Methods for promoting extracellular expression of proteins in <i>Bacillus subtilis </i>using a cutinase

Abstract

Disclosed is a method for promoting extracellular expression of proteins in B. subtilis using cutinase, which belongs to the technical fields of genetic engineering, enzyme engineering and microbial engineering. It teaches co-expressing a cutinase mutant and a target protein in B. subtilis to promote extracellular expression of the target protein which is naturally located inside cells. The target protein includes xylose isomerase, 4,6-α-glucosyltransferase, 4-α-glucosyltransferase, trehalose synthase, branching enzyme and the like. The invention can achieve extracellular expression of intracellularly localized target protein, improve the production efficiency, reduce the production cost and simplify the subsequent extraction process.

Claims

1. A cutinase mutant, which is a cutinase having the amino acid sequence of SEQ ID NO: 1 except having I213A and P214A substitutions.

2. A method for extracellularly producing an exogenous protein, comprising: a) inoculating a recombinant B. subtilis coexpressing the cutinase mutant of claim 1 and an exogenous intracellularly located protein into a seed medium to obtain a seed liquid; b) inoculating the seed liquid into a fermentation medium for performing fermentation to obtain a fermentation broth; and c) centrifuging the fermentation broth and obtaining the exogenous protein in the fermentation supernatant of the centrifuged fermentation broth.

3. The method of claim 2, wherein the recombinant B. subtilis are inoculated into the seed medium and cultured at 35-38° C., 180-220 rpm for 8-10 hours to obtain the seed liquid, and the seed liquid is inoculated into the fermentation medium and cultured at 30-37° C., 180-220 rpm for 20-26 hours.

4. The method of claim 2, wherein the seed medium comprises 8-12 g/L peptone, 4-6 g/L yeast extract and 8-12 g/L sodium chloride.

5. The method of claim 2, wherein the fermentation medium comprises 20-25 g/L yeast extract, 5-10 g/L soy peptone and 4-6 g/L glycerol, and the initial pH of the fermentation medium is 6-7.

Description

DETAILED DESCRIPTION

(1) The media involved in the following examples are as follows:

(2) LB solid medium: 10 g/L peptone, 5 g/L yeast extract, 10 g/L NaCl and 0.2 g/L agar powder.

(3) LB liquid medium: 10 g/L peptone, 5 g/L yeast extract and 10 g/L NaCl.

(4) Seed medium: 10 g/L peptone, 5 g/L yeast extract and 10 g/L sodium chloride.

(5) Fermentation medium: 24 g/L yeast extract, 12 g/L soy peptone, 5 g/L glycerol, 12.54 g/L K.sub.2HPO.sub.4 and 2.31 g/L KH.sub.2PO.sub.4; and the initial pH is 6-7.

(6) The detection methods involved in the following examples are as follows:

(7) Detection Method of Enzyme Activity of Xylose Isomerase

(8) 100 μL of a solution to be tested was added to a reaction system (containing a 3 mol.Math.L.sup.−1 substrate, 100 μL of a glucose solution, 100 μL of a 50 mmol.Math.L.sup.−1 MgSO.sub.4 solution, 100 μL of a 0.3 mol.Math.L.sup.−1 Na.sub.2HPO.sub.4—KH.sub.2PO.sub.4 buffer with pH 7.5, and 600 μL of H.sub.2O). After reacting at 70° C. for 10 min, 1 mL of 0.5 mol.Math.L.sup.−1 HClO.sub.4 was added to stop the reaction. 500 μL of the reaction solution was taken, and 100 μL of a cysteine hydrochloride solution (15 g.Math.L.sup.−1), 3 mL of 75% concentrated H.sub.2SO.sub.4 and 100 μL of a carbazole-alcohol solution were added, and the mixed solution was shaken and mixed well. Color was developed at 60° C. for 10 min. Cooling was performed in an ice bath, and the absorbance was determined at a wavelength of 560 nm (using the inactivated enzyme solution subjected to the same operations as a blank control).

(9) The enzyme activity is defined as the amount of enzyme required to produce 1 μmol of fructose per minute under the above reaction conditions.

Determination Method of Enzyme Activity of 4,6-α-glucosyltransferase

(10) (1) Preparation of substrate: 2 mL of distilled water was added to 40 mg of amylose to fully moisten the amylose, and then 2 mL of a 2 M NaOH solution was added. Vortex shaking was performed to fully dissolve the enzyme to prepare an amylose mother liquor. 500 μL of the amylose mother liquor was added with 250 μL of a 2 M HCl solution, and then 3250 μL of a phosphoric acid-citric acid buffer (pH 7.0) was added to prepare a 0.125% substrate.

