Patent Publication Number: US-2021163962-A1

Title: Method for Constructing Efficient Bacillus Subtilis Promoter

Description:
TECHNICAL FIELD 
     The present disclosure relates to a method for constructing an efficient  Bacillus subtilis  promoter, and belongs to the technical field of gene engineering. 
     BACKGROUND 
       B. subtilis  is a gram positive type strain widely applied to exogenous protein expression, and it is widely applied to the aspect of industrial enzyme preparation production because of the ability to efficiently express exogenous proteins. However, currently-applied  B. subtilis  promoters, especially natural endogenous  B. subtilis  promoters (such as P43 promoters) are relatively low in activity and relatively poor in expression stability of exogenous genes, and this defect seriously restricts application of the  B. subtilis  to the field of efficient expression of the exogenous proteins. In order to overcome this defect, in recent years, on the one hand, people have used the idea of directed evolution to further screen and modify the natural promoters, and on the other hand, they have used the method of gene engineering and the idea of synthetic biology to construct synthetic promoters. Although the activity of the promoters can be greatly improved by modifying the natural promoters, some defects of the natural promoters still cannot be avoided, such as unstable expression and weak incompatibility with other expression regulating and controlling elements. Therefore, the construction of efficient and stable artificial promoters has huge application prospects in the aspect of breaking through the activity bottleneck of the natural promoters and improving the stability and compatibility of promoter elements. 
     At present, there are mainly two strategies for constructing the artificial promoters, one of the strategies is that the natural promoters are used as basic skeletons, the high-activity natural promoters are screened to be simplified, modified, rearranged and combined, and then new efficient artificial promoters including the natural promoter skeletons are constructed. The other strategy is that random DNA sequences of a certain length are fully artificially synthesized to be cloned to promoter screening vectors, and by virtue of a high-throughput screening device and a high-throughput screening method, the fully artificially synthesized sequences with promoter functions are screened out from the numerous random sequences to be used as promoter elements. Although both of the two strategies have significant disadvantages, the former relies on the high-activity natural promoters, people also need to have a deep understanding of the working principle of the promoters, and as for the series promoters, if the new promoters each include a plurality of repeated sequence fragments, the stability of the promoters and expression vectors will be adversely affected; and although the later does not need to deeply understand the working mechanism of the promoters, target promoters screened from the full random sequences need expensive high-throughput screening apparatuses, and the screening efficiency is low. Therefore, it is of great significance to provide a method for constructing an efficient and stable promoter for the efficient expression of the exogenous proteins. 
     SUMMARY 
     The first purpose of the present disclosure is to provide an element for regulating and controlling gene expression, which includes an artificial series promoter and a downstream RBS thereof, the artificial series promoter is formed by connecting at least two of promoters P rpoB , P spoVG  and P sigW  in series and nucleotide sequences of the promoters P rpoB , P spoVG  and P sigW  are respectively shown as SEQ ID NO:1, SEQ ID NO:7 and SEQ ID NO:11. 
     In one implementation of the present disclosure, a nucleotide sequence of the artificial series promoter is shown as any one of SEQ ID NO:17-SEQ ID NO:27. 
     In one implementation of the present disclosure, a nucleotide sequence of the artificial series promoter is shown as any one of SEQ ID NO:29-SEQ ID NO:59. 
     In one implementation of the present disclosure, intervening sequences of 60 bp and 75 bp are respectively inserted between core areas of the promoters P rpoB  and P spoVG , and new promoters P AW-D60  and P AH-D75  shown as SEQ ID NO:32 and SEQ ID NO:33 are respectively obtained. 
     In one implementation of the present disclosure, intervening sequences of 45 bp and 75 bp are respectively inserted between core areas of the first two of the promoters P sigW , P rpoB  and P spoVG , and new promoters P WAH-D45  and P WAH-D75  shown as SEQ ID NO:43 and SEQ ID NO:45 are respectively obtained. 
     In one implementation of the present disclosure, intervening sequences of 30 bp and 90 bp are respectively inserted between core areas of the first two of the promoters P rpoB , P sigW  and P spoVG , and new promoters P AWH-D30  and P WAH-D90  shown as SEQ ID NO:54 and SEQ ID NO:58 are respectively obtained. 
     In one implementation of the present disclosure, a nucleotide sequence of the RBS is shown as any one of SEQ ID NO:60-72. 
     In one implementation of the present disclosure, an expression host of a target gene includes  B. subtilis.    
     In one implementation of the present disclosure, the expression host of the target gene includes  B. subtilis  168,  B. subtilis  WB400,  B. subtilis  WB600 or  B. subtilis  WB800. 
     In one implementation of the present disclosure, the target gene includes an exogenous gene or an endogenous gene. 
     In one implementation of the present disclosure, the target gene includes an enzyme gene or a non-enzyme gene. 
     The second purpose of the present disclosure is to provide a vector containing the above element. 
     The third purpose of the present disclosure is to provide a genetic engineering bacterium for expressing the above vector. 
     The fourth purpose of the present disclosure is to provide a method for regulating and controlling expression of a target gene in  B. subtilis,  and the above regulating and controlling element is co-expressed with the target gene. 
     In one implementation of the present disclosure, a target protein includes an enzyme. 
     In one implementation of the present disclosure, the  B. subtilis  includes  B. subtilis  168,  B. subtilis  WB400,  B. subtilis  WB600 or  B. subtilis  WB800. 
     The fifth purpose of the present disclosure is to provide application of the above regulating and controlling element or genetic engineering bacterium to preparation of the target protein. 
     The sixth purpose of the present disclosure is to provide application of the above regulating and controlling element or genetic engineering bacterium to a field of food, pharmaceuticals or chemical engineering. 
     The present disclosure has the beneficial effects: the promoters identified by different sigma subunits are screened and characterized firstly in the present disclosure, promoters (recombinant plasmids containing different regulating and controlling elements are transformed into the  B. subtilis,  and the expression quantity of the target gene and the activity of the regulating and controlling elements are characterized by the fluorescence intensity of a culture solution cultured by recombinant bacteria) having the highest activity and identified by the subunits of sigA, sigH and sigW are selected therefrom, and through double series connection and triple series connection of the core areas, intervening sequence optimization of the core areas, RBS redesign and other modes, the regulating and controlling element with the activity being further improved is obtained. 
     The fluorescence intensities of P AW-D60  and P AH-D75  are respectively 0.94 time and 1.03 times higher than the fluorescence intensity of P AW  (with the fluorescence intensity of 20262 a.u/OD 600 ) before modification; the fluorescence intensities (18245 a.u/OD 600 ) of P WAH-D45  and P WAH-D75  are respectively 0.87 time and 0.96 time higher than the fluorescence intensity of PwAH (with the fluorescence intensity of 10261 a.u/OD 600 ) before modification; and the fluorescence intensities of P AWH-D30  and P WAH-D90  are respectively 0.78 time and 0.78 time higher than the fluorescence intensity of P AWH  (with the fluorescence intensity of 16879 a.u/OD 600 ) before modification. 
     When P AH-D75  is combined with RBS11 (SEQ ID NO:70), the fluorescence intensity can reach 76216 a.u/OD 600  and is 0.85 time higher than that of P AH-DM ; when P WAH-D75  is combined with RBS13 (SEQ ID NO:72), the fluorescence intensity can reach 77751 a.u/OD 600  and is 1.17 times higher than that of P WAH-D75 ; and when P AWH-D30  is combined with RBS13 (SEQ ID NO:72), the fluorescence intensity can reach 73781 a.u/OD 600  and is 1.45 times higher than that of P AWH-D30 . 
     Through the method provided by the present disclosure, people can obtain the promoters with the higher activity and stronger designability and compatibility through simple and convenient promoter design and modification methods. The method is simple and easy to implement and has wide application prospects in the construction of an exogenous protein efficient expression system and synthetic biology research. 
    
