Patent Publication Number: US-2021163963-A1

Title: Bacillus Subtilis Efficiently-Induced Expression System Based on Artificial Series Promoter

Description:
TECHNICAL FIELD 
     The present disclosure relates to a  Bacillus subtilis  efficiently-induced expression system based on an artificial series promoter, and belongs to the technical field of gene engineering. 
     BACKGROUND 
       B. subtilis  is a gram positive type strain which is 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. Promoters are the most basic elements for constructing an efficient expression system, and the activity of the promoters directly determines the efficiency of the expression system. Functionally, the promoters are mainly divided into constitutive promoters and inducible promoters. The constitutive promoters can express exogenous genes at all growth stages of bacteria, while the inducible promoters show inactivity or low activity when they are not induced, and after an inducer is added, the activity of the promoters is greatly improved, so that the exogenous genes are efficiently expressed. The inducible promoters are widely applied because of their controllable activity, and people can control the expression time and intensity of the exogenous genes by regulating and controlling a reagent and the concentration of the added inducer. At present, what are widely applied to the  B. subtilis  are mainly an IPTG inducible system and a xylose inducible system, wherein the IPTG inducible system is mainly constructed based on a pHT series vector, using non-natural hybrid promoters and Lacl repressors and binding sites of  Escherichia coli.  The xylose inducible system usually uses natural PxylA promoters and XylR repressors. 
     Although IPTG, xylose and other inducible systems have been widely used, the low expression of the exogenous proteins has always been a key problem restricting system application due to limited promoter activity. People usually adopt two strategies of modifying natural promoters and redesigning artificial promoters to improve the activity and stability of the promoters. However, the high-activity promoters are often unable to be used in the construction of an efficiently-induced exogenous gene expression system, mainly because the promoters and other elements of a gene expression regulating and controlling system are often incompatible, and the activity of the high-activity promoters are often unable to be effectively suppressed by the repressors. Therefore, how to construct the strictly regulated and controlled induced exogenous gene expression system based on the efficient promoters is a hot topic in the development of novel expression systems at present. 
     SUMMARY 
     The present disclosure provides an element for regulating and controlling gene expression, including: (1) a vector, (2) an artificial series promoter, (3) a gene for coding a repressor, and (4) a DNA fragment located at downstream of a transcriptional start site of the promoter and capable of being bound to the repressor, and the artificial series promoter includes P AWH-D30-106 , P AH-D75-106  or P WAH-D75-106 , wherein a nucleotide sequence of P AWH-D30-106  is shown as SEQ ID NO:1, a nucleotide sequence of P AH-D75-106  is shown as SEQ ID NO:2, and a nucleotide sequence of P WAH-D75-106  is shown as SEQ ID NO:3. 
     In one implementation of the present disclosure, the repressor is Lacl, XylR, AraC or TetR. 
     In one implementation of the present disclosure, when the repressor is Lacl, expression of a target gene is induced by IPTG. 
     In one implementation of the present disclosure, when the repressor is XylR, expression of a target gene is induced by xylose. 
     In one implementation of the present disclosure, when the repressor is AraC, expression of a target gene is induced by arabinose. 
     In one implementation of the present disclosure, when the repressor is TetR, expression of a target gene is induced by tetracycline. 
     In one implementation of the present disclosure, the vector is pHT-01. 
     In one implementation of the present disclosure, a host for expressing the target gene is  B. subtilis.    
     In one implementation of the present disclosure, a host for expressing the target gene includes, but is not limited to,  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 present disclosure further provides a recombinant plasmid, expressing any foregoing element for regulating gene expression. 
     The present disclosure further provides a genetic engineering bacterium, expressing the recombinant plasmid. 
     The present disclosure further provides a method for regulating and controlling target gene expression, wherein the above element for regulating gene expression regulates and controls expression of a target gene. 
     The present disclosure further provides application of the above element for regulating and controlling gene expression to preparation of a target protein. 
     The present disclosure further provides application of the above element for regulating and controlling gene expression to a field of food, chemical engineering or pharmaceuticals. 
     The present disclosure further provides application of the above genetic engineering bacterium to preparation of a target protein. 
     The present disclosure further provides application of the above genetic engineering bacterium to a field of food, chemical engineering or pharmaceuticals. 
     According to the present disclosure, an efficient artificial series constitutive promoter is used, and by compositely designing the promoter and elements (a repressor and a binding site thereof) relevant to an operon, the activity of the constitutive promoter is regulated and controlled by an inducer to finally construct a  B. subtilis  efficiently-induced expression system induced by the inducer. The result indicates that compared with a P 43  strongly constitutive promoter, the activity of the artificial series promoter in the system is about 15 times higher. The activity of the promoter can be accurately controlled by adding different concentrations of inducers. Therefore, the efficient expression system is simple in structure, high in activity and strict in regulating and controlling and has wide application prospects in heterologous protein efficient expression and synthetic biology research. 
    
