspect of the present invention provides a method for producing arginine, which comprises culturing the aforementioned microorganism, or introducing the aforementioned expression vector into the microorganism for culturing.

[0275] Preferably, the microorganism is R.eutropha HR, into which the above-mentioned expression vector comprising the gdh, argJ, argB, argC, argD, argF, argG, argH and pgl genes is introduced.

[0276] Preferably, the method comprises adding an inducer, and further preferably, the inducer comprises one or more of arabinose, N-(3-hydroxy-tetradecanoyl)-homoserine lactone (OHC14), cumic acid, sodium salicylate, vanillic acid, anhydrotetracycline, N-(3-oxy-hexanoyl)-homoserine lactone (OC6), naringenin and 2,4-diacetylphloroglucinol (DAPG).

[0277] Preferably, the method comprises using arabinose, OHC14, cuminic acid, sodium salicylate, vanillic acid, anhydrotetracycline, OC6, naringenin, or a DAPG inducible expression system.

[0278] Preferably, the method comprises adjusting the concentration of the inducing agent. In the present invention, H.bluephagenesis TDR, H.campaniensis LSR, H.aydingkolgenesis MR, P.entomophila LACR and R.eutropha HR are recombinant microorganisms constructed by inserting the regulatory protein encoding gene araC of the arabinose inducible expression system, the regulatory protein encoding gene cinR of the OHC14 inducible expression system, the regulatory protein encoding gene cymR of the cuminic acid inducible expression system, the regulatory protein encoding gene nahR of the sodium salicylate inducible expression system, the regulatory protein encoding gene vanR of the vanillic acid inducible expression system, the regulatory protein encoding gene tetR of the anhydrotetracycline inducible expression system, the regulatory protein encoding gene luxR of the OC6 inducible expression system, the regulatory protein encoding gene ttgR of the naringenin inducible expression system and the regulatory protein encoding gene phlF of the DAPG inducible expression system into the genomes of H.bluephagenesis TD01, H.campaniensis LS21, H.aydingkolgenesis M1, P.entomophila LAC32 and R.eutropha H16, respectively.

[0279] The "PHA" described in the present invention is a homopolymer PHA and / or a copolymer PHA. Preferably, the PHA is selected from 3-hydroxybutyric acid (3HB) homopolymer PHB, 3-hydroxybutyric acid (3HB) and 4-hydroxybutyric acid (4HB) binary copolymer P3HB4HB, 3-hydroxybutyric acid (3HB), 4-hydroxybutyric acid (4HB) and 3-hydroxyvaleric acid terpolymer P(3HB-co-4HB-co-3HV), 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid binary copolymer PHBHHx, 3-hydroxypropionic acid (3HP) homopolymer or copolymer, preferably, the 3-hydroxypropionic acid (3HP) homopolymer is P3HP, preferably, the 3-hydroxypropionic acid (3HP) copolymer is P(3HB-co-3HP) or PHBHP. In a specific embodiment of the present invention, the PHA is selected from 3-hydroxybutyric acid homopolymer PHB, 3-hydroxybutyric acid and 4-hydroxybutyric acid copolymer P3HB4HB, 3-hydroxybutyric acid, 4-hydroxybutyric acid and 3-hydroxyvaleric acid terpolymer PHBV4HB, 3-hydroxybutyric acid and 3-hydroxyhexanoic acid copolymer PHBHHx, a homopolymer or copolymer of 3-hydroxypropionic acid, the homopolymer of 3-hydroxypropionic acid is P3HP, and the copolymer of 3-hydroxypropionic acid is P(3HB-co-3HP) or PHBHP.

[0280] The term "include" or "comprising" in the present invention is an open description containing the specified components or steps described, as well as other specified components or steps that do not substantially affect them.

[0281] The term "and / or" in this invention includes all combinations of the items connected by the term, and each combination should be considered to have been listed separately herein. For example, "A and / or B" includes "A," "A and B," and "B." For another example, "A, B and / or C" includes "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C." DETAILED DESCRIPTION

[0282] 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 are clearly and completely described below. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

[0283] Unless otherwise specified, the materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources.

[0284] 1. Microorganisms used in the present invention

[0285] (1) Halomonas bluephagenesis TD01 CGMCC.No.4353

[0286] The inventors have screened a salt- and alkali-tolerant, Gram-negative halophilic bacterium that naturally produces PHB. This bacterium naturally accumulates high levels of polyhydroxyalkanoates (PHA), demonstrating excellent prospects for industrial production. The strain, described in the paper "Tan Dan, Wu Qiong, Chen Jinchun, and Chen Guo-Qiang. Engineering Halomonas TD01 for Low-Cost Production of Polyhydroxyalkanoates. Metabolic Engineering 26 (2014) 34–47," is available to the public from the inventors for use solely in replicating the present invention's experiments.

[0287] (2) Halomonas campaniensis LS21 CGMCC No.6593

[0288] See the literature Haitao Yue, Chen Ling, Tao Yang, Xiangbin Chen, Yuling Chen, Haiteng Deng, Qiong Wu, Jinchun Chen and Guo-Qiang Chen. A seawater-based open and continuous process for polyhydroxyalkanoates production by recombinant Halomonas campaniensis LS21 grown in mixed substrates. Biotechnology for Biofuelsc 7 (2014) 108.

[0289] (3) Halomonas aydingkolgenesis M1 CGMCC No.19880

[0290] (4) Pseudomonas entomophila LAC32

[0291] See the literature Mengyi Li, Xiangbin Chenb, Xuemei Che, Haoqian Zhang, Lin-Ping Wu, Hetong Du, Guo-Qiang Chen. Engineering Pseudomonas entomophila for synthesis of copolymers with defined fractions of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates. Metabolic Engineering 52 (2019) 253–262.

[0292] (5) Ralstonia eutropha H16

[0293] See the literature Beom So Kim, Seung Chul Lee, Sang Yup Lee, Ho Nam Chang, Yong Keun Chang, and Seong Ihl Woo Production of poly(3-hydroxybutyric acid) by fed-batch culture of Alcaligenes eutrophus with glucose concentration control. Biotechnology and Bioengineering 43 (1994) 892–898.

