Abstract:
The invention provides an effective method for improving N-acetylglucosamine (GlcNAc) production by engineered  B. subtilis  Deletion of phosphoenolpyruvate carboxykinase encoding gene pckA and encoding pyruvate kinase gene pyK in recombinant GlcNAc-producing strain BSGNK-PxylA-glmS-P43-GNA1 (BSGNK) is first performed to enhance GlcNAc production, followed by overexpression of pyruvate carboxylase encoding gene pycA for facilitating cell growth. Finally, the GlcNAc production of the recombinant strain BPTS3 reached to 11.3 g/L, which was 1.84-fold of BSGNK. This method can be used for improve cellular property of engineered  B. subtilis  for GlcNAc production, which can be further applied to industrial production of GlcNAc.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     The present application claims the benefit of Chinese Patent Application Nos. 201510761678.6, filed on Nov. 10, 2015, 201510762271.5, filed on Nov. 10, 2015, and 201610517961.9, filed on Jul. 1, 2016, all of which are incorporated by reference for all purposes as if fully set forth herein. 
     REFERENCE TO A SEQUENCE LISTING 
     The present application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. The Sequence Listing is an ASCII file named 100150002.txt, and is 11,616 bytes in size. 
     FIELD OF THE INVENTION 
     The present invention relates to the field of genetic engineering, more particularly to a method for enhancing N-acetylglucosamine (GlcNAc) production in genetically modified  B. subtilis  by microbial fermentation. 
     DESCRIPTION OF THE RELATED ART 
     GlcNAc is a pharmaceutically and nutraceutically useful compound, which was widely used for treatment of osteoarthritis and maintaining health of the joints. Biological production of GlcNAc via microbial fermentation by engineered  B. subtilis  emerged as a promising method to produce GlcNAc in a safe and sustainable approach. The phosphoenol-pyruvate (PEP)-sugar phosphotransferase system (PTS) is the main glucose transportation system in  B. subtilis . Despite efficient glucose uptake by the phosphotransferase system (PTS), 1 mol of pyruvate will be produced for each mole of internalized glucose. Rather, pyruvate also can be produced by central metabolism. As a consequence, much glucose would be used to synthesize pyruvate, which leads to low GlcNAc titer and low yield for the conversion of glucose to GlcNAc. Noticeably, low GlcNAc titer of engineered  B. subtilis  restricted the application for industrial production. To move a step forward for microbial GlcNAc fermentation in industrial conditions, GlcNAc titer and yield should be improved. 
     SUMMARY OF THE INVENTION 
     In order to solve the above problems of the prior art, one object of the present application is to provide a method for improving GlcNAc production of recombinant  Bacillus Subtilis.    
     For the above technical purpose and effects, the application utilizes the following technical solutions: 
     A method for improving GlcNAc production of recombinant  Bacillus Subtilis , the method comprises steps of deletion of phosphoenolpyruvate carboxykinase pckA, deletion of pyruvate kinase pyk, as well as overexpression of pyruvate carboxylase pycA. 
     Preferably, the above recombinant  Bacillus Subtilis  is BSGNK which is obtained by overexpressing a glucosamine-6-phosphate synthase glms under the control of an inducible promoter PxylA and GlcN-6-phosphate N-acetyltransferase GNA1 under the control of a constitutive promoter P43 in the basis of deleting nagP, gamP, nagA, nagB, gamA and glck of  Bacillus Subtilis  168. In a specific embodiment, the BSGNK is constructed by the method disclosed in the China Patent Application Ser. No. 201510394205.7. 
     In an embodiment, deletion of phosphoenolpyruvate carboxykinase encoding gene pckA comprises step of constructing a pckA disrupt cassette, the pckA disrupt cassette includes a pckA upstream homology sequence, a zeocin resistant gene expression cassette, and a pckA downstream homology sequence, from  Bacillus Subtilis  168. Wherein DNA sequence of phosphoenolpyruvate carboxykinase encoding gene pckA is as shown in NCBI-Gene ID: 937235. 
     In some embodiments, the length of the pckA upstream homology sequence is 0.5-1.5 kb, preferably 0.5-1.0 kb, and more preferably 1.0 kb. 
     In some other embodiments, the length of the pckA downstream homology sequence is 0.5-1.5 kb, preferably 0.5-1.0 kb, and more preferably 1.0 kb. 
     In a specific embodiment, the DNA sequence of the pckA disrupt cassette is as shown in SEQ ID NO.1. 
     In an embodiment, deletion of pyruvate kinase encoding gene pyk comprises step of constructing a pyk disrupt cassette, the pyk disrupt cassette includes a pyk upstream homology sequence, a zeocin resistant gene expression cassette, and a pyk downstream homology sequence, from  B. subtilis  168. Wherein the DNA sequence of pyruvate kinase encoding gene pyk is as shown in NCBI-Gene ID: 936596. 
