A bidirectional promoter derived from Enterobacter and its applications

Abstract

A bidirectional promoter derived from Enterobacter and its application, belonging to the field of genetic engineering. In order to find a bidirectional promoter that does not require exogenous addition of an inducer. The present invention provides a bidirectional promoter derived from Enterobacter, the sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2, which can simultaneously initiate gene expression from both the forward and reverse directions, and the promoter strengths in the forward and reverse directions of this promoter have time specificity and are regulated by the growth stage of the bacterial cells. The present invention is expected to provide more favorable promoter elements for the field of synthetic biology and promoter elements that do not require induction by an inducer for the fermentation field, and has broad application space and market prospects in the field of fermentation engineering.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and in particular relates to a bidirectional promoter derived from Enterobacter and application thereof. Background Art

[0002] Promoters are important cis-acting elements in gene expression regulation, effectively regulating the onset, shutdown, and abundance of downstream gene expression. Promoters are directional, and unidirectional promoters can specifically initiate the expression of downstream genes. A bidirectional promoter refers to a DNA sequence located between two adjacent genes with opposite transcription directions, which can regulate gene transcription in both directions. Compared with traditional unidirectional promoters, bidirectional promoters can achieve not only the expression of a single exogenous gene in specific applications, but also the simultaneous expression of two exogenous genes. Therefore, they have more extensive applications in fields such as synthetic biology and genetic engineering. Recent studies have shown that bidirectional promoters are widely present in eukaryotic and prokaryotic organisms. Despite this, only a few bidirectional promoters have been identified and applied in transgenic plant and animal research. Currently, there are very few bidirectional promoters in naturally derived bacterial systems.

[0003] Promoters are categorized as constitutive, tissue-specific, and inducible based on their temporal, conditional, and spatial specificity of gene expression. Inducible promoters are those that can significantly increase gene transcription levels in response to certain physical or chemical signals. The greatest advantage of inducible promoters is their ability to rapidly regulate gene expression, making them particularly suitable for expressing toxic exogenous proteins. Consequently, inducible promoters are widely used in transgenic plants and animals, genetically engineered strains, fermentation engineering, synthetic biology, and medical research. For example, in fermentation engineering, cells adapt to environmental changes during fermentation culture by continuously adjusting their metabolic state, resulting in distinct phases such as the lag phase, exponential growth phase, and product production phase. Each phase has distinct requirements for gene protein expression levels, metabolite concentrations, and flux distribution. Consequently, inducible promoters are widely used in fermentation culture to control the expression of target genes at the appropriate time to produce the desired product. Currently, commonly used inducible promoters in bacteria include the lac promoter (lactose promoter), the trc and tac promoters (hybrid promoters of lactose and tryptophan), the T7 phage promoter, the L-arabinose-inducible PBAD promoter, and the L-rhamnose-inducible rha PBAD promoter. However, these promoters generally require expensive chemical reagents as inducers. For example, IPTG (isopropyl β-D-thiogalactoside)-inducible promoters are widely used in fermentation engineering. The high cost of IPTG is a major factor in the high costs of large-scale industrial fermentation. In addition to chemical-inducible promoters, there are also physical signal-inducible promoters such as light-inducible and temperature-inducible promoters. However, in large-scale fermentation systems, these promoters consume a lot of energy and have limited induction effects due to the long heating time and the difficulty of light reaching the fermentation system. Therefore, the development of auto-inducible promoters that can induce target gene expression by changes in the bacterial cell's growth phase without the need for exogenous inducers holds great potential for application. However, there are no reports on bidirectional temporal promoters derived from bacteria. Summary of the Invention

[0004] The purpose of the present invention is to find a bidirectional promoter that does not require exogenous addition of inducers.

[0005] The present invention provides a bidirectional promoter derived from Enterobacter. The nucleotide sequence of the bidirectional promoter is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0006] The present invention provides a recombinant plasmid, which contains the bidirectional promoter.

[0007] It is further defined that the starting vector of the recombinant plasmid is pUC19, pACYCDuet-1, pETDuet1 or pTrcHis2b.