[0294] 2. Culture medium formulation

[0295] (1) LB medium: Contains 5 g / L yeast extract (OXID, UK, catalog number LP0021), 10 g / L peptone (OXID, UK, catalog number LP0042), 10 g / L NaCl, and the remainder is water. Adjust the pH to 7.0-7.2 and sterilize by autoclaving. 60LB medium is LB medium containing 60 g / L NaCl. The remaining ingredients and preparation conditions are the same as those of LB medium.

[0296] (2) 60MM culture medium contains: 60 g / L NaCl, 1 g / L yeast extract (OXID, UK, catalog number LP0021), 30 g / L glucose, 0.5 g / L urea, 0.2 g / L MgSO4, 9.65 g / L Na2HPO4·12H2O, 1.5 g / L KH2PO4, 0.05 g / L ammonium ferric citrate, 0.02 g / L CaCl2, 0.1 g / L ZnSO4·7H2O, 0.03 g / L MnCl2·4H2O, 0.3 g / L H3BO3, 0.2 g / L CoCl2·6H2O, 0.01 g / L CuSO4·5H2O, 0.02 g / L NiCl2·6H2O, 0.03 g / L NaMoO4·2H2O, and the rest is water.

[0297] 3. pSEVA321 plasmid

[0298] European standard series plasmids, containing the chloramphenicol resistance gene, whose specific sequence information is recorded in the article "Silva-Rocha Rafael, Martínez-García Esteban, Calles Belén et al. The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Research 41 (2013) D666–D675", are available to the public from the inventors for use only in repeating the experiments of the present invention.

[0299] 4. Regulatory Proteins and sfGFP

[0300] The genes encoding the regulatory proteins AraC, CinR, CymR, NahR, VanR, TetR, LuxR, TtgR and PhlF, araC, cinR, cymR, nahR, vanR, tetR, luxR, ttgR and phlF, are derived from the literature. The specific sequence information is recorded in the article "Escherichia coli "Marionette" strains with 12 highly optimized small-molecule sensors. Nature Chemical Biology 15(2019)196–204". The protein sequence of AraC is SEQ ID No.1, the protein sequence of CinR is SEQ ID No.2, the protein sequence of CymR is SEQ ID No.3, the protein sequence of NahR is SEQ ID No.4, the protein sequence of VanR is SEQ ID No.5, the protein sequence of TetR is SEQ ID No.6, the protein sequence of LuxR is SEQ ID No.7, and the protein sequence of TtgR is SEQ ID No.8, the protein sequence of PhlF is: SEQ ID No.9; sfGFP, the full name of which is "super fold green fluorescent protein", has a protein sequence of: SEQ ID No.10. It is a green fluorescent protein suitable for use in bacteria, and its encoding gene is sfgfp.

[0301] 5. Plasmid Conjugation Transformation Method

[0302] (1) Transforming the prepared plasmid into Escherichia coli S17-1 to obtain recombinant Escherichia coli;

[0303] (2) E. coli and the bottom plate microorganisms were co-cultured on 20LB solid medium (37°C, 8h), and then the bacterial lawn was picked and spread on 60LB solid medium containing 25mg / L chloramphenicol and cultured at 37°C for 48h;

[0304] (3) After completing step (2), pick a single colony, streak it on 60LB solid culture medium containing 25 mg / L chloramphenicol, and culture it at 37°C overnight.

[0305] 6. Method for measuring green fluorescent protein (sfGFP) fluorescence intensity using flow cytometry

[0306] (1) Add 1 ml of LB medium or 60 LB medium containing 25 mg / L chloramphenicol to each well of a deep-well plate, pick a small amount of H. bluephagenesis, P. entomophila, or R. eutropha carrying the plasmid and add it to the medium. Shake at 37°C, 1000 rpm, for 12 h.

[0307] (2) Add the required amount of inducer to LB medium or 60LB medium containing 25 mg / L chloramphenicol, shake and mix, then add 1 mL to a deep-well plate. Transfer 2 μL of the bacterial solution cultured for 12 h to the medium and shake at 37°C, 1000 rpm, for 12 h.

[0308] (3) 2 μL of bacterial solution was added to 250 μL of PBS buffer and the fluorescence intensity was tested using a flow cytometer (BD bioscience, USA, model LSRFortessa4). The excitation light of the flow cytometer was set to 488 nm, and the fluorescence analysis signal was captured through the FITC, FSC (forward scatter), and SSC (side scatter) channels. The flow rate was 0.5 μL / s, and at least 50,000 cells were recorded for each sample.

[0309] The regulatory proteins and inducible promoters involved in the present invention are shown in Table 1.

[0310] Table 1. English abbreviations and Chinese names of regulatory proteins and inducible promoters used in inducible expression systems

[0311] Example 1: Developing multiple inducible expression systems suitable for microorganisms and controlling gene expression with their corresponding inducers

[0312] Preparation of plasmid P BAD -sfgfp-araC+araE (arabinose transporter encoding gene), P Cin -sfgfp-cinR,P Cym -sfgfp-cymR,P Sal -sfgfp-nahR,P Van -sfgfp-vanR,P Tet -sfgfp-tetR,P Lux -sfgfp-luxR,P Ttg -sfgfp-ttgR and P PhlF -sfgfp-phlF, the plasmid is a circular plasmid with pSEVA321 as the backbone.

[0313] P BAD It is a promoter regulated by AraC, expressing the sfgfp gene, and its sequence is SEQ ID No.11.

[0314] P Cin It is a promoter regulated by CinR, expressing the sfgfp gene, and its sequence is SEQ ID No.12.

[0315] P Cym It is a promoter regulated by CymR, expressing the sfgfp gene, and its sequence is SEQ ID No.13.

[0316] P Sal It is a promoter regulated by NahR, expressing the sfgfp gene, and its sequence is SEQ ID No.14.

[0317] P Van It is a promoter regulated by VanR, expressing the sfgfp gene, and its sequence is SEQ ID No.15.

[0318] P Tet It is a promoter regulated by TetR, expressing the sfgfp gene, and its sequence is SEQ ID No.16.