    
     
       BRIEF DESCRIPTION OF FIGURES 
         FIG. 1 : screening and characterization of core areas of natural endogenous promoters identified by different sigma subunits. 
         FIG. 2 : construction and activity characterization of series promoters. 
         FIG. 3A : a schematic diagram of intervening sequence optimization; 
         FIG. 3B : P AH  intervening sequence optimization of core areas of promoters; 
         FIG. 3C : P WAH  intervening sequence optimization of core areas of promoters; 
         FIG. 3D : P AWH  intervening sequence optimization of core areas of promoters. 
         FIG. 4A : compatibility detection of P rpoB  promoter with an RBS 
         FIG. 4B  compatibility detection of P spoVG  promoter with an RBS; 
         FIG. 4C  compatibility detection of P sigW  promoter with an RBS; 
         FIG. 4D  compatibility detection of P AH-D75  promoter with an RBS; 
         FIG. 4E  compatibility detection of P WAH-D75  promoter with an RBS; 
         FIG. 4F  compatibility detection of P AWH-D30  promoter with an RBS. 
     
    
    
     DETAILED DESCRIPTION 
     1. A cloning method of a promoter: A primer including a promoter sequence is designed. An  Escherichia coli - B. subtilis  shuttle vector pB-sfGFP (i.e., pBSG03, a construction method is shown in Guan C, Cui W, Cheng J, et al. Construction and development of an auto-regulatory gene expression system in  Bacillus subtilis [J]. Microbial Cell Factories, 2015, 14(1):150) with an sfGFP report gene (Genbank ID: AVR55189.1) is taken as a template. PrimeSTAR MAX DNA polymerase (purchased from Takara with an article number of R045Q) is used for full plasmid PCR. PCR procedures are: pre-denaturation at 98° C. for 1 min, circulation including denaturation at 98° C. for 30 s, annealing at 50° C. for 30 s, and extending at 72° C. for 1 min for a total of 30 times, and final extending at 72° C. for 10 min. Then, a plasmid template is digested and removed with a restriction enzyme Dpnl to purify a PCR product. Then, fragments are cyclized through an Infusion reassembling method to be transformed into  E. coli  JM109 competent cells. 
     2. A detection method of an sfGFP fluorescence intensity: A sample is centrifuged at 12000×g for 2 min, and bacteria are collected, washed with PBS 3 times, and then diluted with PBS to a certain concentration to obtain a bacterium suspension. 200 μL of the bacterium suspension is taken to a 96-well ELISA plate, and the 96-well ELISA plate is placed into a Synergy™ H4 fluorescence microplate reader for fluorescence detection. Fluorescence is detected with excitation light of 485 nm and absorbed light of 528 nm. 
     3. A medium: LB medium (g·L −1 ): 10 of Tryptone, 10 of NaCl, 5 of a yeast extract, pH 7.0, and 20 of agar powder added when a solid medium is prepared. 
     4. A transformation method of  B. subtilis  168: Single colonies of the  B. subtilis  168 are picked to be inoculated into a 2 mL SPI medium and subjected to shaking culture at 37° C. for 12-14 h. 100 μL of a culture is taken to be inoculated into a 5 mL SPI medium and subjected to shaking culture at 37° C. for 4-5 h, and then, OD 600  starts to be detected. When OD600 is about 1.0, 200 μL of a bacterium solution is pipetted to be transferred into a 2 mL SPII medium and subjected to shaking incubation at 37° C. and 100 r·min −1  for 1.5 h. 20 μL of a 100×EGTA solution is added into a tube to be cultured in a shaking table at 37° C. and 100 r·min −1  for 10 min, and then each centrifuge tube of 1.5 mL is filled with 500 pi of a mixture. A proper quantity of plasmids verified to be correct by sequencing are added into the tubes, and subjected to blowing-suction uniform mixing to be placed into the shaking table at 37° C. and 100 r·min −1  for 2 h. Culture is completed, and about 200 μL of a bacterium solution is sucked to be uniformly smeared on a corresponding selective plate to be cultured at 37° C. for 12-14 h. 
     EXAMPLE 1 
     Cloning and Characterization of Single Promoters Identified by Different Sigma Subunits 