[0008] The present invention provides a recombinant bacterium, which contains the above-mentioned bidirectional promoter or the above-mentioned recombinant bacterium.

[0009] It is further defined that the recombinant bacteria are based on Enterobacter or Escherichia coli.

[0010] The present invention provides a method for expressing genes without an inducer, which utilizes the above-mentioned recombinant bacteria to express genes.

[0011] Further define the OD 600 The recombinant bacteria with a volume ratio of 4 to 6 are added to the reaction system at a volume ratio of 1 to 5%.

[0012] The present invention provides the use of the bidirectional promoter, the recombinant plasmid or the recombinant bacteria in bacterial fermentation.

[0013] Beneficial Effects: The bidirectional promoter provided by this invention can simultaneously initiate gene expression in both the forward and reverse directions. The activation strength of the promoter in both directions is time-specific and regulated by the bacterial growth period. This invention is expected to provide more advantageous promoter elements in the field of synthetic biology and a promoter element that does not require induction in the fermentation field. It has broad application space and market prospects in the field of fermentation engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of plasmid pLUC.

[0015] Figure 2 Schematic diagrams of plasmids pLUC-KAP1f and pLUC-KAP1r, where A is a schematic diagram of pLUC-KAP1f and B is a schematic diagram of pLUC-KAP1r.

[0016] Figure 3 Schematic diagrams of plasmids pGFP-KAP1-LUC and pKan-GFP-KAP1-LUC, where A is a schematic diagram of pGFP-KAP1-LUC and B is a schematic diagram of pKan-GFP-KAP1-LUC.

[0017] Figure 4 These are the results of LUC expression initiated by the forward promoter KAP1f and the reverse promoter KAP1r, where A is the result of LUC expression initiated by the reverse promoter KAP1r, and B is the result of LUC expression initiated by the forward promoter KAP1f; the horizontal axis is the group, and the vertical axis is the LUC value.

[0018] Figure 5These are the expression results of GFP and LUC initiated by the bidirectional promoter KAP1, where A is the expression result of LUC initiated by the bidirectional promoter KAP1, and B is the expression result of GFP initiated by the bidirectional promoter KAP1 (RFU is fluorescence intensity, which is proportional to the GFP concentration and represents the concentration of GFP protein).

[0019] Figure 6 The temporal expression results of the genes on both sides of the bidirectional promoter KAP1, where A is the temporal expression result of the left gene and B is the temporal expression result of the right gene.

[0020] Figure 7 is the bacterial growth curve, where the horizontal axis is time and the vertical axis is OD 600 . Specific embodiments

[0021] The present invention will be further described below with reference to specific examples, but the present invention is not limited to the examples. The plasmid pLUC, competent cells, primers and reagents used in the examples can be purchased from commercial sources or obtained by conventional means known to those skilled in the art.

[0022] Example 1. Discovery and functional verification of promoters

[0023] (1) Promoter cloning

[0024] Analysis of the Enterobacter sp. CGMCC 5087 genome revealed that two genes are transcribed in opposite directions, with a 185-bp intergenic region. Using the Enterobacter sp. CGMCC 5087 genome (extracted using a microbial extraction kit) as a template, PCR was performed with the primer pair KAP1-1 / 2 to generate this 185-bp DNA fragment, designated KAP1 (sequence shown in SEQ ID NO: 1). PCR was also performed with the primer pair KAP1-3 / 4 to generate this 185-bp DNA fragment, representing the reverse promoter, designated KAP1r (sequence shown in SEQ ID NO: 2).

[0025] (2) Construction of single reporter gene recombinant vector

[0026] Construction of pLUC vector: Using the Pseudomonas syringae genome containing the luciferase gene as a template, the primer pair LUC-1 / 2 was used to obtain the LUC (luxCDABE, sequence shown in SEQ ID NO: 5) fragment by PCR. This fragment was inserted into the BamHI and PstI sites of the pUC19 plasmid to obtain the LUC reporter vector pLUC. The plasmid map is shown in Figure 1 shown.