[0319] P Lux It is a promoter regulated by LuxR, expressing the sfgfp gene, and its sequence is SEQ ID No.17.

[0320] P Ttg It is a promoter regulated by TtgR, expressing the sfgfp gene, and its sequence is SEQ ID No.18.

[0321] P PhlF It is a promoter regulated by PhlF, expressing the sfgfp gene, and its sequence is SEQ ID No.19.

[0322] Each plasmid, any two, three, four, five, six, seven, eight or all nine plasmids (e.g., P Van -sfgfp-vanR,P Lux -sfgfp-luxR,P Cin -sfgfp-cinR,P BAD -sfgfp-araC+araE,P Sal -sfgfp-nahR combination, P Tet -sfgfp-tetR,P Cym -sfGFP-cymR and P PhlF -sfgfp-phlF combination, P Tet -sfgfp-tetR,P BAD -sfgfp-araC+araE and P Cin -sfgfp-cinR combination, P BAD-sfgfp-araC+araE,P Cin -sfgfp-cinR,P Cym -sfgfp-cymR,P Sal -sfgfp-nahR,P Van -sfgfp-vanR,P Tet -sfgfp-tetR,P Lux -sfgfp-luxR,P Ttg -sfgfp-ttgR and P PhlF -sfgfp-phlF combination, P BAD -sfgfp-araC+araE,P Cin -sfGFP-cinR and P Sal -sfgfp-nahR combination) were introduced into Halomonas bluephagenesis TD01, Halomonas campaniensis LS21, Halomonas aydingkolgenesis M1, Pseudomonas entomophila LAC32 and Ralstonia eutropha H16, respectively, to obtain recombinant H.bluephagenesis TD01, H.campaniensis LS21, H.aydingkolgenesis M1, P.entomophila LAC32 and R.eutropha H16.

[0323] In the above-mentioned recombinant H. bluephagenesis TD01, H. campaniensis LS21, H. aydingkolgenesis M1, P. entomophila LAC32, and R. eutropha H16, the expression of the sfgfp gene was induced using arabinose, OHC14, cuminic acid, sodium salicylate, vanillic acid, anhydrotetracycline, OC6, naringenin, and DAPG, respectively. The effect of recombinant H. bluephagenesis TD01, H. campaniensis LS21, H. aydingkolgenesis M1, P. entomophila LAC32, and R. eutropha H16 on sfgfp expression was detected using the aforementioned detection method. The results showed that the expression intensity of the gene was increased under the action of the inducers compared to when no inducer was added. Taking the recombinant H. bluephagenesis TD01 introduced with all nine plasmids as an example, the results are shown in Table 2.

[0324] Table 2. Establishment of various inducible expression systems and their gene expression intensities under induction

[0325] Example 2: Construction of recombinant microorganisms and insertion of regulatory protein encoding genes into the microbial genome

[0326] The arabinose transporter encoding gene araE, the regulatory protein encoding gene araC of the arabinose inducible expression system, the regulatory protein encoding gene cinR of the OHC14 inducible expression system, the regulatory protein encoding gene cymR of the cuminic acid inducible expression system, the regulatory protein encoding gene nahR of the sodium salicylate inducible expression system, the regulatory protein encoding gene vanR of the vanillic acid inducible expression system, the regulatory protein encoding gene tetR of the anhydrotetracycline inducible expression system, the regulatory protein encoding gene luxR of the OC6 inducible expression system, the regulatory protein encoding gene ttgR of the naringenin inducible expression system and the regulatory protein encoding gene phlF of the DAPG inducible expression system are used as the control. Any one, any two, three, four, five, six, seven, eight or all of them (such as the combination of vanR, luxR, cinR, araC+araE, nahR, tetR, cymR and phlF) are used as the control. combination, tetR, araC+araE and cinR combination, araC+araE, cinR, cymR, nahR, vanR, tetR, luxR, ttgR and phlF combination, araC+araE, cinR and nahR combination) were inserted into the genomes of H.bluephagenesis TD01, H.campaniensis LS21, H.aydingkolgenesis M1, P.entomophila LAC32 and R.eutropha H16, respectively, to construct recombinant microorganisms. Among them, the strains introduced with all regulatory protein encoding gene combinations were named H.bluephagenesis TDR, H.campaniensis LSR, H.aydingkolgenesis MR, P.entomophila LACR and R.eutropha HR. Agarose gel electrophoresis and DNA sequencing confirmed the successful gene insertion.

[0327] Example 3: Controlling gene expression using multiple inducible expression systems and their corresponding inducers in recombinant microorganisms

[0328] Preparation of plasmid P BAD -sfgfp,P Cin -sfgfp,P Cym -sfgfp,P Sal -sfgfp,P Van -sfgfp,PTet -sfgfp,P Lux -sfgfp,P Ttg -sfgfp and P PhlF -sfgfp, the plasmid is a circular plasmid with pSEVA321 as the backbone.

[0329] P BAD It is a promoter regulated by AraC, expressing the sfgfp gene, and its sequence is SEQ ID No.11.

[0330] P Cin It is a promoter regulated by CinR, expressing the sfgfp gene, and its sequence is SEQ ID No.12.

[0331] P Cym It is a promoter regulated by CymR, expressing the sfgfp gene, and its sequence is SEQ ID No.13.

[0332] P Sal It is a promoter regulated by NahR, expressing the sfgfp gene, and its sequence is SEQ ID No.14.

[0333] P Van It is a promoter regulated by VanR, expressing the sfgfp gene, and its sequence is SEQ ID No.15.

[0334] P Tet It is a promoter regulated by TetR, expressing the sfgfp gene, and its sequence is SEQ ID No.16.

[0335] P Lux It is a promoter regulated by LuxR, expressing the sfgfp gene, and its sequence is SEQ ID No.17.

[0336] P Ttg It is a promoter regulated by TtgR, expressing the sfgfp gene, and its sequence is SEQ ID No.18.

[0337] P PhlF It is a promoter regulated by PhlF, expressing the sfgfp gene, and its sequence is SEQ ID No.19.