     Promoters (with nucleotide sequences respectively shown as SEQ ID NO:1-SEQ ID NO:6) identified by six SigA subunits of P rpoB , P sucA , P mtnK , P ylbP , P ylxM  and P yydE , promoters (with nucleotide sequences respectively shown as SEQ ID NO:7-SEQ ID NO:10) identified by four SigH subunits of P spoVG , P pspoVS ,P spo0M  and P minC  and promoters (with nucleotide sequences respectively shown as SEQ ID NO:11-SEQ ID NO:16) identified by six SigW subunits of P sigW , P ydbS , P yobJ , P yqeZ , P ythP  and P yuaF  are selected for testing. Core areas of the cloned promoters include −10 areas, −35 areas and transcriptional start sites (TSS) of the above promoters, with a total of 70 bp. To-be-screened-and-identified promoter sequences are designed on primers (shown in Table 2). The promoter sequences are introduced into a vector skeleton pB-sfGFP through a full plasmid PCR method, and then, through Dpnl digestion, purification and assembly, cloned sfGFP expression plasmids containing the single promoters are transformed and constructed. The sfGFP expression plasmids for expressing the single promoters are transformed into strains of  B. subtilis  168, and recombinant  B. subtilis  is constructed. The obtained recombinant  B. subtilis  is cultured in an LB medium at 37° C. and 200 rpm for24 h, the expression level of sfGFP in bacteria is detected, and the degree of the activity of the promoters is judged through the intensity of an sfGFP fluorescence signal. The results are shown in  FIG. 1  and Table 3, in the promoters identified by SigA, the P rpoB  promoter has the highest activity, in the promoters indentified by SigH, P spoVG  has the highest activity, and in the promoters identified by SigW, P sigW  has the highest activity ( FIG. 1 ). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Cloning primers for single promoters 
               
            
           
           
               
               
               
            
               
                 Primer 
                   
                 Sequence table 
               
               
                 number 
                 Sequence (5′-3′) a   
                 number 
               
               
                   
               
               
                 P rpoB -1 
                 CGGTATTTTAACTATGTTAATATTGTAAAATGCCAATGTATTCGAAC 
                 SEQ ID N0: 73 
               
               
                   
                 ATCATATTTAAAGTACGAGGAG 
                   
               
               
                   
               
               
                 P rpoB -2 
                 ACAATATTAACATAGTTAAAATACCGAGTCAAACTTTTTTTGCTTAC 
                 SEQ ID N0: 74 
               
               
                   
                 CTGCCCTCTGCCACC 
                   
               
               
                   
               
               
                 P sucA -1 
                 ACAATCAAGGTAGAATCAAATTGCAAACAGTGGTAAAATATTCGA 
                 SEQ ID NO: 75 
               
               
                   
                 ACATCATATTTAAAGTACGAGGAG 
                   
               
               
                   
               
               
                 P sucA -2 
                 TTGCAATTTGATTCTACCTTGATTGTTCACAAAATAGTAAAAAACAC 
                 SEQ ID NO: 76 
               
               
                   
                 CTGCCCTCTGCCACC 
                   
               
               
                   
               
               
                 P ylbP -1 
                 TTTTTTAAATAAAGCGTTTACAATATATGTAGAAACAACAATCGAA 
                 SEQ ID NO: 77 
               
               
                   
                 CATCATATTTAAAGTACGAGGAG 
                   
               
               
                   
               
               
                 P ylbP -2 
                 ATATTGTAAACGCTTTATTTAAAAAATCCAAATATTTAAACTTTAAC 
                 SEQ ID NO: 78 
               
               
                   
                 CTGCCCTCTGCCACC 
                   
               
               
                   
               
               
                 P ylxM -1 
                 GTGTCATTAAAACCGTGTAAACTAAGTTATCGTAAAGGGATTCGA 
                 SEQ ID NO: 79 
               
               
                   
                 ACATCATATTTAAAGTACGAGGAG 
                   
               
               
                   
               
