Recombinant strain expressing phospholipase D and application thereof

Abstract

The present invention provides a phospholipase D having an amino acid sequence as shown in SEQ ID NO. 1, and further provides a gene sequence encoding phospholipase D, which has a nucleotide sequence as shown in SEQ ID NO. 2. The present invention also provides a method for improving the expression level of phospholipase D by systematically engineering the expression elements. The method comprises screening and replacement of signal peptides, ribosome binding sites and promoters. The constructed recombinant plasmid is transformed into a host cell, and the recombinant strain is capable of successfully expressing phospholipase D. The phospholipase D of the present invention has a good phosphatidyl transferring ability, and can be used for synthesizing the product phosphatidylserine with lecithin and L-serine as substrates. The recombinant strain has good stability of enzyme activity and short fermentation period, which lays the foundation for large-scale industrial production.

Claims

1. A transgenic cell line comprising a polynucleotide that comprises the nucleotide sequence of SEQ ID NO: 2.

2. A recombinant plasmid that comprises a gene encoding a phospholipase D, wherein said gene comprises SEQ ID NO:2, a nucleic acid encoding a signal peptide, wherein said nucleic acid comprises SEQ ID NO:3, a ribosome binding site that comprises SEQ ID NO:4, and a promoter that comprises SEQ ID NO:5.

3. The recombinant plasmid according to claim 2, wherein the recombinant plasmid is obtained by modifying the plasmid pDXW-10a that comprises SEQ ID NO:6 to introduce the gene that comprises SEQ ID NO:2, the nucleic acid encoding the signal peptide that comprises SEQ ID NO:3, the ribosome binding site that comprises SEQ ID NO:4, and the promoter that comprises SEQ ID NO:5.

4. A recombinant strain expressing a phospholipase D, wherein said recombinant strain is obtained by introducing a recombinant plasmid that comprises a gene encoding a phospholipase D, wherein said gene comprises SEQ ID NO:2, a nucleic acid encoding a signal peptide, wherein said nucleic acid comprises SEQ ID NO:3, a ribosome binding site that comprises SEQ ID NO:4, and a promoter that comprises SEQ ID NO:5.

5. The recombinant strain according to claim 4, wherein the recombinant strain is Bacillus subtilis, Pichia pastoris or Corynebacterium glutamicum strain.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is the electrophoretogram after PCR amplification of the phospholipase D coding gene. Lane M: 1,0000 bp DNA marker; and Lane 1: amplified phospholipase D gene.

(2) FIG. 2 is the electrophoretogram after PCR amplification of the WapA signal peptide coding gene containing a RBS site. Lane M: 500 bp DNA Marker; and Lane 1: amplified WapA gene.

(3) FIG. 3 is the electrophoretogram after fusion of the phospholipase D coding gene and the WapA signal peptide coding gene containing a RBS site by PCR. Lane M: 1,0000 bp DNA Marker; and Lane 1: amplified fusion gene.

(4) FIG. 4 is the electrophoretogram after PCR amplification of the pWapA promoter gene. Lane M: 500 bp DNA Marker; and Lane 1: amplified pWapA gene.

(5) FIG. 5 shows the wrpld4 fragment after double enzyme digestion. Lane M: 1,0000 bp DNA Marker; and Lane 1: target gene after double enzyme digestion.

(6) FIG. 6 is the electrophoretogram after double enzyme digestion of the pDXW-10a plasmid obtained by two one-step reverse PCR. Lane M: 1,0000 bp DNA Marker; and Lane 1: pDXW-10a plasmid gene after double enzyme digestion.

(7) FIG. 7 is the electrophoretogram for verifying the amplified wrpld4 fragment on the pDXW-wrpld4 plasmid. Lane M: 1,0000 bp DNA Marker; and Lane 1: amplified wrpld4 fragment.

(8) FIG. 8 is the electrophoretogram for verifying the recombinant plasmid pDXW-wrpld4 after double enzyme digestion. Lane M: 1,0000 bp DNA Marker; and Lane 1: pDXW-10a plasmid and wrpld4 fragment after double enzyme digestion.

(9) FIG. 9 shows the results of the enzyme activity test of phospholipase D after intracellular expression by Bacillus subtilis, Pichia pastoris, and Corynebacterium glutamicum.