[0338] Each plasmid, any two, three, four, five, six, seven, eight or all nine plasmids (e.g., P Van -sfgfp,P Lux -sfgfp,P Cin -sfgfp,P BAD -sfgfp,P Sal -sfgfp-nahR combination, PTet -sfgfp,P Cym -sfgfp and P PhlF -sfgfp combination, P Tet -sfgfp,P BAD -sfgfp and P Cin -sfGFP combination, P BAD -sfgfp,P Cin -sfgfp,P Cym -sfgfp,P Sal -sfgfp,P Van -sfgfp,P Tet -sfgfp,P Lux -sfgfp,P Ttg -sfgfp and P PhlF -sfgfp combination, P BAD -sfgfp,P Cin -sfgfp and P Sal -sfgfp combination) were introduced into H.bluephagenesis TDR, H.campaniensis LSR, H.aydingkolgenesis MR, P.entomophila LACR and R.eutropha HR respectively. Among them, the strain introduced with all plasmid combinations obtained recombinant H.bluephagenesis TDR, H.campaniensis LSR, H.aydingkolgenesis MR, P.entomophila LACR and R.eutropha HR.

[0339] In the above-mentioned recombinant H.bluephagenesis TDR, H.campaniensis LSR, H.aydingkolgenesis MR, P.entomophila LACR and R.eutropha HR, the expression of the sfgfp gene was induced by arabinose, OHC14, cumic acid, sodium salicylate, vanillic acid, anhydrotetracycline, OC6, naringenin and DAPG, respectively. The effect of recombinant H.bluephagenesis TDR, H.campaniensis LSR, H.aydingkolgenesis MR, P.entomophila LACR and R.eutropha HR on expressing sfgfp was detected by the above-mentioned detection method. The results showed that compared with no addition of inducers, the expression intensity of the gene was increased under the action of the inducers. Taking the recombinant H.bluephagenesis TDR as an example, the results are shown in Table 3.

[0340] Table 3. Various inducible expression systems after inserting regulatory protein-encoding genes into the genome and their gene expression intensities under induction

[0341] Example 4: Using promoter core region spacer sequences to modify various inducible expression systems and their corresponding inducers to control gene expression

[0342] Preparation of plasmid P BAD_NNNN -sfgfp,P Cin_NNNN -sfgfp,P Cym_NNNN -sfgfp,P Sal_NNNN -sfgfp,P Van_NNNN -sfgfp,P Tet_NNNN -sfgfp,P Lux_NNNN -sfgfp,P Ttg_NNNN -sfgfp and P PhlF_NNNN -sfgfp, the plasmid is a circular plasmid with pSEVA321 as the backbone.

[0343] P BAD_NNNN is a promoter regulated by AraC, including P BAD and P BAD On the basis of the above, the four bases before the -10 region are replaced with a promoter of any nucleotide to express the sfgfp gene, the sequence of which is SEQ ID No.20.

[0344] P Cin_NNNN CinR-regulated promoters, including P Cin and P Cin On the basis of the above, the four bases before the -10 region are replaced with a promoter of any nucleotide to express the sfgfp gene, the sequence of which is SEQ ID No.21.

[0345] P Cym_NNNN are promoters regulated by CymR, including P Cym and P Cym On the basis of the above, the four bases before the -10 region are replaced with a promoter of any nucleotide to express the sfgfp gene, the sequence of which is SEQ ID No.22.

[0346] P Sal_NNNN is a promoter regulated by NahR, including P Sal and P Sal On the basis of the above, the four bases before the -10 region are replaced with a promoter of any nucleotide to express the sfgfp gene, the sequence of which is SEQ ID No.23.

[0347] P Van_NNNN is a promoter regulated by VanR, including P Van and P VanOn the basis of the above, the four bases before the -10 region are replaced with a promoter of any nucleotide to express the sfgfp gene, the sequence of which is SEQ ID No.24.

[0348] P Tet_NNNN are TetR-regulated promoters, including P Tet and P Tet On the basis of the above, the four bases before the -10 region are replaced with a promoter of any nucleotide to express the sfgfp gene, the sequence of which is SEQ ID No.25.

[0349] P Lux_NNNN is a promoter regulated by LuxR, including P Lux and P Lux On the basis of the above, the four bases before the -10 region are replaced with a promoter of any nucleotide to express the sfgfp gene, the sequence of which is SEQ ID No.26.

[0350] P Ttg_NNNN is a promoter regulated by TtgR, including P Ttg and P Ttg On the basis of the above, the four bases before the -10 region are replaced with a promoter of any nucleotide to express the sfgfp gene, the sequence of which is SEQ ID No.27.

[0351] P PhlF_NNNN are promoters regulated by PhlF, including P PhlF and P PhlF On the basis of the above, the four bases before the -10 region are replaced with a promoter of any nucleotide to express the sfgfp gene, the sequence of which is SEQ ID No.28.

[0352] The prepared plasmids were introduced into H. bluephagenesis TDR, H. campaniensis LSR, H. aydingkolgenesis MR, P. entomophila LACR and R. eutropha HR, respectively, to obtain recombinant H. bluephagenesis TDR, H. campaniensis LSR, H. aydingkolgenesis MR, P. entomophila LACR and R. eutropha HR.

[0353] In the above-mentioned recombinant H.bluephagenesis TDR, H.campaniensis LSR, H.aydingkolgenesis MR, P.entomophila LACR and R.eutropha HR, the expression of the sfgfp gene was induced with arabinose, OHC14, cumic acid, sodium salicylate, vanillic acid, anhydrotetracycline, OC6, naringenin and DAPG, respectively. The effect of recombinant H.bluephagenesis TDR, H.campaniensis LSR, H.aydingkolgenesis MR, P.entomophila LACR and R.eutropha HR on the expression of sfgfp was detected using the aforementioned detection method. The results showed that compared with no addition of inducers, the expression intensity of the gene was increased under the action of the inducers, and promoters with different promoter core region spacer sequences had different expression intensities. Taking the recombinant H.bluephagenesis TDR as an example, the results are shown in Table 4.