               
                 P ylxM -2 
                 CTTAGTTTACACGGTTTTAATGACACTGTCAAGTTTTTATCTTGTAC 
                 SEQ ID NO: 80 
               
               
                   
                 CTGCCCTCTGCCACC 
                   
               
               
                   
               
               
                 P yydE -1 
                 AAAGCAGTTATGCGGTACTATCATATAAAGGTCCAATGTTTTCGAA 
                 SEQ ID NO: 81 
               
               
                   
                 CATCATATTTAAAGTACGAGGAG 
                   
               
               
                   
               
               
                 P yydE -2 
                 ATATGATAGTACCGCATAACTGCTTTTAGAGACAATTAAAACGAGA 
                 SEQ ID NO: 82 
               
               
                   
                 CCTGCCCTCTGCCACC 
                   
               
               
                   
               
               
                 P mtnK -1 
                 CTAACTAAATTACCTGTTACCATGTTCATCAACTGATAAATTCGAAC 
                 SEQ ID N0: 83 
               
               
                   
                 ATCATATTTAAAGTACGAGGAG 
                   
               
               
                   
               
               
                 P mtnK -2 
                 AACATGGTAACAGGTAATTTAGTTAGTTGTCAATATATTTTTTAAAC 
                 SEQ ID N0: 84 
               
               
                   
                 CTGCCCTCTGCCACC 
                   
               
               
                   
               
               
                 P minC -1 
                 GATTTTATCTTTTTTTGACGAAATGAGTATGTTGTTGAGGTTCGAAC 
                 SEQ ID N0: 85 
               
               
                   
                 ATCATATTTAAAGTACGAGGAG 
                   
               
               
                   
               
               
                 P minC -2 
                 TCATTTCGTCAAAAAAAGATAAAATCCTTTTTACTCATCTCTCAAAC 
                 SEQ ID NO: 86 
               
               
                   
                 CTGCCCTCTGCCACC 
                   
               
               
                   
               
               
                 P spoVG -1 
                 TTTCAGAAAAAATCGTGGAATTGATACACTAATGCTTTTATTCGAA 
                 SEQ ID NO: 87 
               
               
                   
                 CATCATATTTAAAGTACGAGGAG 
                   
               
               
                   
               
               
                 P spoVG -2 
                 TATCAATTCCACGATTTTTTCTGAAATCCTGCTCGTTTTTAAAATACC 
                 SEQ ID NO: 88 
               
               
                   
                 TGCCCTCTGCCACC 
                   
               
               
                   
               
               
                 P spoVS -1 
                 GAATATAGCAACTCCTTAGTGAATATAGTAAAAATGGAAGGTCGA 
                 SEQ ID NO: 89 
               
               
                   
                 ACATCATATTTAAAGTACGAGGAG 
                   
               
               
                   
               
               
                 P spVS -2 
                 ATATTCACTAAGGAGTTGCTATATTCCTGCTTTTCTTTTTAATATACC 
                 SEQ ID NO: 90 
               
               
                   
                 TGCCCTCTGCCACC 
                   
               
               
                   
               
               
                 P spo0M -1  
                 GAAAAAAGTATGAATCAAACGAATCTTTTTTTCCTCCTTCTTTCGAAC 
                 SEQ ID NO: 91 
               
               
                   
                 ATCATATTTAAAGTACGAGGAG 
                   
               
               
                   
               
               
                 P spo0M -2 
                 AGATTCGTTTGATTCATACTTTTTTCCTATTATTCGTCTCGGCCTACC 
                 SEQ ID NO: 92 
               
               
                   
                 TGCCCTCTGCCACC 
                   
               
               
                   
               
               
                 P sigW -1  
                 ACCTTTTGAAACGAAGCTCGTATACATACAGACCGGTGAAGTCGA 
                 SEQ ID NO: 93 
               
               
                   
                 ACATCATATTTAAAGTACGAGGAG 
                   
               
               
                   
               
               
                 P sigW -2  
                 TGTATACGAGCTTCGTTTCAAAAGGTTTCAATTTTTTTATAAAATAC 
                 SEQ ID NO: 94 
               
               
                   
                 CTGCCCTCTGCCACC 
                   
               
               
                   
               
               
                 P ydbS -1 
                 ACCTTTCTGTAAAAGAGACGTATAAATAACGACGAAAAAAATCGA 
                 SEQ ID NO: 95 
               
               
                   
                 ACATCATATTTAAAGTACGAGGAG 
                   
               
               
                   
               
               
                 P ydbS -2 
                 TTTATACGTCTCTTTTACAGAAAGGTTTCATTCTTAAGCATACAGAC 
                 SEQ ID NO: 96 
               
               
                   
                 CTGCCCTCTGCCACC 
                   
               
               
                   
               
               
                 P yobJ -1 
                 ACCTTTTTTATTTTAGCCCGTATTAAAAGTAAATTCAGAGATCGAAC 
                 SEQ ID NO: 97 
               
               
                   
                 ATCATATTTAAAGTACGAGGAG 
                   
               
               
                   
               
               
                 P yobJ -2 
                 TTAATACGGGCTAAAATAAAAAAGGTTTCATATAAAACGGGACTA 
                 SEQ ID NO: 98 
               
               
                   
                 ACCTGCCCTCTGCCACC 
                   
               
               
                   
               