(10) FIG. 10 shows the results of the enzyme activity test of phospholipase D after extracellular secretion and expression by Bacillus subtilis, Pichia pastoris, and Corynebacterium glutamicum.

(11) FIG. 11 is an SDS-PAGE electrophoretogram of recombinant C. glutamicum ATCC13032/pDXW-wrpld4. Lane 1: Protein Marker; Lane 2: Blank control; Lane 3: phospholipase D protein band.

(12) FIG. 12 shows the test results of conversion rate for phosphatidylserine produced under different conditions.

(13) FIG. 13 is the HPLC chromatogram of phosphatidylserine catalytically produced with the substrates lecithin and L-serine in the presence of phospholipase D. a: phosphatidylserine standard sample; b: phosphatidylserine in the conversion solution sample.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(14) The specific embodiments of the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments. The following embodiments are intended to illustrate the present invention, instead of limiting the scope of the present invention.

Example 1

(15) The present invention provides a phospholipase D, comprising 541 amino acids. The amino acid sequence is as shown in SEQ ID NO. 1. The gene encoding the phospholipase D has a nucleotide sequence as shown in SEQ ID NO. 2, with a full length of 1,623 nucleotides.

(16) The present invention further provides a recombinant plasmid expressing phospholipase D, which comprises a nucleotide sequence as shown in SEQ ID NO. 2, a signal peptide gene allowing the extracellular expression of phospholipase D, a ribosome binding site for the expression of phospholipase D, and a promoter for the expression of phospholipase D, where the signal peptide gene has a nucleotide sequence as shown in SEQ ID NO. 3, with a full length of 96 nucleotides that encode 32 amino acids; the ribosome binding site (RBS) has a nucleotide sequence as shown in SEQ ID NO. 4, with a total of 15 nucleotides; and the promoter has a nucleotide sequence as shown in SEQ ID NO. 5, with a full length of 372 nucleotides.

Example 2

(17) This example provides the construction of the recombinant plasmid pMA5-pld and its expression method in Bacillus subtilis. The specific steps are as follows:

(18) (1) Amplification of Phospholipase D Coding Sequence

(19) Using the recombinant plasmid pET-28a(+)-spld containing the phospholipase D coding gene as a template, the phospholipase D coding sequence was amplified with the designed primers (P1, P2):

(20) TABLE-US-00001 Primer P1: 5′-CGGGATCCATGGCACGTCATCCGC-3′ (BamHI) Primer P2: 5′-CGACGCGTTTAATCCTGACAAATA-3′ (MluI)

(21) The PCR amplification reaction was carried out in a 50 μL system, in which 25 μL of PrimeSTAR® (Premix), 20 μL of ddH.sub.2O, 2 μL of template DNA, and 1.5 μL of each of the upstream and downstream primers were added. The reaction conditions were as follows: pre-denaturation at 94° C. for 3 min; then 30 cycles of denaturation at 94° C. for 30 s, annealing at 58.4° C. for 30 s, and extension at 72° C. for 1.5 min; and final extension at 72° C. for 10 min. The PCR product was identified by electrophoresis and extracted for recovery and purification. The product recovered in Step (1) was cloned into pMD19-T vector to construct a recombinant cloning plasmid. The plasmid was transformed into E. coli JM 109. Multiple transformants were picked into LB liquid medium (Amp.sup.r) and incubated at 37° C. and 220 rpm for 10-12 h. The plasmid was extracted, and verified by PCR. The plasmid verified to be correct was sequenced.

(22) (2) Construction of Recombinant Plasmid pMA5-pld

(23) The pMA5 plasmid and the correct recombinant cloning plasmid containing the target gene obtained in Step (1) were both cleaved with BamH I and Mlu I at 37° C. for 3 hrs. The electrophoresis was performed for verification and the target gene and target plasmid were extracted and recovered. The recovered products were ligated overnight at 16° C. with T4 DNA ligase, and the ligated product was transformed into E. coli JM 109 competent cells. The cells were cultured overnight on solid LB medium containing ampicillin (100 mg/L). Multiple transformants were picked into LB liquid medium (Amp.sup.r), and cultured at 37° C. and 220 rpm for 10-12 h. The plasmid was extracted and verified by PCR.