[0027] Construction of pLUC-KAP1 recombinant vector: The promoter fragments KAP1 and KAP1r obtained in step (1) were inserted into the BamHI site of the pLUC plasmid to obtain the recombinant vectors pLUC-KAP1 and pLUC-KAP1r. The plasmid maps of this series are shown in Figure 2 shown.

[0028] (3) Promoter activity verification

[0029] The recombinant vectors pLUC-KAP1 and pLUC-KAP1r were respectively transformed into Escherichia coli DH5α strains, and single colonies were picked and shaken to culture overnight. 150 μL of bacterial solution was placed in a 96-well white plate and the luminescence value was detected in a microplate reader. The test results are as follows: Figure 4 The results show that the KAP1 promoter has bidirectional promoter activity and can activate the expression of genes on both sides.

[0030] Take the bacterial solution and measure the OD using a spectrophotometer 600 The primer sequences are shown in Table 1.

[0031] Example 2. A method for constructing a dual-reporter gene recombinant vector for verifying the activity of the KAP1 bidirectional promoter, comprising the following steps:

[0032] (1) Using Enterobacter sp. CGMCC 5087 genomic DNA as a template and KAP1-F and KAP1-R as primers, PCR amplification was performed to obtain the KAP1 promoter fragment; using a plasmid containing the GFP gene as a template and GFP-F and GFP-R as primers, PCR amplification was performed to obtain the GFP fragment (the gene sequence is shown in SEQ ID NO: 3); using GFP-F and KAP1-R as primers, the GFP fragment was fused to the promoter fragment by fusion PCR to obtain a GFP-KAP1 fusion fragment (the sequence is shown in SEQ ID NO: 4), with GFP located on the left side of the promoter.

[0033] (2) The GFP-KAP1 fragment was inserted into the EcoR I and BamH I sites of the pLUC plasmid to obtain the dual reporter gene recombinant vector pGFP-KAP1-LUC containing the bidirectional promoter KAP1. The plasmid map is shown in Figure 2. Figure 3 As shown in A.

[0034] (3) Using primer pair Kan-1 / 2 and plasmid pET28a as template, the Kan gene fragment was obtained by PCR; the plasmid pGFP-KAP1-LUC was digested with BglI to obtain a linear fragment; the Kan fragment was ligated with the linear plasmid to obtain the Kan-resistant recombinant vector pKan-GFP-KAP1-LUC. The plasmid map is shown in Figure 3 As shown in B.

[0035] The primer sequences are shown in Table 1.

[0036] Table 1 Primer sequence list

[0037]

[0038] Functional validation of KAP1 as a bidirectional promoter:

[0039] (1) The recombinant vector pKan-GFP-KAP1-LUC was transformed into the Enterobacter sp. CGMCC5087 strain by electroporation (no need to verify KAP1r: reporter genes are connected on both sides of the KAP1 promoter, so the forward and reverse promoter activities can be directly detected).

[0040] (2) Select the bacteria stored at -80℃, streak them on LB plates for activation, and culture at 37℃ for about 10-12 hours. When a single colony grows, remove the plate, pick the bacteria with a toothpick or a white gun tip, and inoculate them into LB medium (with kanamycin added). Culture overnight at 37℃ and 180 rpm.

[0041] (3) Take 150 μL of bacterial solution and place it in a 96-well white plate. Place it in an enzyme-labeled instrument to detect the luminescence value. The test results are as follows: Figure 5 As shown in A. OD was detected by spectrophotometer 600 .

[0042] Result analysis: This indicated that the promoter could initiate the expression of LUC reporter gene and had positive promoter activity.

[0043] (4) Take 1 mL of bacterial solution, collect the cells by centrifugation, wash once with ddH2O, resuspend the cells with 1 mL of ddH2O, take 150 μL of bacterial solution and place it in a 96-well transparent plate (Costar 3635), and measure the GFP signal with a microplate reader. The test results are as follows: Figure 5 As shown in B. OD was detected by spectrophotometer 600 .

[0044] Result analysis: This indicates that the promoter can initiate the expression of GFP reporter gene and has reverse promoter activity.