(11) (2) Preparation of iodine color solution: 0.26 g of iodine and 2.60 g of potassium iodide were put in a 10 mL volumetric flask, add water to the volumetric flask to the mark (prepared 3 days in advance to ensure that the iodine was completely dissolved) to obtain Lugol's iodine solution. When it is time to perform the assay, 100 μL of the Lugol's iodine solution was added to 50 μL of a 2 M HCl solution, and then water was added to 26 mL to prepare the iodine color solution.

(12) (3) 200 μL of the substrate prepared in step (1) was taken in a 1.5 mL centrifuge tube and placed in a warm bath at 35° C. for 10 min. 200 μL of an enzyme solution to be tested was added and reacted at 35° C. for 10 min. After the reaction, 200 μL of the reaction solution was added to 3800 μL of the iodine color solution for color development for 5 min, and the absorbance at 660 nm was determined by a spectrophotometer.

(13) As a control, 200 μl buffer, instead of the enzyme solution, was added to 3800 μL of the iodine color solution for color development.

(14) The unit of enzyme activity is defined as: the absorbance value decreased by one percent per unit time is a unit of enzyme activity.

(15) Detection Method of Enzyme Activity of Trehalose Synthase:

(16) 400 μL of an enzyme solution diluted to a suitable multiple was taken and 400 μL of a 5% (w/v) maltose solution prepared with a phosphate buffer (20 mmol/L, pH7.0) was added to obtain a mixed solution. The mixed solution was reacted at 30° C. for 30 min, then the enzyme reaction was terminated in a boiling water bath for 10 min, and the content of trehalose produced was determined by HPLC.

(17) The HPLC detection conditions were: a mobile phase contained acetonitrile and water in a ratio of 80:20, the flow rate was 0.8 mL/min, the column temperature was 40° C., and a NH.sub.2 column and a differential detector were used.

(18) Definition of enzyme activity: Under the above reaction conditions, the amount of enzyme required to form 1 μmol of trehalose per minute is defined as 1 unit of enzyme activity.

(19) Detection Method of Enzyme Activity of Branching Enzyme:

(20) (1) Preparation of substrate: 0.01 g of amylose (0.1 g of amylopectin) and 0.2 mL of 96% ethanol were taken, 0.5 mL of a 2 mol.Math.L.sup.−1 NaOH solution was added after 3-4 min, 10 mL of water was added, the mixed solution was stirred for 10 min to dissolve the starch, then 0.5 mL of a 2 mol.Math.L.sup.−1 HCL solution was added, and a phosphate buffer (50 mmol.Math.L.sup.−1, pH 6.5) was added to volume to 10 mL to adjust the pH (prepared when used).

(21) (2) Preparation of termination reaction solution: Lugol's iodine solution (mother liquor): 0.26 g of iodine and 2.60 g of potassium iodide were dissolved in a 10 mL volumetric flask, and stored at room temperature and protected from light. 0.1 mL of the Lugol's iodine solution was added, and 50 μL of a 2 mol.Math.L.sup.−1 hydrochloric acid solution was added, and water was added to volume to 26 mL (prepared when used).

(22) (3) 50 μL of a crude enzyme solution was taken and 50 μL of a substrate was added, and the mixed solution was placed in a water bath at 60° C. for 30 min. After adding 2 mL of the termination reaction solution, the absorbance at 660 nm was determined after being placed at room temperature for 20 min.

(23) Definition of enzyme activity: At room temperature, the absorbance value at 660 nm decreased by 1% per minute is as a unit of enzyme activity.

Detection Method of 4-α-glucosyltransferase

(24) 25 μL of a 0.02% (w.Math.v.sup.−1) potato amylose solution (dissolved in 90% dimethyl sulfoxide) was taken in a test tube, and preheated in a water bath at 70° C. for 10 min. 25 μL of a diluted enzyme solution (dissolved in a 50 mmol.Math.L.sup.−1 Na.sub.2HPO.sub.4-citrate buffer with pH 5.5) was added, and the mixed solution was shaken and mixed well. After reacting at 70° C. for 30 min, 1 mL of an iodine solution (0.1 mL of original iodine solution+0.1 mL of 1 N HCl, diluted to 26 mL) was added to terminate the reaction. The original iodine solution was 26% KI+2.6% I.sub.2.