(24) (3) Transformation of Recombinant Plasmid pMA5-pld into B. Subtilis WB600

(25) The recombinant plasmid obtained in Step (2) was shipped to Shanghai Ruidi Sequencing Company for sequencing. The recombinant plasmid with the correct sequence was designated as pMA5-pld and transformed into B. subtilis WB600. The recombinant strain was cultured in TB medium for 36 h. The fermentation broth was centrifuged for 10 min at 12,000 rpm and 4° C., and then the cells were re-suspended in 4 mL buffer (40 mM Tris-HCl, 0.1% (v/v) Triton X-100, 15 mM CaCl.sub.2). The cells were ultrasonically homogenized in an ice bath, and centrifuged. The supernatant was collected for activity determination. The enzyme activity of the recombinant B. subtilis WB600/pMA5-pld is 0.14 U/mL.

Example 3

(26) This example provides the construction of the recombinant plasmid pMA5-npld and its expression method in Bacillus subtilis. The specific steps are as follows:

(27) (1) Amplification of NprB Signal Peptide Sequence

(28) By using the genome of B. subtilis 168 as a template, the NprB signal peptide sequence was amplified with the designed primers (P3, P4):

(29) TABLE-US-00002 Primer P3: 5′-CGGGATCCCGCAACTTGACCAAGAC-3′ (BamH I) Primer P4: 5′-TTGCGCGGATGACGTGCCATAGCAGCTGAGGCATGTGTTA-3′ 

(30) The PCR amplification reaction was carried out in a 50 μL system, in which 25 μL of PrimeSTAR® (Premix), 20 μL of ddH.sub.2O, 2 μL of template DNA, and 1.5 μL of each of the upstream and downstream primers were added. The reaction conditions were as follows: pre-denaturation at 94° C. for 3 min; then 30 cycles of denaturation at 94° C. for 30 s, annealing at 54.6° C. for 30 s, and extension at 72° C. for 0.5 min; and final extension at 72° C. for 10 min. The PCR product was identified by electrophoresis and extracted for recovery and purification.

(31) (2) Amplification of Phospholipase D Coding Sequence

(32) Using the recombinant plasmid pET-28a(+)-spld containing the phospholipase D coding gene as a template, the phospholipase D coding sequence was amplified with the designed primers (P5, P6):

(33) TABLE-US-00003 Primer P5: 5′-TAACACATGACTAGCAGCTATGGCACGTCATCCGCGCA-3′  Primer P6: 5′-CGACGCGTTTAATCCTGACAAATA-3′ (Mlu I)

(34) The PCR amplification reaction was carried out in a 50 μL system, in which 25 μL of PrimeSTAR® (Premix), 20 μL of ddH.sub.2O, 2 μL of template DNA, and 1.5 μL of each of the upstream and downstream primers were added. The reaction conditions were as follows: pre-denaturation at 94° C. for 3 min; then 30 cycles of denaturation at 94° C. for 30 s, annealing at 53° C. for 30 s, and extension at 72° C. for 1.5 min; and final extension at 72° C. for 10 min. The PCR product was identified by electrophoresis and extracted for recovery and purification.

(35) (3) Fusion of Phospholipase D Gene and NprB Signal Peptide Sequence by Fusion PCR

(36) By using the extracted and purified phospholipase D sequence and NprB signal peptide sequence as templates, the two sequences were fused with Primers P3 and P6:

(37) TABLE-US-00004 Primer P3: 5′-CGGGATCCCGCAACTTGACCAAGAC-3′ (BamH I)   Primer P6: 5′-CGACGCGTTTAATCCTGACAAATA-3′ (Mlu I)

(38) The fusion process included two rounds of PCR reactions. First round: The PCR amplification reaction was carried out in a 47 μL system, in which 25 μL of PrimeSTAR® (Premix), 19 μL of ddH.sub.2O, and 1.5 μL of each of the phospholipase D gene and WapA templates were added. The reaction conditions were as follows: pre-denaturation at 94° C. for 3 min; then 8 cycles of denaturation at 94° C. for 30 s, annealing at 50° C. for 30 s, and extension at 72° C. for 2 min; and final extension at 72° C. for 10 min. Second round: The PCR amplification reaction was carried out in a 50 μL system where 1.5 μL of each of the primers were added to the reaction system of the first round. The reaction conditions were as follows: pre-denaturation at 94° C. for 3 min; then 25 cycles of denaturation at 94° C. for 30 s, annealing at 56.2° C. for 30 s, and extension at 72° C. for 2 min; and final extension at 72° C. for 10 min. The PCR product was identified by electrophoresis and extracted for recovery and purification. The product recovered in Step (3) was cloned into pMD19-T vector to construct a recombinant cloning plasmid. The plasmid was transformed into E. coli JM 109. Multiple transformants were picked into LB liquid medium (Amp.sup.r) and incubated at 37° C. and 220 rpm for 10-12 h. The plasmid was extracted, and verified by PCR.