    
     
       BRIEF DESCRIPTION OF FIGURES 
         FIG. 1 : activity comparison of P AWH-D30-106 , P AH-D75-106  and P WAH-D75-106  with a P 43  promoter. 
         FIG. 2A : IPTG induced system principle; 
         FIG. 2B : IPTG induced system characterization; 
         FIG. 2C : verification of sfGFP expression by SDS-PAGE. 
         FIG. 3A : xylose induced system principle; 
         FIG. 3B : xylose induced system characterization; 
         FIG. 3C : verification of sfGFP expression by SDS-PAGE. 
     
    
    
     DETAILED DESCRIPTION 
     1. A construction method of a plasmid: A primer including a promoter sequence is designed, and PrimeSTAR MAX DNA polymerase (purchased from Takara with an article number of R045Q) is used for full plasmid PCR by taking a pHT01 plasmid as a skeleton template. 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, the plasmid template is digested and removed with a restriction enzyme Dpnl to purify a PCR product. Fragments needing to be cloned are amplified through the same method. Then, the plurality of fragments are assembled through an Infusion reassembling method to be transformed into  E. coli  JM109 competent cells. 
     2. A detection method of an sfGFP fluorescence intensity: A to-be-detected 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 (L −1 ): 10 g of tryptone, 10 g of NaCl, 5 g of a yeast extract, pH 7.0, and 20 g of agar powder added when a solid medium is prepared. 
     4. A transformation method of  B. subtilis  168: Single-colony  B. subtilis  168 is picked to be inoculated into a 2 mL SPI medium and subjected to shaking culture at 37° C. for 12 h-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 till OD 600  is about 1.0, and at this time, 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 (ethylene glycol-bis(α-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) 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 μL 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 h-14 h. 
     5. SDS-PAGE detection: 200 μL of a bacterium solution is taken to be centrifuged at 12000×g for 2 min, and bacteria are collected. The bacteria are resuspended with 200 μL of a 20 mM Tris-HCl (pH8.0) buffer solution containing lysozyme at a concentration of 20 μg/mL, and incubated at 37° C. for 30 min to lyse cell walls. Then, 50 μL of 10× Loading Buffer is added, cooked with boiling water for 10 min and then centrifuged at 12000×g for 5 min. 30 μL of a supernatant sample is taken for electrophoresis detection, dyed with Coomassie brilliant blue R250 and destained with a destaining solution, and then an electrophoresis result is analyzed. 
     EXAMPLE 1 
     Activity Identification of Artificial Series Promoters 
     P AWH-D30-106 , P AH-D75-106  and P WAH-D75-106  promoters are all artificially constructed core area series hybrid promoters. 
     Sequences of the P AWH-D30-106 , P AH-D75-106  and P AH-D75-106  promoters are respectively shown as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. 
     The promoter P AWH-D30-106  shown as SEQ ID NO:1 is cloned onto a pHT01 vector through primers in Table 1 with an sfGFP (Genbank ID: AVR55189.1) expression cassette to substitute a P spac  promoter sequence on an original vector. The expression vector is transformed into  B. subtilis  168, recombinant bacteria are cultured for 20 h, and the expression level of sfGFP is detected. 
     In the same way, the promoter P AH-D75-106  shown as SEQ ID NO:2 and the promoter P WAH-D75-106  shown as SEQ ID NO:3 are respectively adopted to be cloned onto the pHT01 vector with the sfGFP expression cassette to substitute P spac promoter sequences on the original vector. The vector is transformed into the  B. subtilis  168 through the same method, recombinant bacteria are cultured for 24 h, and the expression level of sfGFP of recombinant bacterium culture solutions of recombinant plasmids containing P AH-D75-106  and P WAH-D75-106  is detected. 
     By comparing with the situation that a common strongly constitutive promoter P 43  expresses sfGFP, the result shows that the expression levels of the P AWH-D30-106 , P AH-D75-106  and P WAH-D75-106  promoters for sfGFP are much higher than the expression level of the P 43  promoter, and reach about 14 times the expression level of P 43  ( FIG. 1  and Table 2). It shows that the P AWH-D30-106 , P AH-D75-106  and P WAH-D75-106  promoters are strongly constitutive promoters with very high activity. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Primers for constructing expression vector 
               
            
           
           
               
               
               
            
               
                   
                   
                 Sequence table 
               
               
                 Primer 
                 Sequence (5′-3′) a   
                 number 
               
               
                   
               
               
                 PpHT- 
                 CTAACGGAAAAGGGATTTTTGAGTGATCTTCTCA 
                 SEQ ID NO: 6 
               
               
                 AWH-i1 
                 AAAAATAC 
                   
               
               
                   
               