(25) Definition of unit of enzyme activity: Under the enzyme activity measurement system, the amount of enzyme required to decrease the absorbance value A660 by 0.1 per minute.

Example 1: Construction of Recombinant Plasmid

(26) (1) Plasmid pHYPMLd4P (the plasmid contains pullulanase pμL and chaperone protein prsA genes, and the construction method is recorded in the doctoral dissertation “Modification of Bacillus subtilis Strain, Promoter Optimization and High-Level Expression of Pullulanase”, of Zhang Kang, Jiangnan University, 2018) stored in the laboratory was used as a template to design forward and reverse primers, respectively:

(27) TABLE-US-00001   pHY300PLK-F1: 5′-AAGCTTGGTAATAAAAAAACACCTCC-3′; pHY300PLK-R1: 5′-TCTTGACACTCCTTATTTGATTTTT-3′;

(28) An expression vector pHY300PLK-prsA fragment was amplified.

(29) (2) Plasmid xylA/pET24a (+) (the construction method of the plasmid is recorded in Chinese Patent ZL201210581801.2) stored in the laboratory was used as a template to design forward and reverse primers, respectively:

(30) TABLE-US-00002 xy1A-F: 5′-GGAGTGTCAAGAATGAGCAACTACCAGCCCACAC-3′; xy1A-R: 5′-TTTATTACCAAGCTTTTAGCGCACGCCCAGGAGGTAG-3′;

(31) A xylose isomerase gene fragment was amplified.

(32) (3) The expression vector pHY300PLK-prsA fragment obtained in step (1) and the xylose isomerase gene fragment obtained in step (2) were linked by Infusion. The linked product was transformed into an E. coli JM109 competent cell to obtain a transformed product. The plasmid in the transformed product was extracted and verified by Hind III restriction enzyme digestion and sequenced to obtain the recombinant plasmid pHY300PLK-xylA-prsA.

(33) The recombinant plasmid pHY300PLK-xylA-prsA was used as a template to design forward and reverse primers, respectively:

(34) TABLE-US-00003 pHY300PLK-F2: 5′-GAGCTCGGTACCCTCGAGGG-3′; pHY300PLK-R2: 5′-ACGCGTCCCTCTCCTTTTGC-3′;

(35) An expression vector pHY300PLK-xylA fragment was amplified.

(36) (4) Plasmid pET20b-Tfu_0883 (the construction method of the plasmid is recorded in Chen S, Tong X, Woodard R W, Du G C, Wu J, Chen J, Identification and Characterization of Bacterial Cutinase, Journal of Biological Chemistry, 2008, 283(28):25854-25862) stored in the laboratory was used as a template to design forward and reverse primers, respectively:

(37) TABLE-US-00004 cut-F: 5′-AGGAGAGGGACGCGTATGGCCAACCCCTACGAGCGCGG-3′; cut-R: 5′-GAGGGTACCGAGCTCTTAGAACGGGCAGGTGGAGCG-3′;

(38) A cutinase gene cut was amplified.

(39) The expression vector pHY300PLK-xylA fragment obtained in step (3) and the cutinase gene fragment were linked by Infusion. The linked product was transformed into an E. coli JM109 competent cell to obtain a transformed product. The plasmid in the transformed product was extracted and verified by Hind III restriction enzyme digestion and sequenced to obtain the recombinant plasmid pHY300PLK-xylA-cut.

(40) (5) The recombinant plasmid pHYPMLd4 (the plasmid contains pullulanase pul gene, and the construction method is recorded in the doctoral dissertation “Modification of Bacillus subtilis Strain, Promoter Optimization and High-Level Expression of Pullulanase”, of Zhang Kang, Jiangnan University, 2018) was used as a template, and an expression vector pHY300PLK fragment was amplified using the forward and reverse primers (pHY300PLK-F1 and pHY300PLK-R1). Plasmid xylA/pET24a (+) (disclosed in a patent with the patent number of ZL201210581801.2) stored in the laboratory was used as a template, and a xylose isomerase gene fragment was amplified using the forward and reverse primers (xylA-F and xylA-R). The expression vector pHY300PLK fragment and the xylose isomerase gene fragment were linked by Infusion. The linked product was transformed into an E. coli JM109 competent cell to obtain a transformed product. The plasmid in the transformed product was extracted and verified by Hind III restriction enzyme digestion and sequenced to obtain the recombinant plasmid pHY300PLK-xylA.