               
                 P yqeZ -1 
                 AACCTTTGATACATTTGTTACGTATGAAGAGAAGGCACTTATCGAA 
                 SEQ ID NO: 99 
               
               
                   
                 CATCATATTTAAAGTACGAGGAG 
                   
               
               
                   
               
               
                 P yqeZ -2 
                 CATACGTAACAAATGTATCAAAGGTTTCATTTTTTTATGTATAAAAC 
                 SEQ ID NO: 100 
               
               
                   
                 CTGCCCTCTGCCACC 
                   
               
               
                   
               
               
                 P ythP -1 
                 AAACTTTTTTTATTCTATTTCGTAGTAAATTTTGGAGGTGATCGAAC 
                 SEQ ID NO: 101 
               
               
                   
                 ATCATATTTAAAGTACGAGGAG 
                   
               
               
                   
               
               
                 P ythP -2 
                 ACTACGAAATAGAATAAAAAAAGTTTCTTTAACCATAATAATATTA 
                 SEQ ID NO: 102 
               
               
                   
                 CCTGCCCTCTGCCACC 
                   
               
               
                   
               
               
                 P yuaF -1 
                 ACTTTTCCCGAGGTGTCTCGTATAAATGGTAACGGCAGCCGTCGAA 
                 SEQ ID NO: 103 
               
               
                   
                 CATCATATTTAAAGTACGAGGAG 
                   
               
               
                   
               
               
                 P yuaF -2 
                 TTTATACGAGACACCTCGGGAAAAGTTTCAAAATTTTAAGACAAAA 
                 SEQ ID NO: 104 
               
               
                   
                 CCTGCCCTCTGCCACC 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Total fluorescence intensity of  
               
               
                 recombinant bacteria with sfGFP  
               
               
                 expression plasmids containing single  
               
               
                 promoters after culture for 24 h 
               
            
           
           
               
               
               
            
               
                   
                   
                 Fluorescence 
               
               
                   
                   
                 intensity 
               
               
                   
                 Promoter 
                 (a.u.) 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 P rpoB   
                 83184 
               
               
                   
                 P sucA   
                 49832 
               
               
                   
                 P mtnK   
                 3840 
               
               
                   
                 P ylbP   
                 39028 
               
               
                   
                 P ylxM   
                 2090 
               
               
                   
                 P yydE   
                 13844 
               
               
                   
                 P spoVG   
                 58281 
               
               
                   
                 P spoVS   
                 53313 
               
               
                   
                 P spo0M   
                 1188 
               
               
                   
                 P minC   
                 40567 
               
               
                   
                 P sigW   
                 17181 
               
               
                   
                 P ydbS   
                 11706 
               
               
                   
                 P yobJ   
                 12667 
               
               
                   
                 P yqeZ   
                 13120 
               
               
                   
                 P ythP   
                 4734 
               
               
                   
                 P yuaF   
                 12260 
               
               
                   
                   
               
            
           
         
       
     
     EXAMPLE 2 
     Construction and Characterization of Series Promoters 
     Series design is performed on P rpoB , P spoVG  and P sigW . Full plasmid PCR is conducted by adopting primers in Table 5 and taking three plasmids constructed in Example 1 with promoters P rpoB , P spoVG  and P sigW  as templates. Plasmids containing the series promoters and expressing sfGFP are constructed. The series promoters are named according to the type and order of series core areas, and double-series promoters P AH , P AW , P HA , P HW , P AW , P WA  and P WH  (with nucleotide sequences respectively shown as SEQ ID NO:17-SEQ ID NO:22) and triple-series promoters P AHW , P AWN , P HAW , P HWA , P WAH  and P WHA  (with nucleotide sequences respectively shown as SEQ ID NO:23-SEQ ID NO:28) are obtained. The constructed recombinant plasmids are transformed into  B. subtilis  168, and recombinant  B. subtilis  is obtained. The obtained recombinant  B. subtilis  is cultured in an LB medium at 37° C. and 200 rpm, after 6 h, 12 h and 24 h, a fluorescence signal is detected, and the degree of the activity of the promoters is judged through the intensity of the sfGFP fluorescence signal. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Primers for constructing series promoters 
               
            
           
           
               
               
               
            
               
                   
                   
                 Sequence table 
               
               
                 Primer 
                 Sequence(5′-3′) a   
                 number 
               
               
                   
               
               
                 P rpoB-spoVG -1 
                 CGGTATTTTAACTATGTTAATA 
                 SEQ ID NO: 105 
               
               
                   
                 TTGTAAAATGCCAATGTATATT 
                   
               
               
                   
                 TTAAAAACGAGCAGGATTTCAG 
                   
               
               
                   
               
               
                 P rpoB-sigW -1 
                 CGGTATTTTAACTATGTTAATA 
                 SEQ ID NO: 106 
               
               
                   
                 TTGTAAAATGCCAATGTATATT 
                   
               
               
                   
                 TTATAAAAAAATTGAAACCTTT 
                   
               
               
                   
                 TGAAAC 
                   
               
               
                   
               
               
                 P spoVG-rpoB -1 
                 TTTCAGAAAAAATCGTGGAATT 
                 SEQ ID NO: 107 
               
               
                   
                 GATACACTAATGCTTTTATAAG 
                   
               
               
                   
                 CAAAAAAAGTTTGACTCG 
                   
               
               
                   
               
