Abstract:
The present invention relates to the field of bioengineering and discloses a cutinase-producing genetically engineered microorganism and use thereof. Recombinant plasmid Tfu_0883-hlyAs/pET20b(+) was constructed and transformed into  E. coli  BL21(DE3) to obtain recombinant  E. coli  strain Tfu_0883-hlyAs/pET20b(+)/ E. coli  BL21(DE3). Specific growth rate was maintained at a certain value using fed-batch fermentation mode. After fermenting 30-34 hours, the enzyme activity in the supernatant reached 700-750 U/mL. The present invention uses glycerol as the main raw material and employs semi-synthetic medium, has the advantages of good stability and ease of control, and is suitable for large-scale production.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to the field of bioengineering, particularly to a cutinase-producing genetically engineered microorganism and use thereof. 
       BACKGROUND ART 
       [0002]    Cutinase is a multifunctional enzyme that belongs to the family of serine esterase. It can not only hydrolyze long- and short-chain fatty acid esters, emulsified triglycerides and soluble synthetic esters, but also participate in esterification, transesterification etc. By virtue of its special structure, cutinase can also hydrolyze cutin. Therefore, cutinase finds wide application in a variety of industries, including textile, food, biocatalysis and chemical industries. 
         [0003]    Researches on the fermentation of cutinase are mainly focused on optimization of culturing conditions of wild microorganism strains and highly efficient expression of recombinant fungal cutinase using different genetically engineered host cells (such as  Saccharomyces cerevisiae, E. coli , and  Aspergillus  spp.), aiming to optimize production processes and reducing production cost, among others. However, so far no reports are available on the industrialized production of cutinase, due to the problems of high cost of cultivation and long period of growth of yeasts, as well as poor stability of  F. solani pisi , a recombinant strain for producing cutinase. 
         [0004]    Cheng Sheng from our laboratory reported (Chen S, Tong X, Woodard R W, Du G C, Wu J, Chen J, Identification and Characterization of Bacterial Cutinase, The Journal of Biological Chemistry, 2008, 283 (28) 25854-25862) cutinase from  Thermobifida fusca  which showed a good thermal stability, a wide pH stability range, an optimal temperature of 60° C., and an optimal pH of 8.0, consistent with the requirements of application of cutinase for textile use. Based on this, optimization of fermentation was conducted, achieving an enzyme activity of 500 U/mL in a fermention run in a 3 L fermentor for 30 hours (Chinese Patent Application No. 200910259651.1 to Jing Wu, Dan Wu, Yao Zhang, Jian Chen, and Sheng Chen, titled “A Fermentation Process of Recombinant Cutinase”). However, two disadvantages remain. Firstly, supplementation on the basis of complex media resulted in complexity of the components and inconvenience in the control of fermentation process. Secondly, use of the type II secretion pathway to transport across the outer and inner membranes of  E. coli  in two steps yielded low efficiency of transport, while addition of a certain amount of glycine during fermentation to modify the permeability of cell wall in order to increase the level of extracellular secretion lead to increased production cost. 
       SUMMARY OF THE INVENTION 
       [0005]    A technical problem to be solved by the present invention is to provide a cutinase-producing genetically engineered microorganism. 
         [0006]    The genetically engineered microorganism is a recombinant  E. coli  strain Tfu — 0883-hlyAs/pET20b(+)/ E. coli  BL21(DE3) obtained by constructing recombinant plasmid Tfu — 0883-hlyAs/pET20b(+) and transforming the plasmid into  E. coli  BL21(DE3). 
         [0007]    The cutinase-producing genetically engineered microorganism is constructed as follows:
       1) cutinase Tfu — 0883 gene is amplified using plasmid Tfu — 0883/pET20b(+) previously constructed in our laboratory as template;   2) hlyAs gene is amplified using  E. coli  CFT073 total DNA as template (Chinese Patent Application No. 200910260984.6);   3) PCR amplification is performed using the PCR fragments of Tfu — 0883 gene and hlyAs gene recovered by gel cutting as templates to obtain Tfu — 0883-hlyAs gene;   4) pET20b(+) and Tfu — 0883-hlyAs are subjected to double enzyme digestion, and the products recovered by gel cutting are transformed into  E. coli  JM109 competent cells to obtain plasmid Tfu — 0883-hlyAs/pET20b(+);   5) the recombinant plasmid Tfu — 0883-hlyAs/pET20b(+) is transformed into  E. coli  BL21(DE3) to obtain  E. coli  strain Tfu — 0883-hlyAs/pET20b(+)/ E. coli  BL21(DE3).       
 