Example 2: Construction of Cutinase Mutant

(41) The recombinant plasmid pHY300PLK-xylA-cut obtained in step (4) of Example 1 was used as a template, and according to the gene sequences of cutinase, primers introducing mutations of L175A/T177A, T207A/F209A, I213A/P214A, I178A, L175A, T177A, T207A, F209A, I213A and P214A were designed and synthesized. The cutinase genes were subjected to site-directed mutation and verified by sequencing to obtain recombinant expression vectors containing the cutinase mutant genes: pHY300PLK-xylA-L175A/T177A, pHY300PLK-xylA-T207A/F209A, pHY300PLK-xylA-I213A/P214A, pHY300PLK-xylA-I178A, pHY300PLK-xylA-L175A, pHY300PLK-xylA-T177A, pHY300PLK-xylA-T207A, pHY300PLK-xylA-F209A, pHY300PLK-xylA-I213A, pHY300PLK-xylA-P214A.

(42) The site-directed mutation primer introducing the L175A/T177A mutation was:

(43) TABLE-US-00005 L175A/T177A-F: 5′-GATCATCGGGGCCGACGCAGACGCGATCGCGCCGGTCG-3′ L175A/T177A-R: 5′-CGACCGGCGCGATCGCGTCTGCGTCGGCCCCGATGATC-3′

(44) The site-directed mutation primer introducing the T207A/F209A mutation was:

(45) TABLE-US-00006 T207A/F209A-F: 5′-GGAGCTGGACGGCGCAGCCCACGCAGCCCCGAACATCCCC-3′ T207A/F209A-R: 5′-GGGGATGTTCGGGGCTGCGTGGGCTGCGCCGTCCAGCTCC-3′

(46) The site-directed mutation primer introducing the I213A/P214A mutation was:

(47) TABLE-US-00007 1213A/P214A-F: 5′-CCACTTCGCCCCGAACGCCGCCAACAAGATCATCGG-3′ 1213A/P214A-R: 5′-CCGATGATCTTGTTGGCGGCGTTCGGGGCGAAGTGG-3′

(48) The site-directed mutation primer introducing the I178A mutation was:

(49) TABLE-US-00008 I178A-F: 5′-CCGACCTCGACACGGCAGCGCCGGTCGCCAC-3′ I178A-R: 5′-GTGGCGACCGGCGCTGCCGTGTCGAGGTCGG-3′

(50) The site-directed mutation primer introducing the L175A mutation was:

(51) TABLE-US-00009 L175A-F: 5′-GATCATCGGGGCCGACGCAGACACGATCGCGCCG-3′ L175A-R: 5′-CGGCGCGATCGTGTCTGCGTCGGCCCCGATGATC-3′

(52) The site-directed mutation primer introducing the T177A mutation was:

(53) TABLE-US-00010 T177A-F: 5′-GGGCCGACCTCGACGCGATCGCGCCGGTCG-3′ T177A-R: 5′-CGACCGGCGCGATCGCGTCGAGGTCGGCCC-3′

(54) The site-directed mutation primer introducing the T207A mutation was:

(55) TABLE-US-00011 T207A-F: 5′-GGAGCTGGACGGCGCAGCCCACTTCGCCCCGAAC-3′ T207A-R: 5′-GTTCGGGGCGAAGTGGGCTGCGCCGTCCAGCTCC-3′

(56) The site-directed mutation primer introducing the F209A mutation was:

(57) TABLE-US-00012 F209A-F: 5′-GCTGGACGGCGCAACCCACGCAGCCCCGAACATCCCC-3′ F209A-R: 5′-GGGGATGTTCGGGGCTGCGTGGGTTGCGCCGTCCAGC-3′

(58) The site-directed mutation primer introducing the I213A mutation was:

(59) TABLE-US-00013 I213A-F: 5′-CCACTTCGCCCCGAACGCCCCCAACAAGATCATCGG-3′ I213A-R: 5′-CCGATGATCTTGTTGGGGGCGTTCGGGGCGAAGTGG-3′

(60) The site-directed mutation primer introducing the P214A mutation was:

(61) TABLE-US-00014 P214A-F: 5′-CCACTTCGCCCCGAACATCGCCAACAAGATCATCGG-3′ P214A-R: 5′-CCGATGATCTTGTTGGCGATGTTCGGGGCGAAGTGG-3′

Example 3: Construction of Recombinant Bacteria Co-Expressing Cutinase Mutant and Xylose Isomerase