     In some embodiments, the length of the pyk upstream homology sequence is 0.5-1.5 kb, preferably 0.5-1.0 kb, and more preferably 1.0 kb. 
     In some other embodiments, the length of the pyk downstream homology sequence is 0.5-1.5 kb, preferably 0.5-1.0 kb, and more preferably 1.0 kb. 
     In a specific embodiment, the DNA sequence of the pyk disrupt cassette is as shown in SEQ ID NO.2. 
     In an embodiment, the pyruvate carboxylase pycA is overexpressed under the control of a constitutive promoter P43 and replacing a start codon GTG with ATG. The DNA sequence of pyruvate carboxylase encoding gene pycA is shown in NCBI-Gene ID: 935920. 
     In another embodiment, overexpression of pyruvate carboxylase encoding gene pycA comprises step of constructing a pycA overexpressed cassette which includes a pycA upstream homology sequence, a zeocin resistant gene expression cassette, a P43 strong promoter, and a pycA sequence with replacing the start codon GTG with ATG, from  B. subtilis  168. 
     In some embodiments the length of the pycA upstream homology sequence is 0.5-5 kb, preferably 0.5-1.0 kb, and more preferably 1.0 kb. 
     In a specific embodiment, the DNA sequence of the pycA overexpressed cassette is shown as SEQ ID NO.3. 
     In a preferable embodiment of the invention, the method comprises the following steps: 
     (1) deleting phosphoenolpyruvate carboxykinase encoding gene pckA of recombinant  Bacillus Subtilis  BSGNK to block the anaplerosis from PEP to oxaloacetate, to obtain a recombinant strain BPTS1; 
     (2) deleting pyruvate kinase encoding gene pyk of the recombinant strain BPTS1 to block the synthesis from PEP to pyruvate by glycolysis pathway, to obtain a recombinant strain BPTS2; and 
     (3) overexpressing pyruvate carboxylase encoding gene pycA of recombinant strain BPTS2 to facilitate cell growth, to obtain a recombinant strain BPTS3. 
     In a more preferable embodiment, the step (1) specifically comprises: 
     utilizing a primer pckA-F/pckA-R to amplify a pckA disrupt cassette which includes a pckA upstream homology sequence, a zeocin resistant gene expression cassette, and a pckA downstream homology sequence, from  Bacillus Subtilis  168; 
     transforming the amplified pckA disrupt cassette into BSGNK, and selecting a positive transformant; 
     introducing a vector pTSC into the positive transformant to delete the resistance marker cassette; and 
     evicting the plasmid pTSC to obtain the recombinant strain BPTS1. 
     In a more preferable embodiment, the step (2) specifically comprises: 
     utilizing a primer pyk-F/pyk-R to amplify a pyk disrupt cassette which includes a pyk upstream homology sequence, a zeocin resistant gene expression cassette, and a pyk downstream homology sequence, from  Bacillus Subtilis  168; 
     transforming the amplified pyk disrupt cassette into the recombinant strain BPTS1, and selecting a positive transformant; 
     introducing a vector pTSC into the positive transformant to delete the resistance marker cassette; and 
     evicting the plasmid pTSC to obtain the recombinant strain BPTS2. 
     In a still more preferable embodiment, the step (3) specifically comprises: 
     utilizing a primer pycA-F/pycA-R to amplify a pycA overexpressed cassette which includes a pycA upstream homology sequence, a zeocin resistant gene expression cassette, a P43 strong promoter, and a pycA sequence with replacing the start codon GTG with ATG, from  Bacillus Subtilis  168; 
     transforming the amplified pycA overexpressed cassette into the recombinant strain BPTS2, and selecting a positive transformant; 
     introducing a vector pTSC into the positive transformant to delete the resistance marker cassette; and 
     evicting the plasmid pTSC to obtain the recombinant strain BPTS3. 
     Due to the above technical solutions, the embodiments of the present invention have the following advantages as compared with the prior art: the method disclosed in the specification is more effective for improving production of N-acetylglucosamine (GlcNAc). Specially, the GlcNAc production of the recombinant strain BPTS3 reached to 11.3 g/L, which was 1.84-fold of that of the starting strain BSGNK. Furthermore, the method also greatly increases the yield for the conversion of glucose to GlcNAc and the GlcNAc Productivity. So, this method can be used for improving cellular property of engineered  B. subtilis  for GlcNAc production, which can be further applied to industrial production of GlcNAc. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a graph showing the comparison of cell growth of the strains BSGNK, BPTS1, BPTS2 and BPTS3 during fermentation; 
         FIG. 2  is a graph showing the comparison of GlcNAc titer of the strains BSGNK, BPTS1, BPTS2 and BPTS3 during fermentation; 
         FIG. 3  is a graph showing the comparison of the residual glucose concentration of the strains BSGNK, BPTS1, BPTS2 and BPTS3 during fermentation. 
     