         [0013]    The above-said Tfu — 0883/pET20b(+) can be obtained using conventional means (Chen S, Tong X, Woodard R W, Du G C, Wu J, Chen J, Identification and Characterization of Bacterial Cutinase, The Journal of Biological Chemistry, 2008, 283 (28) 25854-25862). 
         [0014]    The above-said plasmid pET20b(+) and  E. coli  BL21(DE3) strain are commercially available from Novagen Inc., and  E. coli  CFT073 strain (ATCC 700928) is available from ATCC. 
         [0015]    Another technical problem to be solved by the present invention is to provide a method for the production of cutinase by fermenting the above-said cutinase-producing genetically engineered microorganism. 
         [0016]    The above-said problem is addressed by the following specific production processes: 
         [0017]    1) during fermentation, the temperature is maintained at 36-38° C., the dissolved oxygen is maintained at 20-40% by increasing or decreasing stirring rotation speed or supplying oxygen-rich air, and the pH is maintained at 7.0-7.2 in the growth stage and at 6.4-6.6 in the stage of induction of production of the enzyme by supplementing ammonia water; 
         [0018]    2) after fermenting for 5-6 hours or when the concentration of dissolved oxygen rises to more than 70%, 500 g/L of glycerol is supplemented at an initial flow rate of 3.5-5.0 mL·L −1 ·h −1 , and subsequently, glycerol is fed in exponential feeding mode to control the specific growth rate of the microorganism in the range of 0.15-0.22 h −1 ; 
         [0019]    3) after fermenting for 12-13 hours or when OD 600  reaches 25-35, IPTG is added at a final concentration of 0.02-0.04 mM/L for induction, and at the same time 50 g/L of lactose is supplemented at a rate of 8-10 mL·L −1 ·h −1  and the supplementation of ammonia water is stopped until the pH drops to 6.4-6.6, at which time the supplementation of ammonia water is resumed to control the pH at 6.4-6.6; 
         [0020]    4) after fermenting for 15-17 hours or inducing for 3-4 hours, glycerol is fed at a constant flow rate of about 25-35 mL·L −1 ·h −1 ; 
         [0021]    5) after fermenting for 19-21 hours or when OD 600  no longer increases, the flow rate of lactose is decreased to 2-4 mL·L −1 ·h −1 , and the flow rate of glycerol is gradually decreased to half of that in step 4) over 6-8 hours. 
         [0022]    Seed culture is obtained by inoculating the inoculum having been stored at −80° C. in a seed culture medium at an initial pH of 7.0-7.2 and culturing on a constant-temperature rotating shaking bed at 37° C., 200 rpm for 7-8 hours. The seed culture medium consists of 10 g/L of peptone, 5 g/L of yeast powder, 10 g/L of NaCl and 100 mg/L of ampicillin. 
         [0023]    The seed culture is inoculated at an amount of 4-8% for the fermentation. 
         [0024]    The fermentation medium consists of 1 g/L of peptone, 2 g/L of yeast powder, 4 g/L of (NH 4 ) 2 HPO 4 , 13.5 g/L of KH 2 PO 4 , 4.1 g/L of MgSO 4 .7H 2 O, 0.85 g/L of citric acid, 8 g/L of glycerol, 5 mL/L of a solution of trace elements and 100 mg/L of ampicillin. The seed culture is inoculated at an amount of 4-8% for the fermentation. 
         [0025]    The solution of trace elements consists of 5 M/L of HCl, 10 g/L of FeSO 4 .7H 2 O, 2.25 g/L of ZnSO 4 .7H 2 O, 1.0 g/L of CuSO 4 .5H 2 O, 0.5 g/L of MnSO 4 .4H 2 O, 0.23 g/L of Na 2 B 4 O 7 .10H 2 O, 2.0 g/L of CaCl 2 .2H 2 O and 0.1 g/L of (NH 4 ) 6 Mo 7 O 24 . 
         [0026]    The cutinase-producing genetically engineered microorganism Tfu — 0883-hlyAs/pET20b(+)/BL21(DE3) constructed according to the present invention, when cultured in a flask for 60 hours, achieved a yield of cutinase of up to 274 U/mL, and when fermented using the method provided in the present invention for 30-34 hours, achieved an activity of cutinase of 700-750 U/mL. The present invention also has the advantages of low cost of the fermentation medium and easy control over the microorganism, and is adapted for application in the industrialized production of cutinase. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0027]    The present invention is further described by the following examples, which should be considered as illustrative and not limitative of the scope of the present invention. In the following examples, those experimental procedures for which specific conditions are not specified are essentially performed in accordance with the conditions described in commonly used manuals for molecular cloning. 
         [0028]    Strains and plasmid: plasmid pET20b(+), and strains  E. coli  BL21(DE3),  E. coli  CFT073 and  E. coli  JM109. 
         [0029]    Materials and methods: restriction endonucleases, T4 DNA ligase, pMD18-T simple vector, PCR reagents and DNA Markers were purchased from Takara Co., Ltd.; competent  E. coli  JM109 cells, primers, plasmid extraction kit and PCR products purification kit were purchased from Sangon Biotech (Shanghai) Co., Ltd. The electroporator was purchased from Bio-Rad Co., Ltd. 
       EXAMPLE 1 
     Construction of Tfu — 0883-hlyAs-pET20b(+)/ E. coli  BL21 (DE3) 
       [0030]    Based on the genes of cutinase and hlyAs, two pairs of primers P1, P2 and P3, P4 were designed. 
         [0000]    
       