[0045] KAP1 promoter timing function detection:

[0046] (1) (Wild-type Enterobacter sp. CGMCC 5087 strain, without any plasmid) Activation of strains: Take the strain stored at -80℃, streak it on an LB plate, culture it in a 37℃ incubator overnight, pick a single colony and inoculate it into liquid LB medium, and culture the activated strain in a shaking incubator at 37℃ and 180 rpm.

[0047] (2) Collect the cells, wash them twice with ddH2O, resuspend them in 1 mL ddH2O, transfer them to 5 mL M9 medium, and adjust the OD 600 = 0.1, and incubate the samples on a shaker at 37°C and 180 rpm.

[0048] (3) Samples were collected at 4h, 12h, 15h, 18h, 21h, and 24h, RNA was extracted, and gene expression on both sides of the KAP1 promoter was detected. Figure 6 As shown. OD was detected by spectrophotometer. 600 , detect bacterial growth, the results are as follows Figure 7 shown.

[0049] Results: Under natural conditions, the KAP1 promoter can activate the expression of genes on both sides. The expression of the left gene increases with bacterial growth, reaches the highest point when it reaches the stable phase, and then decreases. The right gene is mainly expressed in the logarithmic phase and its expression decreases with bacterial growth. SEQUENCE LISTING <110> Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences <120> A bidirectional promoter derived from Enterobacter and its application <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 185 <212> DNA <213> artificial synthesis <400> 1 gaacaggtat ccttctaatg ttgacctcac cttgagtatt aaataagcgt gatgactgtc 60 cagaattggc gataagaaga gcatggaaag aatgctttac aaaaaaggct gcgctatgct 120 gcttttttcc gcgttaatgt aaacgcatac actgattttt ttcttcagcg ccagaggtca 180 tgaga 185 <210> 2 <211> 185 <212> DNA <213> artificial synthesis <400> 2 tctcatgacc tctggcgctg aagaaaaaaa tcagtgtatg cgtttacatt aacgcggaaa 60 tctcatgacc tctggcgctg aagaaaaaaa tcagtgtatg cgtttacatt aacgcggaaa 60 aaagcagcat agcgcagcct tttttgtaaa gcattctttc catgctcttc ttatcgccaa 120 aaagcagcat agcgcagcct tttttgtaaa gcattctttc catgctcttc ttatcgccaa 120 ttctggacag tcatcacgct tatttaatac tcaaggtgag gtcaacatta gaaggatacc 180 ttctggacag tcatcacgct tatttaatac tcaaggtgag gtcaacatta gaaggatacc 180 tgttc 185 tgttc 185 <210> 3<210> 3 <211> 717<211> 717 <212> DNA<212> DNA <213> 人工合成<213> Synthetic <400> 3<400> 3 ttatttgtag agctcatcca tgccatgtgt aatcccagca gcagttacaa actcaagaag 60 ttatttgtag agctcatcca tgccatgtgt aatcccagca gcagttacaa actcaagaag 60 gaccatgtgg tctctctttt cgttgggatc tttcgaaagg gcagattgtg tggacaggta 120 gaccatgtgg tctctctttt cgttgggatc tctttcgaaagg gcagattgtg tggacaggta 120 atggttgtct ggtgaaagga cagggccatc gccaattgga gtattttgtt gataatggtc 180 atggttgtct ggtgaaagga cagggccatc gccaattgga gtattttgtt gataatggtc 180 tgctagttga acgcttccat cttcaatgtt gtgtctaatt ttgaagttaa ctttgattcc 240 tgctagttga acgcttccat