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The invention will be further illustrated in more detail with reference to accompanying drawings. It is noted that, the following embodiments are intended for purposes of illustration only and are not intended to limit the scope of the invention. 
     The present invention provides a g method for improving GlcNAc production in engineered  B. subtilis . Specifically, the method comprises the steps of deletion of phosphoenolpyruvate carboxykinase encoding gene pckA, deletion of pyruvate kinase encoding gene pyk and overexpression of pyruvate carboxylase encoding gene pycA in a recombinant  Bacillus Subtilis . In a preferable embodiment, the starting strain is BSGNK-PxylA-glmS-P43-GNA1 (BSGNK), and the finally obtained strain with improved GlcNAc production and yield is BPTS3. 
     Embodiment 1 
     Deletion of Phosphoenolpyruvate Carboxykinase Encoding Gene pckA of the Strain BSGNK to Block the Anaplerosis from PEP to Oxaloacetate, to Obtain the Recombinant Strain BPTS1, Wherein BSGNK is Obtained by the Method Disclosed in China Patent Application Ser. No. 201510394205.7. 
     Deletion of phosphoenolpyruvate carboxykinase pckA was first performed to block the anaplerosis from PEP to oxaloacetate. Specifically, a primer pckA-F (ACGGACTTCACTTAGGCGGC)/pckA-R (GACGGATTTTTATATTTGCGCG) was used to amplify a pckA disrupt cassette, which included a pckA upstream homology sequence (1 kb), a zeocin resistant gene expression cassette, and a pckA downstream homology sequence (1 kb), from  B. subtilis  168. DNA sequence of the pckA disrupt cassette is as shown in SEQ ID NO.1. The amplified pckA disrupt cassette was transformed into the strain BSGNK, and transformants were selected on LB plate with 30 μg/mL zeocin. Positive transformants with pckA gene deletion were further verified by colony PCR with primers pckA-F/pckA-R. The vector pTSC was introduced into the Positive transformants to promote the recombination between lox71 and lox66, thereby deleting the resistance marker cassette. Plasmid pTSC was then evicted by incubating at 50° C. for 12 h to obtain the strain without the selected marker and plasmid, naming BPTS1. 
     Embodiment 2 
     Deletion of Pyruvate Kinase Encoding Gene pyK in the Strain BPTS1 to Block the Synthesis from PEP to Pyruvate by Glycolysis Pathway. 
     Deletion of pyruvate kinase pyK was performed to block the synthesis from PEP to pyruvate by glycolysis pathway. Specifically, the primer pyK-F (ACGAATAGGGGTATTAACGAGCG)/pyK-R(CAGCTAACAGCAAAGCAATCAGC) was used to amplify a pyK disrupt cassette, which included a pyK upstream homology sequence (1 kb), a zeocin resistant gene expression cassette, and a pyK downstream homology sequence (1 kb), from  B. subtilis  168. DNA sequence of the pyK disrupt cassette is as shown in SEQ ID NO.2. The amplified pyk disrupt cassette was transformed into the strain BPTS1, and transformants were selected for on LB plate with 30 μg/mL zeocin. Positive transformants with pyK gene deletion were further verified by colony PCR with primers pyK-F/pyK-R. The vector pTSC was introduced into the Positive transformants to promote the recombination between lox71 and lox66, thereby deleting the resistance marker cassette. Plasmid pTSC was then evicted by incubating at 50° C. for 12 h to obtain the strain without the selected marker and plasmid, naming BPTS2. 
     Embodiment 3 
     Overexpression of Pyruvate Carboxylase Encoding Gene pycA of the Strain BPTS2 to Facilitate Cell Growth. 