(62) (1) Preparation of Competent Cells

(63) Cryopreserved B. subtilis WS5 was taken by dipping with an inoculating loop, then streaked on an LB solid medium, and cultured overnight at 37° C. for activation. A single colony was picked, inoculated in 10 mL of LB liquid medium, and cultured overnight at 37° C. and 200 rpm for 8 h to obtain a culture solution. 2.5 mL of the culture solution was transferred to 40 mL of LB liquid medium containing 0.5 M sorbitol, and cultured at 37° C. and 200 rpm for 4-5 h to obtain a bacterial solution. The obtained bacterial solution was placed in an ice-water bath for 10 min, and then centrifuged at 4° C. and 5000 rpm for 5 min, and bacterial cells were collected. The bacterial cells were resuspended in 50 mL of a pre-cooled electroporation transformation buffer, and centrifuged at 4° C. and 5000 rpm for 5 min. The supernatant was removed, and the bacterial cells were rinsed 4 times according to the above steps. The washed bacterial cells were resuspended in 1 mL of the electroporation transformation medium and dispensed into 1.5 mL EP tubes with 200 μL per tube to obtain the competent cells.

(64) (2) Transformation of Competent Cells

(65) The recombinant plasmids obtained in Examples 1 and 2 were added to the competent cells obtained in step (1). After being placed in an ice bath for 18 min, the competent cells and the recombinant plasmids were added to a pre-cooled electroporation cuvette (2 mm) and shocked (at 2.4 kv, 25 μF, 200Ω) once. After the electric shock is completed, 1 mL of a pre-cooled RM medium (RM medium components: peptone 10 g/L, yeast powder 5 g/L, NaCl 10 g/L, sorbitol 91 g/L and mannitol 69 g/L) was added immediately. After resuscitating at 37° C. and 200 rpm for 3 h, the competent cells were applied to a plate containing tetracycline resistance (50 μg/mL) to obtain recombinant bacteria:

(66) Bacillus subtilis WS5/pHY300PLK-xylA, Bacillus subtilis WS5/pHY300PLK-xylA-cut, Bacillus subtilis WS5/pHY300PLK-xylA-L175A/T177A, Bacillus subtilis WS5/pHY300PLK-xylA-T207A/F209A, Bacillus subtilis WS5/pHY300PLK-xylA-I213A/P214A, Bacillus subtilis WS5/pHY300PLK-xylA 4178A, Bacillus subtilis WS5/pHY300PLK-xylA-L175A, Bacillus subtilis WS5/pHY300PLK-xylA-T177A, Bacillus subtilis WS5/pHY300PLK-xylA-T207A, Bacillus subtilis WS5/pHY300PLK-xylA-F209A, Bacillus subtilis WS5/pHY300PLK-xylA4213A, Bacillus subtilis WS5/pHY300PLK-xylA-P214A.

Example 4: Production of Xylose Isomerase by Shake Flask Fermentation

(67) (1) The recombinant B. subtilis strains obtained in Example 3 were inoculated into the seed culture media, and cultured at 35-38° C. and 180-220 rpm for 8-10 h to obtain the seed liquids.

(68) (2) The seed liquids obtained in step (1) were transferred to the fermentation media at an inoculum concentration of 5% (v/v), and cultured at 33° C. and 200 rpm for 24 h. Then the culture solutions were centrifuged at 12000 r.Math.min.sup.−1 for 10 min to obtain fermentation supernatant. The fermentation supernatant was tested for the enzyme activity of xylose isomerase. The test results are shown in Table 1:

(69) TABLE-US-00015 TABLE 1 Enzyme activity of xylose isomerase in fermentation supernatant Enzyme activity of xylose isomerase Recombinant bacteria expressed (U/mL) Bacillus subtilis WS5/pHY300PLK-xylA 0 Bacillus subtilis WS5/pHY300PLK-xylA-cut 0.8 Bacillus subtilis WS5/pHY300PLK-xylA-L175A/ 4.2 T177A Bacillus subtilis WS5/pHY300PLK-xylA-T207A/ 5.3 F209A Bacillus subtilis WS5/pHY300PLK-xylA-I213A/ 5.6 P214A Bacillus subtilis WS5/pHY300PLK-xylA-I178A 3.8 Bacillus subtilis WS5/pHY300PLK-xylA-L175A 3.3 Bacillus subtilis WS5/pHY300PLK-xylA-T177A 2.1 Bacillus subtilis WS5/pHY300PLK-xylA-T207A 3.2 Bacillus subtilis WS5/pHY300PLK-xylA-F209A 4.5 Bacillus subtilis WS5/pHY300PLK-xylA-I213A 3.6 Bacillus subtilis WS5/pHY300PLK-xylA-P214A 3.8

(70) It can be seen from the test results that when the xylose isomerase was expressed alone, the extracellular enzyme activity of the xylose isomerase was not detected. When co-expressed with the cutinase or mutants thereof, the extracellular enzyme activity was detected, proving that the technical solution of the disclosure realizes the extracellular secretion of the xylose isomerase in B. subtilis. At the same time, the enzyme activity when the xylose isomerase and cutinase mutant I213A/P214A were co-expressed is 7 times the enzyme activity when the xylose isomerase and wild-type cutinase were co-expressed.