[0354] Table 4. Various inducible expression systems with altered promoter core region spacer sequences after inserting regulatory protein-encoding genes into the genome and their gene expression intensities under induction

[0355] Example 5: Controlling the synthesis of lycopene by Halomonas bluephagenesis using various inducible expression systems

[0356] Preparation of plasmid pP Van -crtE-P Lux_CTGT -crtI-P Cin_GAAC -crtB-P BAD_ACAC -idi-P Sal -dxs, the plasmid is a circular plasmid with pSEVA321 as the backbone. Van (DNA sequence: SEQ ID No.15) expressing crtE, P Lux_CTGT (DNA sequence: SEQ ID No. 29) expressing crtI, P Cin_GAAC (DNA sequence: SEQ ID No. 30) expressing crtB, P BAD_ACAC (DNA sequence: SEQ ID No.31) expressing idi, P Sal (DNA sequence: SEQ ID No. 14) Expression of dxs, crtE, crtI, crtB, idi and dxs are genes related to lycopene synthesis.

[0357] The prepared plasmid was introduced into Halomonas bluephagenesis TDR to obtain recombinant H. bluephagenesis TDR. The recombinant H. bluephagenesis TDR was inoculated into 60LB medium and cultured for 12 hours. After that, the inoculum was transferred to fresh 60LB medium at a 1% inoculum size. After 10 hours of culture, 5 μL of the bacterial solution was inoculated into 1 mL of 60LB medium. Deep-well plate culture experiments were performed at a rotation speed of 1000 rpm for 24 hours. Simultaneously with the inoculation of the bacterial solution, five combinations of inducers of varying concentrations were added to induce expression of the crtE, crtI, crtB, idi, and dxs genes.

[0358] Inducer combination 1: 10 -4 M vanillic acid, 10 -9 M OC6, 10 -8 M OHC14,10 -4 M arabinose and 10 -7 M sodium salicylate.

[0359] Inducer combination 2: 10 -7 M vanillic acid, 10 -6 M OC6, 10 -6 M OHC14,10 -3 M arabinose and 10 -8 M sodium salicylate.

[0360] Inducer combination three: 10 -6 M vanillic acid, 10 -8 M OC6, 10 -7 M OHC14,10 -2 M arabinose and 10 -9 M sodium salicylate.

[0361] After the culture, lycopene in the cells was extracted with acetone, and the absorbance at 472 nm was analyzed. The lycopene production was calculated based on the standard curve. The results showed that under the induction of five different types and concentrations of inducers, the cells synthesized lycopene, and the lycopene production was different in different inducer combinations. The lycopene production was closely related to the inducer combination, as shown in Table 5.

[0362] Table 5. Lycopene production induced by three inducer concentration combinations

[0363] Example 6: Controlling PHB synthesis by Halomonas campaniensis using various inducible expression systems

[0364] Preparation of plasmid pP Tet -phaA-PCym_GCTG -phaB-P PhlF -phaC, the plasmid is a circular plasmid with pSEVA321 as the backbone. Tet (DNA sequence: SEQ ID No.16) expressing phaA, P Cym_GCTG (DNA sequence: SEQ ID No.32) expressing phaB, P PhlF (DNA sequence: SEQ ID No. 19) phaC is expressed. phaA, phaB and phaC are genes related to PHB synthesis.

[0365] The prepared plasmid was introduced into a Halomonas LSR to obtain a recombinant H. campaniensis LSR. The recombinant H. campaniensis LSR was inoculated into 60LB medium and cultured for 12 hours. After 12 hours, the inoculum was transferred to fresh 60LB medium at a 1% inoculum size. After 10 hours of culture, 2.5 μL of the bacterial solution was inoculated into 50 mL of 60 MM medium. Shake flask cultures were performed at 200 rpm for 48 hours. Simultaneously with the inoculation, three combinations of inducers at varying concentrations were added to induce expression of the phaA, phaB, and phaC genes.

[0366] Inducer combination 1: 5×10 -9 M anhydrotetracycline, 5×10 -6 M cuminic acid and 10 -6 M DAPG.

[0367] Inducer combination 2: 10 -8 M anhydrotetracycline, 10 -4 M cuminic acid and 10 -4 M DAPG.

[0368] Inducer combination three: 5×10 -8 M anhydrotetracycline, 10 -5 M cuminic acid and 10 -5 M DAPG.

[0369] After the culture, the bacteria were centrifuged, freeze-dried, weighed, esterified, and analyzed by gas chromatography. The results showed that the cells synthesized PHB under the induction of three inducers of the same type and different concentrations. In addition, the cell dry weight and PHB content were different in different inducer combinations, and the cell dry weight and PHB content were closely related to the inducer combination, as shown in Table 6.

[0370] Table 6. Cell dry weight and PHB content induced by three inducer concentration combinations

[0371] Example 7: Controlling the synthesis of ectoine by Halomonas aydingkolgenesis using various inducible expression systems

[0372] Preparation of plasmid pP Ttg -ectA-P BAD_TTGC -ectB-P Cin -ectC, the plasmid is a circular plasmid with pSEVA321 as the backbone. Tet (DNA sequence: SEQ ID No.16) expressing ectA, P BAD_TTGC (DNA sequence: SEQ ID No.33) expressing ectB, P PhlF (DNA sequence: SEQ ID No. 19) Expression of ectC, ectA, ectB and ectC are genes related to ectoine synthesis.

[0373] The prepared plasmid was introduced into Halomonas Haydingkolgenesis MR to obtain recombinant Haydingkolgenesis MR. The recombinant Haydingkolgenesis MR was inoculated into 60LB medium and cultured for 12 hours. After that, the culture was transferred to fresh 60LB medium with an inoculum size of 1%. After another 10 hours of culture, 2.5 μL of the bacterial solution was inoculated into 50 mL of 60MM medium. Shake flask culture was performed at a rotation speed of 200 rpm for 48 hours. Simultaneously with the inoculation of the bacterial solution, three combinations of inducers at different concentrations were added to induce expression of the ectA, ectB, and ectC genes.

[0374] Inducer combination 1: 10 -9 M Naringenin, 10 -9 M arabinose and 10 -9 M OHC14.

[0375] Inducer combination 2: 10 -3 M Naringenin, 10 -4 M arabinose and 10 -6 M OHC14.

[0376] Inducer combination three: 10 -5 M Naringenin, 10 -3 M arabinose and 10 -5 M OHC14.