         
               
               
             
           
               
                   
                 P1: 5′-GTAATCCATATGGCCAACCCCTACGAGCGC-3′ 
               
               
                   
                   
               
               
                   
                 P2: 5′-GACTTCCATAGGCTAAGAACGGGCAGGTGGAG-3′ 
               
               
                   
                   
               
               
                   
                 P3: 5′-CTCCACCTGCCCGTTCTTAGCCTATGGAAGTC-3′ 
               
               
                   
                   
               
               
                   
                 P4: 5′-CCGCTCGAGTTATGCTGATGCTGTCAAAG-3′ 
               
             
          
         
       
     
         [0031]    The gene of cutinase, Tfu — 0883, was amplified by PCR using plasmid Tfu — 0883/pET20b(+)DNA as template and P1, P2 as primers. The gene of hlyAs was amplified by PCR using  E. coli  CFT073 total DNA as template and P3, P4 as primers. 
         [0032]    The PCR reactions were performed in a 50 μL system under the conditions of: denaturing at 94° C. for 1 minute, and then 30 cycles of denaturing at 94° C. for 30 seconds, annealing at 60° C. for 30 seconds, and elongation at 72° C. for 1 minute and 20 seconds respectively, followed by elongation at 72° C. for 10 minutes. The amplification yielded PCR fragments of 783 by and 180 by respectively, which were recovered by gel cutting. 
         [0033]    Again, PCR reaction was performed using the PCR fragments of Tfu — 0883 gene and hlyAs gene recovered by gel cutting as templates and P1, P4 as primers. The amplification yielded a PCR fragment of 963 bp, which was recovered by gel cutting. The recovered fragment was ligated with pMD18-T simple vector and the ligation product was transformed into  E. coli  JM109. The transformed cells were plated on an LB solid plate containing 100 mg/L of ampicillin and cultured at 37° C. overnight. Single clones were picked and inoculated into an LB liquid medium containing 100 mg/L of ampicillin and cultured at 37° C., 200 rpm for 8-10 hours followed by extraction of the plasmid. The plasmid was sequenced, indicating that this gene had 963 nucleotides in full length and was completely consistent with the sequences of both Tfu — 0883 and hlyAs genes. 
         [0034]    The pET20b(+) plasmid and the Tfu — 0883-hlyAs gene were subjected to double enzyme digestion with NdeI and XhoI. The enzyme digestion products were recovered by gel cutting and then ligated using T4 ligase at 16° C. overnight. The ligation products were transformed into competent  E. coli  JM109 cells. The transformed cells were plated on an LB solid plate containing 100 mg/L of ampicillin and cultured at 37° C. overnight. The transformants were picked into an LB liquid medium containing 100 mg/L of ampicillin and cultured, followed by extraction of the plasmid to obtain the enriched Tfu — 0883-hlyAs/pET20b(+) plasmid. 
         [0035]    The recombinant plasmid Tfu — 0883-hlyAs/pET20b(+) was transformed into  E. coli  BL21 (DE3) host strain. The transformed cells were cultured on an LB plate containing 100 mg/L of ampicillin at 37° C. for 8-10 hours and the transformants (Tfu — 0883-hlyAs-pET20b(+)/ E. coli  BL21 (DE3)) were picked. 
       EXAMPLE 2 
     Shake-flask Fermentation of  E. coli  Strain Tfu — 0883-hlyAs/pET20b(+)/ E. coli  BL21(DE3) 
       [0036]    Strain:  E. coli  strain Tfu — 0883-hlyAs/pET20b(+)/ E. coli  BL21(DE3). 