               
                 PpHT- 
                 CCTCGTATGTTTCAAAGAGTGCACCATATGCGG 
                 SEQ ID NO: 7 
               
               
                 AWH-i2 
                   
                   
               
               
                   
               
               
                 PpHT- 
                 CATATGGTGCACTCTTTGAAACATACGAGGCTAA 
                 SEQ ID NO: 8 
               
               
                 AWH-v1 
                 TATCGG 
                   
               
               
                   
               
               
                 PpHT- 
                 AAGATCACTCAAAAATCCCTTTTCCGTTAGCTTTT 
                 SEQ ID NO: 9 
               
               
                 AWH-v2 
                 TC 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Activity of artificial series promoters 
               
            
           
           
               
               
               
            
               
                   
                   
                 Fluorescence intensity 
               
               
                   
                 Promoter 
                 (a.u./OD 600 ) 
               
               
                   
                   
               
               
                   
                 P 43   
                  473 
               
               
                   
                 P AWH-D30-106   
                 6635 
               
               
                   
                 P AH-D75-106   
                 6782 
               
               
                   
                 P WAH-D75-106   
                 6910 
               
               
                   
                   
               
            
           
         
       
     
     EXAMPLE 2 
     Construction of an IPTG Induced Expression System 
     A sequence of a binding site of lacl is GGAATTGTGAGCGGATAACAATTCC (SEQ ID NO:4), which is designed on primers P AWH -lacO-1 and P AWH -lacO-2 in Table 3. The sequence of the Lacl protein binding site is cloned to the downstream of a transcriptional start site of a promoter P AWH-D30-106  through a full plasmid PCR method. Meanwhile, a repressor protein Lacl is expressed through an lad module on a pHT01 vector skeleton. The IPTG induced expression system ( FIG. 2 ) is constructed. 
     sfGFP serves as a target protein to characterize the magnitude of an expression quantity to construct a plasmid pHT-AWH-lac-sfGFP. The recombinant plasmid pHT-AWH-lac-sfGFP is transformed into  B. subtilis  168. Recombinant bacteria are cultured in an LB medium at 37° C. and 200 rpm. After 24 h, the expression level of sfGFP is detected. 
     In the absence of an IPTG inducer, the repressor protein Lac! can be effectively bound to the downstream of a transcriptional start site of a promoter to prevent transcription of downstream genes by RNA polymerase, so that expression of the downstream genes is inhibited. When IPTG is added, the activity of the promoter is released. Within the range of not more than 1 mM, the higher the concentration of the added IPTG is, the stronger the activity of the promoter is. Therefore, expression intensities of the downstream genes with different intensities can be obtained by regulating the usage amount of the IPTG ( FIG. 2  and Table 4). SDS-PAGE detection also proves that sfGFP expression with different intensities is obtained through addition of inducers IPTG of different concentrations ( FIG. 2 ). 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Primers for constructing IPTG induced expression vector 
               
            
           
           
               
               
               
            
               
                   
                   
                 Sequence table 
               
               
                 Primer 
                 Sequence (5′-3′) a   
                 number 
               
               
                   
               
               
                 PAWH-lacO- 
                 GGAATTGTGAGCGGATAACAATTCCATGC 
                 SEQ ID NO: 10 
               
               
                 1 
                 TTTTATTCGAACATCATATTTAAAG 
                   
               
               
                   
               
               
                 PAWH-lacO- 
                 GGAATTGTTATCCGCTCACAATTCCTAGT 
                 SEQ ID NO: 11 
               
               
                 2 
                 GTATCAATTCCACGATTTTTTC 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Expression situations of sfGFP induced by  
               
               
                 IPTG of different concentrations 
               
            
           
           
               
               
               
            
               
                   
                   
                 Fluorescence intensity 
               
               
                   
                 Condition 
                 (a.u./OD 600 ) 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 pHT-01 
                 179 
               
               
                   
                 pHT-AWH  
                 6635 
               
               
                   
                 (uncombined operon) 
                   
               
               
                   
                   0 mM IPTG 
                 516 
               
               
                   
                 0.01 mM IPTG 
                 929 
               
               
                   
                 0.05 mM IPTG 
                 1993 
               
               
                   
                  0.1 mM IPTG 
                 2665 
               
               
                   
                  0.5 mM IPTG 
                 4132 
               
               
                   
                   1 mM IPTG 
                 4163 
               
               
                   
                   
               
            
           
         
       