(39) (4) Construction of Recombinant Plasmid pMA5-npld

(40) The pMA5 plasmid and the correct recombinant cloning plasmid containing the target gene were both cleaved with BamH I and Mlu I at 37° C. for 3 hrs. The electrophoresis was performed for verification and the target gene and target plasmid were extracted and recovered. The recovered products were ligated overnight at 16° C. with T4 DNA ligase, and the ligated product was transformed into E. coli JM 109 competent cells. The cells were cultured overnight on solid LB medium containing ampicillin (100 mg/L). Multiple transformants were picked into LB liquid medium containing ampicillin, and cultured at 37° C. and 220 rpm for 10-12 h. The plasmid was extracted. The recombinant plasmid was shipped to Shanghai Ruidi Sequencing Company for sequencing. The recombinant plasmid with the correct sequence was designated as pMA5-npld.

(41) (5) Transformation of Recombinant Plasmid pMA5-npld into B. Subtilis WB600

(42) The recombinant plasmid obtained in Step (4) was transformed into B. subtilis WB600. The recombinant strain was cultured in TB medium for 36 h. The fermentation broth was centrifuged for 10 min at 12000 rpm and 4° C. The fermentation supernatant containing phospholipase D was determined for enzyme activity. The enzyme activity of recombinant B. subtilis WB600/pMA5-pld was 0.16 U/mL.

Example 4

(43) This example provides the construction of the recombinant plasmid pPIC3.5K-pld and its expression method in Pichia pastoris. The specific steps are as follows:

(44) (1) Amplification of Phospholipase D Coding Sequence

(45) Using the recombinant plasmid pET-28a(+)-spld containing the phospholipase D coding gene as a template, the phospholipase D coding sequence was amplified with the designed primers (P7, P8):

(46) TABLE-US-00005 Primer P7: 5′-CGGGATCCATGGCACGTCATCCGC-3′ (BamHI) Primer P8: 5′-CGGAATTCTTAATCCTGACAAATA-3′ (EcoRI)

(47) The PCR amplification reaction was carried out in a 50 μL system, in which 25 μL of PrimeSTAR® (Premix), 20 μL of ddH.sub.2O, 2 μL of template DNA, and 1.5 μL of each of the upstream and downstream primers were added. The reaction conditions were as follows: pre-denaturation at 94° C. for 3 min; then 30 cycles of denaturation at 94° C. for 30 s, annealing at 58.4° C. for 30 s, and extension at 72° C. for 1.5 min; and final extension at 72° C. for 10 min. The PCR product was identified by electrophoresis and extracted for recovery and purification. The product recovered in Step (1) was cloned into pMD19-T vector to construct a recombinant cloning plasmid. The plasmid was transformed into E. coli JM 109. Multiple transformants were picked into LB liquid medium (Amp.sup.r) and incubated at 37° C. and 220 rpm for 10-12 h. The plasmid was extracted, and verified by PCR. The plasmid verified to be correct was sequenced.

(48) (2) Construction of Recombinant Plasmid pPIC3.5K-pld

(49) The pPIC3.5K plasmid and the correct recombinant cloning plasmid containing the target gene were both cleaved with BamH I and EcoRI at 37° C. for 3 h. The electrophoresis was performed for verification and the target gene and target plasmid were extracted and recovered. The recovered products were ligated overnight at 16° C. with T4 DNA ligase, and the ligated product was transformed into E. coli JM 109 competent cells. The cells were cultured overnight on solid LB medium containing ampicillin (100 mg/L). Multiple transformants were picked into LB liquid medium containing ampicillin, and cultured at 37° C. and 220 rpm for 10-12 h. The plasmid was extracted.