[0377] After the culture was completed, the bacteria were broken and centrifuged, and the supernatant after centrifugation was analyzed by liquid chromatography, and the production of ectoine was calculated according to the standard curve. The results showed that under the induction of three inducers of the same type and different concentrations, the cells synthesized ectoine, and the production of ectoine was different in different inducer combinations. The ectoine production was closely related to the inducer combination, as shown in Table 7.

[0378] Table 7. Ectohydropyrimidine production under the induction of three inducer concentration combinations

[0379] Example 8: Controlling Arginine Synthesis in Ralstonia eutropha Using Various Inducible Expression Systems

[0380] P BAD_GGCG -gdh-P Cin_GCCC -argJ-P Cym_AGGA -argB-P Sal -argC-P Van -argD-P Tet -argF-P Lux_GGTG -argG-P Ttg -argH-P PhlF -pgl, this plasmid is a circular plasmid with pSEVA321 as the backbone. BAD_GGCG (DNA sequence: SEQ ID No.34) expressing gdh, P Cin_GCCC (DNA sequence: SEQ ID No.35) expressing argJ, P Cym_AGGA (DNA sequence: SEQ ID No.36) expressing argB, P Sal Expression of argC (DNA sequence: SEQ ID No. 14), P Van Expression of argD (DNA sequence: SEQ ID No. 15), P Tet Expression of argF (DNA sequence: SEQ ID No. 16), P Lux_GGTG Expression of argG (DNA sequence: SEQ ID No. 37), P Ttg Expression of argH (DNA sequence: SEQ ID No. 18), P PhlF pgl (DNA sequence: SEQ ID No. 19) is expressed. gdh, argJ, argB, argC, argD, argF, argG, argH and pgl are genes related to arginine synthesis.

[0381] The prepared plasmid was introduced into R. eutropha HR to obtain recombinant R. eutropha HR. The recombinant R. eutropha HR was inoculated into LB medium and cultured for 12 hours. After that, the culture was transferred to fresh LB medium at a 1% inoculum size. After 10 hours of culture, 2.5 μL of the bacterial solution was inoculated into 50 mL of LB medium. Shake flask cultures were performed at 200 rpm for 48 hours. Simultaneously with the inoculation of the bacterial solution, nine combinations of inducers at varying concentrations were added to induce expression of the gdh, argJ, argB, argC, argD, argF, argG, argH, and pgl genes.

[0382] Inducer combination 1: 10 -6 M arabinose, 10 -9 M OHC14,10 -3 M cuminic acid, 5×10 -4 M sodium salicylate, 10 - 4 M vanillic acid, 5×10 -6 M anhydrotetracycline, 10 -9 M OC6, 10 -3 M Naringenin, 10 -3 M DAPG.

[0383] Inducer combination 2: 10 -4 M arabinose, 10 -8 M OHC14,10 -5 M cuminic acid, 10 -6 M sodium salicylate, 10 - 7 M vanillic acid, 10 -6 M anhydrotetracycline, 10 -8 M OC6, 10 -4 M Naringenin, 10 -5 M DAPG.

[0384] Inducer combination three: 10 -3 M arabinose, 10 -6 M OHC14,10 -4 M cuminic acid, 10 -5 M sodium salicylate, 10 - 5 M vanillic acid, 10 -7 M anhydrotetracycline, 10 -6 M OC6, 10 -5 M Naringenin, 10 -5 M DAPG.

[0385] After the culture was completed, the bacteria were centrifuged, and the supernatant was taken for liquid chromatography analysis. The arginine production was calculated based on the standard curve. The results showed that under the induction of nine different types and concentrations of inducers, the cells synthesized arginine, and the arginine production was different in different inducer combinations. The arginine production was closely related to the inducer combination, as shown in Table 8.

[0386] Table 8. Arginine production induced by three inducer concentration combinations

[0387] Example 9: Controlling PHB synthesis by Pseudomonas entomophila using various inducible expression systems

[0388] Preparation of plasmid pP Sal -phaA-P Cin -phaB-P BAD -phaB, the plasmid is a circular plasmid with pSEVA321 as the backbone. Sal (DNA sequence: SEQ ID No.14) expressing phaA, P Cin (DNA sequence: SEQ ID No.12) expressing phaB, P BAD (DNA sequence: SEQ ID No. 11) phaC is expressed. phaA, phaB and phaC are genes related to PHB synthesis.

[0389] The prepared plasmid was introduced into Pseudomonas entomophila LACR to obtain recombinant P. entomophila LACR. The recombinant P. entomophila LACR was inoculated into LB medium and cultured for 12 hours. After 12 hours, the inoculum was transferred to fresh LB medium at a 1% concentration. After 10 hours of culture, 2.5 μL of the bacterial solution was inoculated into 50 mL of MM medium. Shake flask cultures were performed at 200 rpm for 48 hours. Simultaneously with the inoculation, three combinations of inducers at varying concentrations were added to induce expression of the phaA, phaB, and phaC genes.

[0390] Inducer combination 1: 5×10 -4 M sodium salicylate, 10 -7 MOHC14 and 10 -8 M arabinose.

[0391] Inducer combination 2: 10 -7 M sodium salicylate, 10 -5 MOHC14 and 10 -6 M arabinose.

[0392] Inducer combination three: 10-6 M sodium salicylate, 10 -6 MOHC14 and 10 -5 M arabinose.

[0393] After the culture, the bacteria were centrifuged, freeze-dried, weighed, esterified and analyzed by gas chromatography. The results showed that the cells synthesized PHB under the induction of three inducers of the same type but different concentrations. In addition, the cell dry weight and PHB content were different in different inducer combinations, and the cell dry weight and PHB content were closely related to the inducer combination, as shown in Table 9.

[0394] Table 9. Cell dry weight and PHB content induced by three inducer concentration combinations

[0395] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and substance 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 readily 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 microorganism for regulating the expression of multiple genes, characterized in that: The microorganism comprises an expression vector, which contains a promoter acting on a regulatory protein, a nucleotide sequence encoding the regulatory protein and a product synthesis gene.