     
     EXAMPLE 3 
     Construction of a Xylose Induced Expression System 
     A binding site of a repressor protein XylR is cloned to the downstream of a transcriptional start site of a promoter P AWH-D30-106  through primers in Table 5. Meanwhile, an lacl gene on a pHT01 vector skeleton is substituted with an xylR gene. An XylR expression module and the xylose induced expression system are constructed. Specific steps are as follows: 
     The plasmid pHT-AWH-lac-sfGFP constructed in Example 2 is taken as a template, a vector skeleton is amplified through the primer PAWH-xylR-v1/PAWH-xylR-v2 (Table 5), the xylR gene is amplified through the primer PAWH-xylR-i1/PAWH-xylR-i2, two amplified fragments are recombined, and a plasmid with xylR substituting lacl is obtained; then, through the primer PAWH-xylO-1/PAWH-xylO-2, a binding site of lad is substituted with the binding site AGTTAGTTTATTGGATAAACAAACTAACT (SEQ ID NO:5) of xylR through a full plasmid PCR method; and finally, the plasmid pHT-AWH-xyl-sfGFP is constructed. 
     The recombinant plasmid is transformed into  B. subtilis  168. Recombinant bacteria are cultured in an LB medium at 37° C. and 200 rpm. After 24 h, the expression level of sfGFP is detected. 
     In the absence of an inducer xylose, a repressor protein xylR can be effectively bound to the downstream of a transcriptional start site of a promoter to prevent transcription of the promoter by RNA polymerase, so that expression of downstream genes is inhibited ( FIG. 3 ). When xylose of different concentrations is added, the activity of the promoter is released gradually, and therefore, expression intensities of the downstream genes with different intensities can be obtained by regulating the concentration of xylose. With the gradual increase of the concentration of xylose, a fluorescence signal generated through sfGFP expression is enhanced accordingly. When the concentration of xylose exceeds 1% (W/V), the expression level of sfGFP is even higher than a control pHT-AWH ( FIG. 3  and Table 6) of constitutive expression sfGFP. Further, through SDS-PAGE detection, it is proven that sfGFP expression with different intensities is obtained through addition of inducers xylose of different concentrations ( FIG. 3 ). 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Primers for constructing xylose induced expression vector 
               
            
           
           
               
               
               
            
               
                   
                   
                 Sequence table 
               
               
                 Primer 
                 Sequence (5′-3′) a   
                 number 
               
               
                   
               
               
                 PAWH-xylO-1 
                 AGTTAGTTTATTGGATAAACAAACTAACTATGCTTT 
                 SEQ ID NO: 12 
               
               
                   
                 TATTCGAACATCATATTTAAAG 
                   
               
               
                   
               
               
                 PAWH-xylO-2 
                 AGTTAGTTTGTTTATCCAATAAACTAACTTAGTGTA 
                 SEQ ID NO: 13 
               
               
                   
                 TCAATTCCACGATTTTTTC 
                   
               
               
                   
               
               
                 PAWH-xylR-i1 
                 TTAATTGCGTTGCGCCTAACTTATAGGGGTAACAC 
                 SEQ ID NO: 14 
               
               
                   
                 TTAAAAAAG 
                   
               
               
                   
               
               
                 PAWH-xylR-i2 
                 AGGGAGACGATTTTGATGGTTATTATTCAAATTGC 
                 SEQ ID NO: 15 
               
               
                   
                 AGATC 
                   
               
               
                   
               
               
                 PAWH-xylR-v1 
                 TTGAATAATAACCATCAAAATCGTCTCCCTCC 
                 SEQ ID NO: 16 
               
               
                   
               
               
                 PAWH-xylR-v2 
                 ACCCCTATAAGTTAGGCGCAACGCAATTAATG 
                 SEQ ID NO: 17 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Expression situations of sfGFP under different  
               
               
                 concentrations of xylose 
               
            
           
           
               
               
               
            
               
                   
                   
                 Fluorescence intensity 
               
               
                   
                 Condition 
                 (a.u./OD 600 ) 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 pHT-01 
                 179 
               
               
                   
                 pHT-AWH  
                 6635 
               
               
                   
                 (uncombined operon) 
                   
               
               
                   
                   0% xylose 
                 393 
               
               
                   
                 0.01% xylose 
                 2263 
               
               
                   
                 0.05% xylose 
                 3808 
               
               
                   
                  0.1% xylose 
                 4322 
               
               
                   
                  0.5% xylose 
                 5480 
               
               
                   
                   1% xylose 
                 7326 
               
               
                   
                   2% xylose 
                 9556 
               
               
                   
                   
               
            
           
         
       
     
     EXAMPLE 4 
     Expression of a Target Gene by a Xylose Induced Expression System 
     The same strategies as Examples 1-3 are adopted, and the difference is that sfGFP is substituted with other target genes, such as an endogenous gene or an exogenous gene participating in microbial metabolism, to construct a recombinant microorganism containing the xylose induced expression system. The constructed recombinant microorganism is cultured under a suitable condition and induced by inducers xylose of different concentrations. By detecting the gene expression level, it is found that target gene expression results at different intensities can be obtained through addition of the inducers xylose of different concentrations. 
     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.