Example 5: Co-Expression of Cutinase Mutants Promotes Extracellular Expression of 4,6-α-glucosyltransferase

(71) (1) Recombinant plasmids pHY300PLK-gtfB, pHY300PLK-gtfB-cut, pHY300PLK-gtfB-L175A/T177A, pHY300PLK-gtfB-T207A/F209A, pHY300PLK-gtfB-I213A/P214A, pHY300PLK-gtfB-I178A, pHY300PLK-gtfB-L175A, pHY300PLK-gtfB-T177A, pHY300PLK-gtfB-T207A, pHY300PLK-gtfB-F209A, pHY300PLK-gtfB-I213A and pHY300PLK-gtfB-P214A were constructed by the methods of Examples 1-3 and transformed into B. subtilis WS5 to obtain recombinant bacteria:

(72) Bacillus subtilis WS5/pHY300PLK-gtfB, Bacillus subtilis WS5/pHY300PLK-gtfB-cut, Bacillus subtilis WS5/pHY300PLK-gtfB-L175A/T177A, Bacillus subtilis WS5/pHY300PLK-gtfB-T207A/F209A, Bacillus subtilis WS5/pHY300PLK-gtfB-I213A/P214A, Bacillus subtilis WS5/pHY300PLK-gtfB-I178A, Bacillus subtilis WS5/pHY300PLK-gtfB-L175A, Bacillus subtilis WS5/pHY300PLK-gtfB-T177A, Bacillus subtilis WS5/pHY300PLK-gtfB-T207A, Bacillus subtilis WS5/pHY300PLK-gtfB-F209A, Bacillus subtilis WS5/pHY300PLK-gtfB-I213A, Bacillus subtilis WS5/pHY300PLK-gtfB-P214A.

(73) (2) The recombinant B. subtilis strains were inoculated into the seed culture media, and cultured at 35-38° C. and 180-220 rpm for 8-10 h to obtain the seed liquids.

(74) (3) The seed liquids obtained in step (2) were transferred to the fermentation media at an inoculum concentration of 5% (v/v), and cultured at 33° C. and 200 rpm for 24 h. Then the culture solutions were centrifuged at 12000 r.Math.min.sup.−1 for 10 min to obtain fermentation supernatant. The fermentation supernatant was tested for the enzyme activity of 4,6-α-glucosyltransferase. The test results are shown in Table 2:

(75) TABLE-US-00016 TABLE 2 Enzyme activity of 4,6-a-glucosyltransferase in fermentation supernatant Enzyme activity of 4,6-a-glucosyltransferase Recombinant bacteria expressed (U/mL) Bacillus subtilis WS5/pHY300PLK-gtfB 0 Bacillus subtilis WS5/pHY300PLK-gtfB-cut 123.6 Bacillus subtilis WS5/pHY300PLK-gtfB-L175A/ 458.9 T177A Bacillus subtilis WS5/pHY300PLK-gtfB-T207A/ 652.8 F209A Bacillus subtilis WS5/pHY300PLK-gtfB-I213A/ 745.2 P214A Bacillus subtilis WS5/pHY300PLK-gtfB-I178A 428.6 Bacillus subtilis WS5/pHY300PLK-gtfB-L175A 136.2 Bacillus subtilis WS5/pHY300PLK-gtfB-T177A 325.8 Bacillus subtilis WS5/pHY300PLK-gtfB-T207A 232.8 Bacillus subtilis WS5/pHY300PLK-gtfB-F209A 465.3 Bacillus subtilis WS5/pHY300PLK-gtfB-I213A 389.4 Bacillus subtilis WS5/pHY300PLK-gtfB-P214A 486.6

(76) It can be seen from the test results that when the 4,6-α-glucosyltransferase was expressed alone (Bacillus subtilis WS5/pHY300PLK-gtfB), the extracellular enzyme activity of the 4,6-α-glucosyltransferase was not detected.