     Overexpression of pyruvate carboxylase encoding gene pycA was performed to facilitate cell growth. Specifically, a primer pycA-F (GCAGAGCTGGTTTAAAATCGG)/pycA-R(CCCAAGTTGAAAGCTTAACGAGA) was used to amplify a pycA overexpressed cassette, which included a pycA upstream homology sequence (1 Kb), a zeocin resistant gene expression cassette, a P43 strong promoter, a pycA sequence with replacing the start codon GTG with ATG, from  B. subtilis  168. DNA sequence of pycA overexpressed cassette is as shown in SEQ ID NO.3. The amplified pycA overexpressed cassette was transformed into the strain BPTS2, and transformants were selected on LB plate with 30 μg/mL zeocin. Positive transformants with pycA gene overexpression were further verified by colony PCR with primers pycA-F/pycA-R. The vector pTSC was introduced into the positive transformants to promote the recombination between lox71 and lox66, thereby deleting the resistance marker cassette. Plasmid pTSC was then evicted by incubating at 50° C. for 12 h to obtain the strain without the selected marker and plasmid, naming BPTS3. 
     Shake-Flask Fermentation of the Strains BSGNK, BPTS1, BPTS2 and BPTS3. 
     The seed medium was Luria-Bertani broth or agar plates containing (g/L): tryptone 10, yeast extract 5, and NaCl 10. The fermentation medium contained (g/L): tryptone 6, yeast extract 12, (NH 4 )SO 4  6, K 2 HPO 4 .3H 2 O 12.5, KH 2 PO 4  2.5, MgSO 4 .7H2O 3, CaCO 3  5, glucose 60, and 15 ml of trace metal solution. The trace metal solution contained (per liter of 5M HCl) (g/L): FsSO 4 .7H 2 O 4.0, CaCl 2  4.0, MnSO 4 .5H 2 O 1.0, CoCl 2 .6H 2 O 0.4, NaMnO 4 .2H 2 O 0.2, ZnSO 4 .7H 2 O 0.2, AlCl 3 .6H 2 O 0.1, CuCl 2 .H2O 0.1, and H 3 BO 4  0.05. Seed culture was carried out in 250-mL shake flasks each containing 20 ml of seed medium with shaking at 200 rpm and 37° C. for 12 h. The seed culture (5 ml) was inoculated into 500-mL shake flasks containing 95 mL of fermentation medium. And then, fermentation was carried out at 220 rpm and 37° C. for 48 h on rotary shakers. When the optical density at 600 nm (OD600) reached 0.4, xylose was added to the medium to a final concentration of 5 g/L to induce gene expression under the control of the xylose-inducible P xyla  promoter. 
     Embodiment 4 
     Effects of Deletion of Phosphoenolpyruvate Carboxykinase Encoding Gene pckA on Cell Growth and GlcNAc Production 
     To determine the effects of deletion of phosphoenolpyruvate carboxykinase encoding gene pckA on cell growth and GlcNAc production, the strain BPTS1 and BSGNK were inoculated with an inoculum size of 5% (v/v) into 500-mL shake flasks each containing 95 mL of fermentation medium. And then, fermentation was carried out at 220 rpm and 37° C. for 48 h on rotary shakers. 
     It can be seen from  FIGS. 1-2  that the strain BPTS1 produced GlcNAc to a higher concentration than BSGNK by 7.14 g/L in the flask cultivation, which was 1.16-fold of that of BSGNK, and deletion of phosphoenolpyruvate carboxykinase encoding gene pckA had little influence on cell growth. The results indicate that deletion of phosphoenolpyruvate carboxykinase encoding gene pckA is benefit for the GlcNAc synthesis. 
     Embodiment 6 
     Effects of Deletion of Pyruvate Kinase Encoding Gene pyk on Cell Growth and GlcNAc Production 
     To determine the effects of deletion of pyruvate kinase encoding gene pyk on cell growth and GlcNAc production, the strains BPTS2 and BPTS1 were inoculated with an inoculum size of 5% (v/v) into 500-mL shake flasks each containing 95 mL of fermentation medium. And then, fermentation was carried out at 220 rpm and 37° C. for 48 h on rotary shakers. 