               
                 P spoVG-sigW -1 
                 TTTCAGAAAAAATCGTGGAATT 
                 SEQ ID NO: 108 
               
               
                   
                 GATACACTAATGCTTTTATATT 
                   
               
               
                   
                 TTATAAAAAAATTGAAACCTTT 
                   
               
               
                   
                 TGAAACG 
                   
               
               
                   
               
               
                 P sigW-rpoB -1 
                 ACCTTTTGAAACGAAGCTCGTA 
                 SEQ ID NO: 109 
               
               
                   
                 TACATACAGACCGGTGAAGAAG 
                   
               
               
                   
                 CAAAAAAAGTTTGACTCG 
                   
               
               
                   
               
               
                 P sigW-spoVG -1 
                 ACCTTTTGAAACGAAGCTCGTA 
                 SEQ ID NO: 110 
               
               
                   
                 TACATACAGACCGGTGAAGATT 
                   
               
               
                   
                 TTAAAAACGAGCAGGATTTCAG 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Total fluorescence intensity of recombinant bacteria  
               
               
                 with sfGFP expression plasmids containing  
               
               
                 double-series and triple-series promoters after culture 
               
            
           
           
               
               
               
               
            
               
                   
                 Fluorescence 
                 Fluorescence 
                 Fluorescence 
               
               
                   
                 intensity 
                 intensity  
                 intensity  
               
               
                 Primer 
                 (a.u.)-6 h 
                 (a.u.)-12 h 
                 (a.u.)-24 h 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 P rpoB   
                 12262 
                 42849 
                 83184 
               
               
                 P spoVG   
                 11581 
                 36157 
                 58281 
               
               
                 P sigW   
                 3421 
                 9383 
                 17181 
               
               
                 P AH   
                 20062 
                 71167 
                 110576 
               
               
                 P AW   
                 25264 
                 53255 
                 72453 
               
               
                 P HA   
                 21630 
                 53088 
                 76553 
               
               
                 P HW   
                 16424 
                 36954 
                 65127 
               
               
                 P WA   
                 5999 
                 43618 
                 91459 
               
               
                 P WH   
                 15518 
                 60354 
                 102747 
               
               
                 P AHW   
                 28506 
                 61632 
                 80954 
               
               
                 P AWN   
                 26367 
                 76719 
                 109470 
               
               
                 P HAW   
                 9267 
                 44867 
                 74699 
               
               
                 P HWA   
                 9830 
                 49937 
                 85714 
               
               
                 P WAH   
                 10261 
                 71036 
                 101361 
               
               
                   
               
            
           
         
       
     
     The fluorescence intensity of the recombinant bacteria with the sfGFP expression plasmids containing the double-series and triple-series promoters after culture for 6, 12 and 24 h is shown in  FIG. 2  and Table 6. Most of the activity of the double-series promoters and the triple-series promoters is improved to different degrees compared with the activity of single promoters, and most of the activity of the triple-series promoters is higher than the activity of the double-series promoters, wherein the PwHA promoter plasmid is transformed into  B. subtilis  unsuccessfully. P AH  with relatively high activity in the double-series promoters, and P WAH  and P AWH  with relatively high activity in the triple-series promoters are selected as further modification materials. 
     EXAMPLE 3 
     Intervening Sequence Optimization of Series Promoters 
     Intervening sequences ( FIG. 3 a   ) of different lengths are inserted between core areas of the promoters, the lengths of the intervening sequences are set to be 15 bp, 30 bp, 45 b, 60 bp, 75 bp and 90 bp, and AH-D15, AH-D30, AH-D45, AH-D60, AH-D75, AH-D90, WAH-U15, WAH-U30, WAH-U45, WAH-U60, WAH-U75, WAH-U90, WAH-D15, WAH-D30, WAH-D45, WAH-D60, WAH-D75, WAH-D90, AWH-U15, AWH-U30, AWH-U45, AWH-U60, AWH-U75, AWH-U90, AWH-D15, AWH-D30, AWH-D45, AWH-D60, AWH-D75, AWH-D90 and AWH-DU30 (with nucleotide sequences respectively shown as SEQ ID NO:29-SEQ ID NO:59) are obtained. Promoter sequences shown as SEQ ID NO:29-SEQ ID NO:59 are respectively cloned onto a pB-sfGFP vector. Then, recombinant plasmids are transformed into  B. subtilis  168 for detection. After the obtained recombinant  B. subtilis  is cultured in an LB medium at 37° C. and 200 rpm for 24 h, a fluorescence signal is detected, and the degree of the activity of the promoters is judged through the intensity of the fluorescence signal of sfGFP. 
     The results show that for example, the fluorescence intensities of P AW-D60  and P AH-D75  are respectively 0.94 time and 1.03 times higher than the fluorescence intensity of PAW (with the fluorescence intensity of 20262 a.u/OD 600 ) before modification; the fluorescence intensities (18245 a.u/OD 600 ) of P WAH-D45  and P WAH-D75  are respectively 0.87 time and 0.96 time higher than the fluorescence intensity of P WAH  (with the fluorescence intensity of 10261 a.u/OD 600 ) before modification; and the fluorescence intensities of P AWH-D30  and P WAH-D90  are respectively 0.78 time and 0.78 time higher than the fluorescence intensity of P AWH  (with the fluorescence intensity of 16879 a.u/OD 600 ) before modification. 
     ( FIGS. 3 b , 3 c  and 3 d   ; and Table 8). The result shows that the activity of the series promoters can be further effectively improved by inserting the intervening sequences of the proper lengths between the series core areas. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Fluorescence intensity of recombinant bacteria  
               