(77) The enzyme activity when the 4,6-α-glucosyltransferase and cutinase mutant I213A/P214A were co-expressed (Bacillus subtilis WS5/pHY300PLK-gtfB-I213A/P214A) is 6 times the enzyme activity when the 4,6-α-glucosyltransferase and wild-type cutinase were co-expressed (Bacillus subtilis WS5/pHY300PLK-gtfB-cut).

Example 6: Co-Expression of Cutinase Mutants Promotes Extracellular Expression of 4-α-glucosyltransferase

(78) (1) Recombinant plasmids pHY300PLK-4GT, pHY300PLK-4GT-cut, pHY300PLK-4GT-L175A/T177A, pHY300PLK-4GT-T207A/F209A, pHY300PLK-4GT-I213A/P214A, pHY300PLK-4GT-I178A, pHY300PLK-4GT-L175A, pHY300PLK-4GT-T177A, pHY300PLK-4GT-T207A, pHY300PLK-4GT-F209A, pHY300PLK-4GT 4213A and pHY300PLK-4GT-P214A were constructed by the methods of Examples 1-3 and transformed into B. subtilis WS5 to obtain recombinant bacteria:

(79) Bacillus subtilis WS5/pHY300PLK-4GT, Bacillus subtilis WS5/pHY300PLK-4GT-cut, Bacillus subtilis WS5/pHY300PLK-4GT-L175A/T177A, Bacillus subtilis WS5/pHY300PLK-4GT-T207A/F209A, Bacillus subtilis WS5/pHY300PLK-4GT I213A/P214A, Bacillus subtilis WS5/pHY300PLK-4GT-I178A, Bacillus subtilis WS5/pHY300PLK-4GT-L175A, Bacillus subtilis WS5/pHY300PLK-4GT-T177A, Bacillus subtilis WS5/pHY300PLK-4GT-T207A, Bacillus subtilis WS5/pHY300PLK-4GT-F209A, Bacillus subtilis WS5/pHY300PLK-4GT 4213A, Bacillus subtilis WS5/pHY300PLK-4GT-P214A.

(80) (2) The recombinant B. subtilis strains were inoculated into the seed culture media, and cultured at 35-38° C. and 180-220 rpm for 8-10 h to obtain the seed liquids.

(81) (3) The seed liquids obtained in step (2) were transferred to the fermentation media at an inoculum concentration of 5% (v/v), and cultured at 33° C. and 200 rpm for 24 h. Then the culture solutions were centrifuged at 12000 r.Math.min.sup.−1 for 10 min to obtain fermentation supernatant. The fermentation supernatant was tested for the enzyme activity of 4-α-glucosyltransferase. The test results are shown in Table 3:

(82) TABLE-US-00017 TABLE 3 Enzyme activity of 4-a-glucosyltransferase in fermentation supernatant Enzyme activity of 4-a-glucosyltransferase Recombinant bacteria expressed (U/mL) Bacillus subtilis WS5/pHY300PLK-4GT 0 Bacillus subtilis WS5/pHY300PLK-4GT-cut 2.5 Bacillus subtilis WS5/pHY300PLK-4GT-L175A/ 6.6 T177A Bacillus subtilis WS5/pHY300PLK-4GT-T207A/ 11.4 F209A Bacillus subtilis WS5/pHY300PLK-4GT-I213A/ 10.5 P214A Bacillus subtilis WS5/pHY300PLK-4GT-I178A 8.2 Bacillus subtilis WS5/pHY300PLK-4GT-L175A 6.0 Bacillus subtilis WS5/pHY300PLK-4GT-T177A 4.2 Bacillus subtilis WS5/pHY300PLK-4GT-T207A 7.8 Bacillus subtilis WS5/pHY300PLK-4GT-F209A 7.3 Bacillus subtilis WS5/pHY300PLK-4GT-I213A 7.4 Bacillus subtilis WS5/pHY300PLK-4GT-P214A 8.6

(83) It can be seen from the test results that when the 4-α-glucosyltransferase was expressed alone (Bacillus subtilis WS5/pHY300PLK-4GT), the extracellular enzyme activity of the 4-α-glucosyltransferase was not detected.

(84) The enzyme activity when the 4-α-glucosyltransferase and cutinase mutant T207A/F209A were co-expressed (Bacillus subtilis WS5/pHY300PLK-4GT-T207A/F209A) is 4.6 times the enzyme activity when the 4-α-glucosyltransferase and wild-type cutinase were co-expressed (Bacillus subtilis WS5/pHY300PLK-4GT-cut).