2. The microorganism according to claim 1, characterized in that The regulatory protein is selected from one or more of AraC, CinR, CymR, NahR, VanR, TetR, LuxR, TtgR, PhlF and proteins having the same functions as the above regulatory proteins.

3. The microorganism according to claim 2, characterized in that The regulatory protein comprises any one or more than two of SEQ ID No. 1-9 or comprises an amino acid sequence having more than 80% homology with any one or more than two of SEQ ID No. 1-9 and having the same or similar activity.

4. The microorganism according to any one of claims 1 to 3, characterized in that The promoter that acts with the regulatory protein contains a binding site for the regulatory protein.

5. The microorganism according to claim 4, characterized in that The binding site of AraC comprises the nucleotide sequence shown in any one of SEQ ID NO.38-41; the binding site of CinR comprises the nucleotide sequence shown in SEQ ID NO.42; the binding site of CymR comprises the nucleotide sequence shown in SEQ ID NO.43; the binding site of NahR comprises the nucleotide sequence shown in SEQ ID NO.44; the binding site of VanR comprises the nucleotide sequence shown in SEQ ID NO.45; the binding site of TetR comprises the nucleotide sequence shown in SEQ ID NO.46; the binding site of LuxR comprises the nucleotide sequence shown in SEQ ID NO.47; the binding site of TtgR comprises the nucleotide sequence shown in SEQ ID NO.48; the binding site of PhlF comprises the nucleotide sequence shown in SEQ ID NO.

49.

6. The microorganism according to any one of claims 1 to 5, characterized in that The promoter is selected from a promoter P with a regulatory protein AraC binding site. BAD_NNNN , promoter P with a binding site for the regulatory protein CinR Cin_NNNN , promoter P with a binding site for the regulatory protein CymR Cym_NNNN , promoter P with a binding site for the regulatory protein NahR Sal_NNNN , promoter P with a binding site for the regulatory protein VanR Van_NNNN , promoter P with a binding site for regulatory protein TetR Tet_NNNN , promoter P with a binding site for the regulatory protein LuxR Lux_NNNN , promoter P with a binding site for regulatory protein TtgR Ttg_NNNN , promoter P with binding site for regulatory protein PhlF PhlF_NNNN One or more of the following: The NNNN is located at least one mutation in the first 4 bases of the -10 region of the original promoter.

7. The microorganism according to claim 6, characterized in that The promoter P BAD_NNNN comprising SEQ ID No.20, wherein NNNN is ACAC, TTGC, GGCG, AGAG, ACTC or GCTT; or, the promoter P Cin_NNNN comprising SEQ ID No.21, wherein NNNN is GAAC, GCCC, GGGG, TGGG, TCAT or TTAA; or, the promoter P Cym_NNNN Including SEQ ID No.22, wherein NNNN is GCTG, AGGA, GAGA, TCTG, CCGG or CCAC; or, the promoter P Sal_NNNN comprising SEQ ID No.14; or, the promoter P Van_NNNN comprising SEQ ID No.15; or, the promoter P Tet_NNNN comprising SEQ ID No.16; or, the promoter P Lux_NNNN comprising SEQ ID No.26, wherein NNNN is CTGT, GGTG, GTGG, ATGT, TTGT or CATG; or, the promoter P Ttg_NNNN comprising SEQ ID No.18; or, the promoter P PhlF_NNNN Including SEQ ID No.

19.

8. The microorganism according to claim 1, characterized in that The microorganism is a eukaryotic microorganism and / or a prokaryotic microorganism, wherein the prokaryotic microorganism includes any one of the genus Escherichia, Ralstonia, Bacillus Cohn, Corynebacterium, Alcaligenes, Pseudomonas, Aeromonas or Halomonas, and the eukaryotic microorganism includes any one of yeast, fungi or algae.

9. The microorganism according to claim 8, characterized in that The genus Escherichia includes Escherichia coli, the genus Ralstonia includes Ralstonia eutropha, the genus Bacillus includes Bacillus subtilis, the genus Corynebacterium includes Corynebacterium glutamicum, the genus Alcaligenes includes Alcaligenes faecalis, the genus Pseudomonas includes Pseudomonas entomophila, the genus Aeromonas includes Aeromonas hydrophila, and the genus Halomonas includes any one of Halomonas bluephagenesis, Halomonas campaniensis, and Halomonas aydingkolgenesis.

10. The microorganism according to any one of claims 1 to 9, characterized in that The nucleotide sequence encoding the regulatory protein and the product synthesis gene can be on one expression vector or on two expression vectors.

11. The microorganism according to claim 1, characterized in that The products include intracellular products, enzymes secreted outside the cell, proteins related to cell morphology, or proteins or RNA related to gene regulation in the cell.

12. The microorganism according to claim 11, characterized in that The product comprises one or more of PHA, ectoine, amino acids, inositol, 1,3-propanediol, 3-hydroxypropionic acid, 1,5-pentanediamine, 5-aminolevulinic acid, lipase, amylase, saccharifying enzyme, protease, cellulase, esterase, glucanase, pectinase, phospholipase, gelatinase, nuclease, microtubule protein FtsZ, cytoskeleton protein MreB, cell division regulatory factor MinCD, lactose operon repressor protein LacI, tetracycline operon repressor protein TetR, sgRNA used in CRISPRi / CRISPRa or lycopene.

13. The microorganism according to claim 1, characterized in that The product synthesis gene includes one or more of dxs, crtE, crtI, crtB, idi, dxs, phaA, phaB, phaC, ectA, ectB, ectC, gdh, argJ, argB, argC, argD, argF, argG, argH and pgl.

14. The microorganism according to claim 1, characterized in that The microorganism is added with an inducer during the culture process, and the inducer includes one or more of arabinose, OHC14, cuminic acid, sodium salicylate, vanillic acid, anhydrotetracycline, OC6, naringenin and DAPG.

15. The microorganism according to any one of claims 1 to 14, characterized in that The regulation of multi-gene expression uses a variety of inducible expression systems, which include inducers, regulatory proteins, and promoters that act on the regulatory proteins; Preferably, the inducible expression system includes one or more of an arabinose inducible expression system, an OHC14 inducible expression system, a cuminic acid inducible expression system, a sodium salicylate inducible expression system, a vanillic acid inducible expression system, anhydrotetracycline inducible expression system, an OC6 inducible expression system, a naringenin inducible expression system and a DAPG inducible expression system.