cttcaatgtt gtgtctaatt ttgaagttaa ctttgattcc 240 attcttttgt ttgtctgcca tgatgtatac attgtgtgag ttatagttgt attccaattt 300 attcttttgt ttgtctgcca tgatgtatac attgtgtgag ttatagttgt attccaattt 300 gtgtccaaga atgtttccat cttctttaaa atcaatacct tttaactcga ttctattaac 360 gtgtccaaga atgtttccat cttctttaaa atcaatacct tttaactcga ttctattaac 360 aagggtatca ccttcaaact tgacttcagc acgtgtcttg tagttcccgt catctttgaa 420 aagggtatca ccttcaaact tgacttcagc acgtgtcttg tagttcccgt catctttgaa 420 aaatatagtt ctttcctgta cataaccttc gggcatggca ctcttgaaaa agtcatgctg 480 tttcatatga tctgggtatc ttgaaaagca ttgaacacca taagtcagag tagtgacaag 540 tgttggccat ggaacaggta gttttccagt agtgcaaata aatttaaggg taagttttcc 600 gtatgttgca tcaccttcac cctctccact gacagaaaat ttgtgcccat taacgtcacc 660 atctaattca acaagaattg ggacaactcc agtgaaaagt tcttctcctt tactcat 717 <210> 4 <211> 908 <212> DNA <213> Synthetic <400> 4 ttatttgtag agctcatcca tgccatgtgt aatcccagca gcagttacaa actcaagaag 60 gaccatgtgg tctctctttt cgttgggatc tttcgaaagg gcagattgtg tggacaggta 120 atggttgtct ggtgaaagga cagggccatc gccaattgga gtattttgtt gataatggtc 180 tgctagttga acgcttccat cttcaatgtt gtgtctaatt ttgaagttaa ctttgattcc 240 attcttttgt ttgtctgcca tgatgtatac attgtgtgag ttatagttgt attccaattt 300 gtgtccaaga atgtttccat cttctttaaa atcaatacct tttaactcga ttctattaac 360 aagggtatca ccttcaaact tgacttcagc acgtgtcttg tagttcccgt catctttgaa 420 aaatatagtt ctttcctgta cataaccttc gggcatggca ctcttgaaaa agtcatgctg 480 tttcatatga tctgggtatc ttgaaaagca ttgaacacca taagtcagag tagtgacaag 540 tgttggccat ggaacaggta gttttccagt agtgcaaata aatttaaggg taagttttcc 600 gtatgttgca tcaccttcac cctctccact gacagaaaat ttgtgcccat taacgtcacc 660 atctaattca acaagaattg ggacaactcc agtgaaaagt tcttctcctt tactcatctc 720 gaggaacagg tatccttcta atgttgacct caccttgagt attaaataag cgtgatgact 780 gtccagaatt ggcgataaga agagcatgga aagaatgctt tacaaaaaag gctgcgctat 840 gctgcttttt tccgcgttaa tgtaaacgca tacactgatt tttttcttca gcgccagagg 900 tcatgaga 9​​​​​​​​​​​​​gatttagtgc aatccattaa ttttggtgat aatagtgttt acctgccaat attgaatgac 120 tctcatgtaa aaaacattat tgattgtaat ggaataacg aattacggtt gcataacatt 180 gtcaattttc tctatacggt agggcaaaga tggaaaaatg aagaatactc aagacgcagg 240 acatacattc gtgacttaaa aaaatatatg ggatattcag aagaaatggc taagctagag 300 gccaattgga tatctatgat tttatgttct aaaggcggcc tttatgatgt tgtagaaaat 360 gaacttggtt ctcgccatat catggatgaa tggctacctc aggatgaaag ttatgttcgg 420 gcttttccga aaggtaaatc tgtacatctg ttggcaggta atgttccatt atctgggatc 480 atgtctatat tacgcgcaat tttaactaag aatcagtgta ttataaaaaac atcgtcaacc 540 gatcctttta ccgctaatgc attagcgtta agttttattg atgtagaccc taatcatccg 600 ataacgcgct ctttatctgt tatatattgg ccccaccaag gtgatacatc actcgcaaaa 660 gaaattatgc gacatgcgga tgttattgtc gcttggggag ggccagatgc gattaattgg 720 gcggtagagc atgcgccatc ttatgctgat gtgattaaat ttggttctaa aaagagtctt 780 tgcattatcg ataatcctgt tgatttgacg tccgcagcga caggtgcggc tcatgatgtt 840 