Example 7: Co-Expression of Cutinase Mutants Promotes Extracellular Expression of Trehalose Synthase

(85) (1) Recombinant plasmids were constructed by the methods of Examples 1-3 and transformed into B. subtilis WS5 to obtain recombinant bacteria:

(86) Bacillus subtilis WS5/pHY300PLK-treS, Bacillus subtilis WS5/pHY300PLK-treS-cut, Bacillus subtilis WS5/pHY300PLK-treS-L175A/T177A, Bacillus subtilis WS5/pHY300PLK-treS-T207A/F209A, Bacillus subtilis WS5/pHY300PLK-treS-I213A/P214A, Bacillus subtilis WS5/pHY300PLK-treS-I178A, Bacillus subtilis WS5/pHY300PLK-treS-L175A, Bacillus subtilis WS5/pHY300PLK-treS-T177A, Bacillus subtilis WS5/pHY300PLK-treS-T207A, Bacillus subtilis WS5/pHY300PLK-treS-F209A, Bacillus subtilis WS5/pHY300PLK-treS-I213A, Bacillus subtilis WS5/pHY300PLK-treS-P214A (wherein the literature involved in plasmid construction is: doctoral dissertation “Study on B. subtilis Strain Modification, Promoter Optimization and Efficient Preparation of Pullulanase” of Zhang Kang, Jiangnan University, 2018; Luo Feng, Duan Xuguo, Su Lingqia, Wu Jing, Cloning Expression and Fermentation Optimization of Thermobifida fusca Trehalose Synthase Gene, Journal of Chinese Biotechnology, 2013, 33 (8): 98-104).

(87) (2) The recombinant B. subtilis strains were inoculated into the seed culture media, and cultured at 35-38° C. and 180-220 rpm for 8-10 h to obtain the seed liquids.

(88) (3) The seed liquids obtained in step (2) were transferred to the fermentation media at an inoculum concentration of 5% (v/v), and cultured at 33° C. and 200 rpm for 24 h. Then the culture solutions were centrifuged at 12000 r.Math.min.sup.−1 for 10 min to obtain fermentation supernatant. The fermentation supernatant was tested for the enzyme activity of trehalose synthase. When the trehalose synthase was expressed alone, the extracellular enzyme activity of the trehalose synthase was not detected. When co-expressed with the cutinase or mutants thereof, the extracellular enzyme activity was detected.

Example 8: Co-Expression of Cutinase Mutants Promotes Extracellular Expression of Branching Enzyme

(89) (1) Recombinant plasmids were constructed by the methods of Examples 1-3 and transformed into B. subtilis WS5 to obtain recombinant bacteria:

(90) Bacillus subtilis WS5/pHY300PLK-TtSBE, Bacillus subtilis WS5/pHY300PLK-TtSBE-cut, Bacillus subtilis WS5/pHY300PLK-TtSBE-L175A/T177A, Bacillus subtilis WS5/pHY300PLK-TtSBE-T207A/F209A, Bacillus subtilis WS5/pHY300PLK-TtSBE-I213A/P214A, Bacillus subtilis WS5/pHY300PLK-TtSBE-1178A, Bacillus subtilis WS5/pHY300PLK-TtSBE-L175A, Bacillus subtilis WS5/pHY300PLK-TtSBE-T177A, Bacillus subtilis WS5/pHY300PLK-TtSBE-T207A, Bacillus subtilis WS5/pHY300PLK-TtSBE-F209A, Bacillus subtilis WS5/pHY300PLK-TtSBE-1213A, Bacillus subtilis WS5/pHY300PLK-TtSBE-P214A (wherein the literature involved in plasmid construction is: Master's thesis of Liu Jun, Jiangnan University, 2017).

(91) (2) The recombinant B. subtilis strains were inoculated into the seed culture media, and cultured at 35-38° C. and 180-220 rpm for 8-10 h to obtain the seed liquids.

(92) (3) The seed liquids obtained in step (2) were transferred to the fermentation media at an inoculum concentration of 5% (v/v), and cultured at 33° C. and 200 rpm for 24 h. Then the culture solutions were centrifuged at 12000 r.Math.min.sup.−1 for 10 min to obtain fermentation supernatant. The fermentation supernatant was tested for the enzyme activity of branching enzyme. When the branching enzyme was expressed alone, the extracellular enzyme activity of the branching enzyme was not detected. When co-expressed with the cutinase or mutants thereof, the extracellular enzyme activity was detected.