               
                 of recombinant plasmids of double-series and  
               
               
                 triple-series promoters containing inserted  
               
               
                 intervening sequences after culture for 24 h 
               
            
           
           
               
               
               
            
               
                   
                   
                 Fluorescence 
               
               
                   
                   
                 intensity 
               
               
                   
                 Promoter 
                 (a.u./OD 600 ) 
               
               
                   
                   
               
               
                   
                 P AH   
                 20262 
               
               
                   
                 P AH-D15   
                 32865 
               
               
                   
                 P AH-D30   
                 33033 
               
               
                   
                 P AH-D45   
                 34869 
               
               
                   
                 P AH-D60   
                 39476 
               
               
                   
                 P AH-D75   
                 41154 
               
               
                   
                 P AH-D90   
                 28592 
               
               
                   
                 P WAH   
                 18245 
               
               
                   
                 P WAH-U15   
                 18278 
               
               
                   
                 P WAH-U30   
                 18553 
               
               
                   
                 P WAH-U45   
                 17198 
               
               
                   
                 P WAH-U60   
                 18161 
               
               
                   
                 P WAH-U75   
                 19295 
               
               
                   
                 P WAH-U90   
                 18419 
               
               
                   
                 P WAH-D15   
                 24711 
               
               
                   
                 P WAH-D30   
                 25307 
               
               
                   
                 P WAH-D45   
                 34100 
               
               
                   
                 P WAH-D60   
                 32029 
               
               
                   
                 P WAH-D75   
                 35819 
               
               
                   
                 P WAH-D90   
                 24355 
               
               
                   
                 P AWH   
                 16879 
               
               
                   
                 P AWH-U15   
                 16707 
               
               
                   
                 P AWH-U30   
                 15762 
               
               
                   
                 P AWH-U45   
                 16157 
               
               
                   
                 P AWH-U60   
                 15852 
               
               
                   
                 P AWH-U75   
                 16770 
               
               
                   
                 P AWH-U90   
                 18045 
               
               
                   
                 P AWH-D15   
                 27627 
               
               
                   
                 P AWH-D30   
                 30084 
               
               
                   
                 P AWH-D45   
                 28409 
               
               
                   
                 P AWH-D60   
                 25195 
               
               
                   
                 P AWH-D75   
                 26580 
               
               
                   
                 P AWH-D90   
                 29999 
               
               
                   
                 P AWH-DU30   
                 24562 
               
               
                   
                   
               
            
           
         
       
     
     EXAMPLE 4 
     Compatibility Research of Promoters and RBSs 
     The promoters P rpoB , P spoVG , P sigW , P AH-D75 , P WAH-D75  and P AWH-D30  in Example 1 and Example 3 are respectively combined with 13 RBSs. RBS1-13 sequences (with nucleotide sequences respectively shown as SEQ ID NO:60-SEQ ID NO:72) are respectively cloned onto a vector containing series promoters. Then, recombinant plasmids are transformed into  B. subtilis  168. After the obtained recombinant  B. subtilis  is cultured in an LB medium at 37° C. and 200 rpm for 24 h, a fluorescence signal is detected, and the degree of the activity of the promoters is judged through the intensity of the fluorescence signal of sfGFP. Then, correlation analysis is performed on RBS theoretical intensities of different combinations and actually-measured fluorescence values, and correlations are evaluated through r values. The result is shown in  FIG. 4 , the correlation of single promoter P ropB  and RBS combined design is low, while each of the correlations after the series promoters and the RBSs are combined is higher than combination of the single promoter P ropB  and the RBSs, which shows that the compatibility of combined use of the promoters and RBS elements can be improved through the design of the RBSs by the series promoters, that is, the designability and predictability during exogenous protein expression are enhanced. 
     As shown in Table 8, when P AH-D75  is combined with RBS11 (SEQ ID NO:70), the fluorescence intensity can reach 76216 a.u/OD 600  and is 0.85 time higher than that of P AH-D75 ; when P WAH-D75  is combined with RBS13 (SEQ ID NO:72), the fluorescence intensity can reach 77751 a.u/OD 600  and is 1.17 times higher than that of P WAH-D75 ; and when P AWH-D30  is combined with RBS13 (SEQ ID NO:72), the fluorescence intensity can reach 73781 a.u/OD 600  and is 1.45 times higher than that of P AWH-D30 . It shows that the expression of a target gene can be further enhanced through the design of the RBSs by the series promoters. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Translation initiation rate and fluorescence intensity after 
               
               
                 respective combination of promoters with RBS1-13 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Translation 
                   
               
               
                   
                   
                 initiation 
                 Fluorescence 
               
               
                   
                   
                 rate  
                 intensity 
               
               
                 Promoter 
                 RBS 
                 (a.u.) 
                 (a.u./OD 600 ) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 P rpoB−   
                 RBS1 
                 993 
                 506 
               
               
                   
                 RBS2 
                 5675 
                 490 
               
               
                   
                 RBS3 
                 8164 
                 744 
               
               
                   
                 RBS4 
                 30770 
                 1224 
               
               
                   
                 RBS5 
                 56900 
                 8983 
               
               
                   