tgtttttacg atcagcgagc ttgtttttct gcccaaaaca tatattacat gggaaatcat 900 tatgagaat ttaagttagc gttgatagaa aaacttaatc tatatgcgca tatattaccg 960 aatgccaaaa aagattttga tgaaaaggcg gcctattctt tagttcaaaa agaaagcttg 1020 1080 gcaggtgtgg aatttaatca accacttggc agatgtgtgt accttcatca cgtcgataat 1140 attgagcaaa tattgcctta tgttcaaaaa aataagacgc aaaccatatc tatttttcct 1200 tgggagtcat catttaaata tcgagatgcg ttagcattaa aaggtgcgga area 1260 gaagcaggaa tgaataacat atttcgagtt ggtggatctc atgacggaat gagaccgttg 1320 caacgattag tgacatatat ttctcatgaa aggccatcta actatacggc tagaggatgtt 1380 gcggttgaaa tagaacagac tcgattcctg gaagagata agttccttgt atttgtccca 1440 taataggtaa aagtatggaa aatgaatcaa aatataaaac catcgaccac gttatttgtg 1500 ttgaaggaaa taaaaaatt catgtttggg aaacgctgcc agaagaaaac agcccaaaga 1560 gaaagaatgc cattattatt gcgtctggtt ttgcccgcag gatggatcat tttgctggtc 1620 tggcggaata tttatcgcgg aatggattc atgtgatccg ctatgattcg cttcaccacg 1680 ttggattgag ttcagggaca attgatgaat ttacaatgtc tataggaaag cagagcttgt 1740 tagcagtggt tgattggtta actacacgaa aaataaataa cttcggtatg ttggcttcaa 1800 gcttatctgc gcggatagct tatgcaagcc tatctgaaat caatgcttcg ttttaatca 1860 ccgcagtcgg tgttgttaac ttaagatatt ctcttgaaag agctttaggg tttgattatc 1920 tcagtctacc cattaatgaa ttgccggata atctagattt tgaaggccat aaattgggtg 1980 ctgaagtctt tgcgagagat tgtcttgatt ttggttggga agatttagct tctacaatta 2040 ataacatgat gtatcttgat ataccgttta ttgcttttac tgcaaataac gataattggg 2100 tcaagcaaga tgaagttatc acattgttat caaatattcg tagtaatcga tgcaagatat 2160 attctttgtt aggaagttcg catgacttga gtgaaaattt agtggtcctg cgcaattttt 2220 atcaatcggt tacgaaagcc gctatcgcga tggataatga tcatctggat attgatgttg 2280 atattactga accgtcattt gaacatttaa ctattgcgac agtcaatgaa cgccgaatga 2340 gaattgagat tgaaaatcaa gcaatttctc tgtcttaaaa tctattgaga tattctatca 2400 ctcaaatagc aatataagga ctctctatga aatttggaaa ctttttgctt acataccaac 2460 ctccccaatt ttctcaaaca gaggtaatga aacgtttggt taaattaggt cgcatctctg 2520 aggagtgtgg ttttgatacc gtatggttac tggagcatca tttcacggag tttggtttgc 2580 ttggtaaccc ttatgtcgct gctgcatatt tacttggcgc gactaaaaaa ttgaatgtag 2640 gaactgccgc tattgttctt cccacagccc atccagtacg ccaacttgaa gatgtgaatt 2700 tattggatca aatgtcaaaa ggacgatttc ggtttggtat ttgccgaggg ctttacaaca 2760 aggactttcg cgtattcggc acagatatga ataacagtcg cgccttagcg gaatgctggt 2820 acgggctgat aaagaatggc atgacagagg gatatatgga agctgataat gaacatatca 2880 agttccataa ggtaaaagta aaccccgcgg cgtatagcag aggtggcgca ccggtttatg 2940 tggtggctga atcagcttcg acgactgagt gggctgctca atttggccta ccgatgatat tagttggat fatheract aacgaaaaga aagcacaact tgagctttat aatgaagtgg ctcaagaata tgggcacgat attcataata tcgaccattg cttatcatat attacatctg 3180. tagatcatga ctcaattaaa gcgaaagaga tttgccgga atttctgggg cattggtatg attcttatgt gaatgctacg actatttttg atgattcaga ccaaacaaga ggttatgatt tcaataaagg gcagtggcgt gactttgtat taaaaggaca taaagatact aatcgccgta ttgattacag ttacgaaatc aatcccgtgg gaacgccgca