         [0037]    Seed culture: Inoculum having been stored at −80° C. was inoculated in a seed culture medium at an initial pH of 7.0-7.2 and cultured on a constant-temperature rotating shaking bed at 37° C., 200 rpm for 7-8 hours. The seed culture medium consisted of 10 g/L of peptone, 5 g/L of yeast powder, 10 g/L of NaCl and 100 mg/L of ampicillin. 
         [0038]    Fermention to produce the enzyme: The seed culture was inoculated at an amount of 5% for the fermentation; the fermentation medium consisted of 5 g/L of glycerol, 12 g/L of peptone, 24 g/L of yeast extract, 12.54 g/L of K 2 HPO 4  and 2.31 g/L of KH 2 PO 4 ; after culturing at 37° C. for 2 hours, IPTG was added at a final concentration of 0.4 mM for induction, and culturing was continued at a lowered temperature of 25° C., with the yield of the enzyme reaching 274 U/mL at 60 hours. 
       EXAMPLE 3 
     Fermentation of  E. coli  Strain Tfu — 0883-hlyAs/pET20b(+)/ E. coli  BL21(DE3) 
       [0039]    Strain:  E. coli  strain Tfu — 0883-hlyAs/pET20b(+)/ E. coli  BL21(DE3). 
         [0040]    Seed culture: Inoculum having been stored at −80° C. was inoculated in a seed culture medium at an initial pH of 7.0-7.2 and cultured on a constant-temperature rotating shaking bed at 37° C., 200 rpm for 7-8 hours. The seed culture medium consisted of 10 g/L of peptone, 5 g/L of yeast powder, 10 g/L of NaCl and 100 mg/L of ampicillin. 
         [0041]    The seed culture was inoculated at an amount of 4-8% for the fermentation. 
         [0042]    The fermentation medium consisted of 1 g/L of peptone, 2 g/L of yeast powder, 4 g/L of (NH 4 ) 2 HPO 4 , 13.5 g/L of KH 2 PO 4 , 4.1 g/L of MgSO 4 .7H 2 O, 0.85 g/L of citric acid, 8 g/L of glycerol, 5 mL/L of a solution of trace elements and 100 mg/L of ampicillin. The solution of trace elements consisted of 5 M/L of HCl, 10 g/L of FeSO 4 .7H 2 O, 2.25 g/L of ZnSO 4 .7H 2 O, 1.0 g/L of CuSO 4 .5H 2 O, 0.5 g/L of MnSO 4 .4H 2 O, 0.23 g/L of Na 2 B 4 O 7 .10H 2 O, 2.0 g/L of CaCl 2 .2H 2 O and 0.1 g/L of (NH 4 ) 6 Mo 7 O 24 . 
         [0043]    1) during fermentation, the temperature was maintained at 36-38° C., the dissolved oxygen was maintained at 20-40% by increasing or decreasing stirring rotation speed or supplying oxygen-rich air, and the pH was maintained at 7.0-7.2 in the growth stage and at 6.4-6.6 in the stage of induction of production of the enzyme by supplementing ammonia water; 
         [0044]    2) after fermenting for 5-6 hours or when the concentration of dissolved oxygen rose to more than 70%, 500 g/L of glycerol was supplemented at an initial flow rate of 3.5-5.0 mL·L −1 ·h −1 , and subsequently, glycerol was fed in exponential feeding mode to control the specific growth rate of the microorganism in the range of 0.15-0.22 h −1 ; 
         [0045]    3) after fermenting for 12-13 hours or when OD 600  reached 25-35, IPTG was added at a final concentration of 0.02-0.04 mM/L for induction, and at the same time 50 g/L of lactose was supplemented at a rate of 8-10 mL·L −1 ·h −1  and the supplementation of ammonia water was stopped until the pH dropped to 6.4-6.6, at which time the supplementation of ammonia water was resumed to control the pH at 6.4-6.6; 
         [0046]    4) after fermenting for 15-17 hours or inducing for 3-4 hours, glycerol was fed at a constant flow rate of 25-35 mL·L −1 ·h −1 ; 
         [0047]    5) after fermenting for 19-21 hours or when OD 600  no longer increased, the flow rate of lactose was decreased to 2-4 mL·L −1 ·h −1 , and the flow rate of glycerol was gradually decreased to half of that in step 4) over 6-8 hours. 
         [0048]    When fermenting for 30-34 hours, the enzyme activity reached 700-750 U/mL.