                 RBS6 
                 77621 
                 3665 
               
               
                   
                 RBS7 
                 109196 
                 48675 
               
               
                   
                 RBS8 
                 280412 
                 47717 
               
               
                   
                 RBS9 
                 507918 
                 50055 
               
               
                   
                 RBS10 
                 818340 
                 65439 
               
               
                   
                 RBS11 
                 1011200 
                 892 
               
               
                   
                 RBS12 
                 1489100 
                 27493 
               
               
                   
                 RBS13 
                 2031360 
                 41641 
               
               
                 P spoVG−   
                 RBS1 
                 993 
                 258 
               
               
                   
                 RBS2 
                 5675 
                 279 
               
               
                   
                 RBS3 
                 8164 
                 390 
               
               
                   
                 RBS4 
                 30770 
                 434 
               
               
                   
                 RBS5 
                 56900 
                 6024 
               
               
                   
                 RBS6 
                 77621 
                 3272 
               
               
                   
                 RBS7 
                 109196 
                 5417 
               
               
                   
                 RBS8 
                 280412 
                 43386 
               
               
                   
                 RBS9 
                 507918 
                 19364 
               
               
                   
                 RBS10 
                 818340 
                 53879 
               
               
                   
                 RBS11 
                 1011200 
                 56203 
               
               
                   
                 RBS12 
                 1489100 
                 51777 
               
               
                   
                 RBS13 
                 2031360 
                 70532 
               
               
                 P sigW−   
                 RBS1 
                 993 
                 270 
               
               
                   
                 RBS2 
                 5675 
                 317 
               
               
                   
                 RBS3 
                 8164 
                 312 
               
               
                   
                 RBS4 
                 30770 
                 312 
               
               
                   
                 RBS5 
                 56900 
                 2213 
               
               
                   
                 RBS6 
                 77621 
                 1385 
               
               
                   
                 RBS7 
                 109196 
                 2142 
               
               
                   
                 RBS8 
                 280412 
                 25573 
               
               
                   
                 RBS9 
                 507918 
                 6991 
               
               
                   
                 RBS10 
                 818340 
                 48885 
               
               
                   
                 RBS11 
                 1011200 
                 32878 
               
               
                   
                 RBS12 
                 1489100 
                 32409 
               
               
                   
                 RBS13 
                 2031360 
                 37704 
               
               
                 P AH-D75−   
                 RBS1 
                 993 
                 373 
               
               
                   
                 RBS2 
                 5675 
                 700 
               
               
                   
                 RBS3 
                 8164 
                 1078 
               
               
                   
                 RBS4 
                 30770 
                 1822 
               
               
                   
                 RBS5 
                 56900 
                 31182 
               
               
                   
                 RBS6 
                 77621 
                 506 
               
               
                   
                 RBS7 
                 109196 
                 484 
               
               
                   
                 RBS8 
                 280412 
                 73457 
               
               
                   
                 RBS9 
                 507918 
                 58264 
               
               
                   
                 RBS10 
                 818340 
                 3976 
               
               
                   
                 RBS11 
                 1011200 
                 76216 
               
               
                   
                 RBS12 
                 1489100 
                 75723 
               
               
                   
                 RBS13 
                 2031360 
                 73351 
               
               
                 P WAH-D75−   
                 RBS1 
                 993 
                 447 
               
               
                   
                 RBS2 
                 5675 
                 704 
               
               
                   
                 RBS3 
                 8164 
                 1229 
               
               
                   
                 RBS4 
                 30770 
                 2416 
               
               
                   
                 RBS5 
                 56900 
                 38253 
               
               
                   
                 RBS6 
                 77621 
                 28222 
               
               
                   
                 RBS7 
                 109196 
                 34329 
               
               
                   
                 RBS8 
                 280412 
                 2539 
               
               
                   
                 RBS9 
                 507918 
                 60937 
               
               
                   
                 RBS10 
                 818340 
                 6350 
               
               
                   
                 RBS11 
                 1011200 
                 76533 
               
               
                   
                 RBS12 
                 1489100 
                 79375 
               
               
                   
                 RBS13 
                 2031360 
                 77751 
               
               
                 P AWH-D30−   
                 RBS1 
                 993 
                 310 
               
               
                   
                 RBS2 
                 5675 
                 590 
               
               
                   
                 RBS3 
                 8164 
                 960 
               
               
                   
                 RBS4 
                 30770 
                 1465 
               
               
                   
                 RBS5 
                 56900 
                 31627 
               
               
                   
                 RBS6 
                 77621 
                 18187 
               
               
                   
                 RBS7 
                 109196 
                 27310 
               
               
                   
                 RBS8 
                 280412 
                 73026 
               
               
                   
                 RBS9 
                 507918 
                 59282 
               
               
                   
                 RBS10 
                 818340 
                 73459 
               
               
                   
                 RBS11 
                 1011200 
                 74559 
               
               
                   
                 RBS12 
                 1489100 
                 61201 
               
               
                   
                 RBS13 
                 2031360 
                 73781 
               
               
                   
               
            
           
         
       
     
     Although the present disclosure has been disclosed as above as exemplary examples, it is not intended to limit the present disclosure. Any of those skilled in the art may make various alterations and modifications without departing from the spirit and scope of the present disclosure. Therefore, the protection scope of the present disclosure shall be as defined in the claims.