ggaatgtatt gacataattc aaaaagacat tgatgctaca ggaatatca atatttgttg tggatttga gctaatgga cagtagga aattattgct tccatgaagc tcttccagtc tgatgtcatg ccatttctta aagaaaaaca acgttcgcta ttattatagc tagged ttggattgtt cttccttaac ttcatcaatt caacaactgt tcaagaacaa agtatagttc gcatgcagga aatacggag tatgttgata agttgaatt tgaacagatt ttagtgtatg aaaatcattt ttcagataat ggtgttgtcg gcgctcctct gactgtttct ggttttctgc tcggtttaac 3720 agaagaaaatt aaaattggtt cattaaatca catcattaca actcatcatc ctgtcgccat 3780 agcggaggaa gcttgcttat tggatcagtt aagtgaaggg agatttattt tagggtttag 3840 tgattgcgaa aaaaaagatg aaatgcattt tttaatcgc ccggttgaat atcaacagca 3900 actatttgaa gagtgttatg aaatcattaa cgatgcttta acaacaggct attgtaatcc 3960 agataacgat ttttatagct tccctaaat atctgtaaat ccccatgctt atacgccagg 4020 cggacctcgg aaatatgtaa cagcaaccag tcatcatatt gttgagtggg cggccaaaaa 4080 aggtattcct ctcatcttta agtgggatga ttctaatgat gttagatatg aatatgctga 4140 aagatataaa gccgttgcgg ataaatatga cgttgaccta tcagagatag accatcagtt 4200 aatgatatta gttaactata acgaagatag taataaagct aaacaagaga cgcgtgcatt 4260 tattagtgat tatgttcttg aaatgcaccc taatgaaaat ttcgaaaata aacttgaaga 4320 aataattgca gaaaacgctg tcggaaatta tacggagtgt ataactgcgg ctaagttggc 4380 aattgaaaag tgtggtgcga aaagtgtatt gctgtccttt gaaccaatga atgatttgat 4440 gagccaaaaa aatgtaatca atattgttga tgataatatt aagaagtacc acatggaata 4500 tacctaatag atttcgagtt gcagcgaggc ggcaagtgaa cgaatcccca ggagcataga 4560 taactatgtg actggggtga gtgaaagcag ccaacaaagc agcagcttga aagatgaagg 4620 gtataaaaga gtatgacagc agtgctgcca tactttctaa tattatcttg aggagtaaaa 4680 caggtatgac ttcatatgtt gataaacaag aaattacagc aagctcagaa attgatgatt 4740 tgatttttc gagcgatcca ttagtgtggt cttacgacga gcaggaaaaa atcagaaaga 4800 aacttgtgct tgatgcattt cgtaatcatt ataaacattg tcgagaatat cgtcactact 4860 gtcaggcaca caaagtagat gacaatatta cggaaattga tgacatacct gtattcccaa 4920 catcggtttt taagtttact cgcttattaa cttctcagga aaacgagatt gaaagttggt 4980 ttaccagtag cggcacgaat ggtttaaaaa gtcaggtggc gcgtgacaga ttaagtattg 5040 agagactctt aggctctgtg agttatggca tgaaatatgt tggtagttgg tttgatcatc 5100 aaatagaatt agtcaatttg ggaccagata gatttaatgc tcataatatt tggttttaat 5160 atgttatgag ttggtggaa ttgttatatc ctacgacatt taccgtaca gagaacgaa 5220 tagattgt taaaacattg atagctctg aacgaataa aaatcaaggg aagatcttt 5280 gtcttattgg ttcgccatac tttattt tactctgcca ttatatgaaa gataaaaaaa 5340 tctcatttc tggataa agcctttata tcataccgg agggcggctgg aaagttacg 5400 aaaaagaatc tctgaaacgt gatgatttca atcatcttttt atttgatact ttcaatctca 5460 gtgatattag tcagatccga gatatattta atcaagttga actcacact tgtttctttg 5520 aggatgaaat gcagcgtaaa catgttccgc cgtgggtata tgcgcgagcg cttgatcctg 5580 aaacgttgaa acctgtacct gatgaacgc cggggttgat gagttatatg gatgcgtcag 5640 caccagtta tccagcattt attgttaccg atgatgtcgg gataattagc agagaatatg 5700 gtaagtatcc cggcgtgctc gttgaaattt tacgtcgcgt caatacgagg acgcagaaag 5760 ggtgtgcttt aagctttacc gaagcgtttg atagttga 5798 <210> 6 <211> 40 <212> DNA <213> artificial synthesis <400> 6 cgtaatcatg gtcatctcga ggaacaggta tccttctaat 40 <210> 7 <211> 40 <212> DNA <213> artificial synthesis <400> 7 aattttttta gtcatggatc ctctcatgac ctctggcgct 40 <210> 8 <211> 40 <212> DNA <213> artificial synthesis <400> 8 cgtaatcatg gtcatctcga gtctcatgac ctctggcgct 40 <210> 9 <211> 40 <212> DNA <213> artificial synthesis <400> 9 aattttttta gtcatggatc cgaacaggta tccttctaat 40 <210> 10 <211> 79 <212> DNA <213> artificial synthesis <400> 10 gagctcggta cccggggatc catgactaaa aaaatttcac caagcttgca tgcctgcagt 60 caactatcaa acgcttcgg 79 <210> 11 <211> 38 <212> DNA <213> artificial synthesis <400> 11 gttcttctcc tttactcatg aacaggtatc cttctaat 38 <210> 12 <211> 28 <212> DNA <213> Synthetic <400> 12 aatggatcct ctcatgacct ctggcgct 28 <210> 13 [[ID=十六]]<211> 30 <212> DNA 十六 [[ID=二十]]<213> Synthetic <400> 13 aatgaattct tatttgtaga gctcatccat 30 <210> 14 <211> 38 <212> DNA <213> Synthetic <40oac> 14 attagaagga tacctgttca tgagtaaagg agaagaac 38 <2(oac> 15 .<211> 41 <212> DNA <213> Synthetic <400> 15 taaaccagcc agccggaagg gcgaagatcc tttgatcttt t 41 <210> 16 <211> 44 <212> DNA <213> Synthetic <400> 16 ccaacagttg cgcagcctga atggccaggt ggcacttttc gggg 44 Note: There seem to be some incorrect tags like "<2(oac>" and "<40oac>" in the original which are likely errors. I've translated them as best as possible while keeping the incorrect tags as they are. You may want to check and correct those if they are indeed mistakes in the original text.

Claims

1. A bidirectional promoter derived from Enterobacter Enterobacter and Characterized in that, The nucleotide sequence of the bidirectional promoter is as shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. A recombinant plasmid, Characterized in that, The recombinant plasmid contains the bidirectional promoter described in claim 1.

3. The recombinant plasmid according to claim 2, Characterized in that, The starting vector of the recombinant plasmid is pUC19, pACYCDuet-1, pETDuet1 or pTrcHis2b.

4. A recombinant bacterium, Characterized in that, The recombinant bacterium contains the bidirectional promoter described in claim 1 or the recombinant plasmid described in claim 2; the recombinant bacterium is derived from a bacterium of Enterobacter Enterobacter or Escherichia coli.

5. A method for expressing a gene without an inducer, Characterized in that, The recombinant bacterium described in claim 4 is used to express the gene.

6. The application of the bidirectional promoter described in claim 1, the recombinant plasmid described in claim 2 or 3 in bacterial fermentation, Characterized in that, The strain is Enterobacter Enterobacter bacteria or Escherichia coli.

7. The application of the recombinant bacterium described in claim 4 in bacterial fermentation.

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