     It can be seen from  FIG. 2  that the strain BSPT2 produced GlcNAc to a higher concentration than BPTS1 by 8.5 g/L in the flask cultivation, which was 1.18-fold of that of BPTS1. However, deletion of pyruvate kinase encoding gene pyk leaded to the low glucose consumption rate and decreased DCW.  FIG. 3  shows the time profile of glucose consumption of BSGNK, BPTS1, BPTS2, BPTS3 in shake flask. The residual glucose concentration of BPTS2 was 12.2 g/L. This was thought to be the non-optimal TCA flux and reduced glycolytic flux. 
     Embodiment 7 
     Overexpression of Pyruvate Carboxylase Encoding Gene pycA to Facilitate Cell Growth 
     It is possible that the overexpression of pycA can lead more pyruvate to synthesize OAA and facilitates the glutamine synthesis. Finally, we tested the effects of overexpression of pyruvate carboxylase to cell growth and GlcNAc production. It can be seen from  FIGS. 1-2  that the overexpression of pycA can facilitate cell growth, and the glucose consumption rate of BPTS3 was improved than that of BPTS2. Finally, the GlcNAc production of BPTS3 reached 11.3 g/L, which was 32.8% higher than that of BPTS2 and 1.84-fold of that of BSGNK. 
       FIGS. 1-3  show the effects of deletion of phosphoenolpyruvate carboxykinase encoding gene pckA and pyruvate kinase encoding gene pyk and overexpression of pyruvate carboxylase encoding gene pycA on cell growth, GlcNAc production and glucose consumption. 
     Table 1 shows the comparison of the maximum GlcNAc titer, the maximum DCW and the GlcNAc productivity of BSGNK, BPTS1, BPTS2, BPTS3 in shake flask fermentation system. 
     
       
         
               
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                   
                 The maximum 
                 The maximum 
                 GlcNAc 
               
               
                   
                   
                 GlcNAc titer 
                 DCW 
                 productivity 
               
               
                   
                 Strains 
                 (g/L) 
                 (g/L) 
                 (g/L/h) 
               
               
                   
                   
               
             
             
               
                   
                 BSGNK 
                 6.17 
                 7.17 
                 0.036 
               
               
                   
                 BPTS1 
                 7.14 
                 6.81 
                 0.044 
               
               
                   
                 BPTS2 
                 8.48 
                 6.08 
                 0.030 
               
               
                   
                 BPTS3 
                 11.3 
                 6.03 
                 0.052 
               
               
                   
                   
               
             
          
         
       
     
     The above preferred embodiments are described for illustration only, and are not intended to limit the scope of the invention. It should be understood, for a person skilled in the art, that various improvements or variations can be made therein without departing from the spirit and scope of the invention, and these improvements or variations should be covered within the protecting scope of the invention.