16. The microorganism according to claim 15, characterized in that The inducible expression system comprises the following combination: 1) Vanillic acid, OC6, OHC14, arabinose and sodium salicylate inducible expression systems; 2) Anhydrotetracycline, cuminic acid and DAPG inducible expression system; 3) Naringenin, arabinose and OHC14 inducible expression system; 4) Arabinose, OHC14, cuminic acid, sodium salicylate, vanillic acid, anhydrotetracycline, OC6, naringenin and DAPG inducible expression system; or, 5) Sodium salicylate, OHC14 and arabinose inducible expression system.

17. A method for regulating the expression of multiple genes in a microorganism, characterized in that: The method comprises culturing the microorganism according to any one of claims 1-16.

18. The method according to claim 17, characterized in that The method further comprises adding an inducer, wherein the inducer comprises one or more of arabinose, OHC14, cuminic acid, sodium salicylate, vanillic acid, anhydrotetracycline, OC6, naringenin and DAPG.

19. The method according to any one of claims 17-18, characterized in that: The regulation of multi-gene expression uses a variety of inducible expression systems, which include inducers, regulatory proteins, and promoters that act on the regulatory proteins; Preferably, the inducible expression system includes one or more of an arabinose inducible expression system, an OHC14 inducible expression system, a cuminic acid inducible expression system, a sodium salicylate inducible expression system, a vanillic acid inducible expression system, anhydrotetracycline inducible expression system, an OC6 inducible expression system, a naringenin inducible expression system and a DAPG inducible expression system.

20. The method according to claim 19, characterized in that The inducible expression system comprises the following combination: 1) Vanillic acid, OC6, OHC14, arabinose and sodium salicylate inducible expression systems; 2) Anhydrotetracycline, cuminic acid and DAPG inducible expression system; 3) Naringenin, arabinose and OHC14 inducible expression system; 4) Arabinose, OHC14, cuminic acid, sodium salicylate, vanillic acid, anhydrotetracycline, OC6, naringenin and DAPG inducible expression system; or, 5) Sodium salicylate, OHC14 and arabinose inducible expression system.

21. A method for producing a product, characterized in that The method comprises culturing the microorganism according to any one of claims 1-16.

22. The method according to claim 21, characterized in that The method further comprises adding an inducer, wherein the inducer comprises one or more of arabinose, OHC14, cuminic acid, sodium salicylate, vanillic acid, anhydrotetracycline, OC6, naringenin and DAPG.

23. The method according to any one of claims 21-22, characterized in that: The regulation of multi-gene expression uses a variety of inducible expression systems, which include inducers, regulatory proteins, and promoters that act on the regulatory proteins; Preferably, the inducible expression system includes one or more of an arabinose inducible expression system, an OHC14 inducible expression system, a cuminic acid inducible expression system, a sodium salicylate inducible expression system, a vanillic acid inducible expression system, anhydrotetracycline inducible expression system, an OC6 inducible expression system, a naringenin inducible expression system and a DAPG inducible expression system.

24. The method according to claim 23, characterized in that The inducible expression system comprises the following combination: 1) Vanillic acid, OC6, OHC14, arabinose and sodium salicylate inducible expression systems; 2) Anhydrotetracycline, cuminic acid and DAPG inducible expression system; 3) Naringenin, arabinose and OHC14 inducible expression system; 4) Arabinose, OHC14, cuminic acid, sodium salicylate, vanillic acid, anhydrotetracycline, OC6, naringenin and DAPG inducible expression system; or, 5) Sodium salicylate, OHC14 and arabinose inducible expression system.

25. The method according to any one of claims 21 to 24, characterized in that: The products include intracellular products, enzymes secreted outside the cell, proteins related to cell morphology, or proteins or RNA related to gene regulation in the cell.

26. The method according to claim 25, characterized in that The product comprises one or more of PHA, ectoine, amino acids, inositol, 1,3-propanediol, 3-hydroxypropionic acid, 1,5-pentanediamine, 5-aminolevulinic acid, lipase, amylase, saccharifying enzyme, protease, cellulase, esterase, glucanase, pectinase, phospholipase, gelatinase, nuclease, microtubule protein FtsZ, cytoskeleton protein MreB, cell division regulatory factor MinCD, lactose operon repressor protein LacI, tetracycline operon repressor protein TetR, sgRNA used in CRISPRi / CRISPRa or lycopene.

27. A promoter, characterized in that The promoter interacts with the regulatory protein, and the promoter comprises P BAD The mutant, P Cin The mutant, P Cym The mutant, P Sal The mutant, P Van The mutant, P Tet The mutant, P Lux The mutant, P Ttg mutants or P PhlF One or a combination of two or more mutants; The mutant comprises a mutation of at least one base in the first 4 bases of the original promoter -10 region.

28. The promoter according to claim 27, characterized in that The original promoter comprises the nucleotide sequence shown in SEQ ID No.11-19.

29. The promoter according to claim 27, characterized in that The promoter is shown in SEQ ID No.20, wherein NNNN is ACAC, TTGC, GGCG, AGAG or GCTT; the promoter is shown in SEQ ID No.21, wherein NNNN is GCCC, GGGG, TGGG, TCAT or TTAA; the promoter is shown in SEQ ID No.22, wherein NNNN is GCTG, AGGA, GAGA, CCGG or CCAC; the promoter is shown in SEQ ID No.26, wherein NNNN is CTGT, GGTG, GTGG, ATGT or CATG.

30. The promoter according to any one of claims 27 to 29, characterized in that The promoter includes any nucleotide sequence shown in SEQ ID No.29-37.

31. An expression vector or cell, characterized in that: The expression vector or cell comprises the promoter according to any one of claims 27-30.

32. Use of the microorganism described in any one of claims 1 to 16, the method described in any one of claims 17 to 26, the promoter described in any one of claims 27 to 30 and / or the expression vector or cell described in claim 31 in gene editing or product synthesis.

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