(50) (3) Transformation of Recombinant Plasmid pPIC3.5K-pld into P. Pastoris GS115

(51) The recombinant plasmid obtained in Step (2) was shipped to Shanghai Ruidi Sequencing Company for sequencing. The recombinant plasmid with the correct sequence was designated as pPIC3.5K-pld. After linearization with Sal I restriction endonuclease, the plasmid was transformed into P. pastoris GS115 at 1500 v for 5 ms. The electroporated cell suspension was coated on MD medium, and incubated upside down in a constant-temperature incubator at 30° C. until the transformants were grown out. Then the transformants were transferred to YPD solid medium with different concentrations of antibiotic G418 sulfate and cultured for additional three days, to screen out high-copy strains on the plate with high concentration of antibiotic. The recombinant strain was inoculated into 10 mL YPD medium and incubated at 30° C. for 12 h and then inoculated into BMGY medium. After culturing for 24 h, the cells were harvested by centrifugation at 4500 rpm for 10 min and then resuspended in BMMY medium. Methanol with a final concentration of 5 g/L was added to the culture every 24 h for induction. After induction for 96 h, the fermentation broth was centrifuged for 10 min at 12000 rpm and 4° C. The cells were re-suspended in 4 mL buffer (40 mM Tris-HCl, 0.1% (v/v) Triton X-100, 15 mM CaCl.sub.2). The cells were ultrasonically homogenized in an ice bath, and centrifuged. The supernatant was collected for activity determination. The enzyme activity of the recombinant P. pastoris GS115/pPIC3.5K-pld is 0.22 U/mL.

Example 5

(52) This example provides the construction of the recombinant plasmid pPIC9K-pld and its expression method in Pichia pastoris. The specific steps are as follows:

(53) (1) Amplification of Phospholipase D Coding Sequence

(54) Using the recombinant plasmid pET-28a(+)-spld containing the phospholipase D coding gene as a template, the phospholipase D coding sequence was amplified with the designed primers (P9, P10):

(55) TABLE-US-00006 Primer P9: 5′-CGGAATTCATGGCACGTCATCCGCGCAAA-3′ (EcoRI) Primer P10: 5′-AAGGAAAAAAGCGGCCGCTTAATCCTGACAAAT-3′ (NotI)

(56) The PCR amplification reaction was carried out in a 50 μL system, in which 25 μL of PrimeSTAR® (Premix), 20 μL of ddH.sub.2O, 2 μL of template DNA, and 1.5 μL of each of the upstream and downstream primers were added. The reaction conditions were as follows: pre-denaturation at 94° C. for 3 min; then 30 cycles of denaturation at 94° C. for 30 s, annealing at 59° C. for 30 s, and extension at 72° C. for 1.5 min; and final extension at 72° C. for 10 min. The PCR product was identified by electrophoresis and extracted for recovery and purification. The product recovered in Step (1) was cloned into pMD19-T vector to construct a recombinant cloning plasmid. The plasmid was transformed into E. coli JM 109. Multiple transformants were picked into LB liquid medium (Amp.sup.r) and incubated at 37° C. and 220 rpm for 10-12 h. The plasmid was extracted, and verified by PCR. The plasmid verified to be correct was sequenced.

(57) (2) Construction of Recombinant Plasmid pPIC9K-pld

(58) The pPIC9K plasmid and the correct recombinant cloning plasmid containing the target gene were both cleaved with EcoRI and NotI at 37° C. for 3 h. The electrophoresis was performed for verification and the target gene and target plasmid were extracted and recovered. The recovered products were ligated overnight at 16° C. with T4 DNA ligase, and the ligated product was transformed into E. coli JM 109 competent cells. The cells were cultured overnight on solid LB medium containing ampicillin (100 mg/L). Multiple transformants were picked into LB liquid medium containing ampicillin, and cultured at 37° C. and 220 rpm for 10-12 h. The plasmid was extracted.

(59) (3) Transformation of Recombinant Plasmid pPIC9K-pld into P. Pastoris GS115

(60) The recombinant plasmid obtained in Step (2) was shipped to Shanghai Ruidi Sequencing Company for sequencing. The recombinant plasmid with the correct sequence was designated as pPIC9K-pld. After linearization with Sal I restriction endonuclease, the plasmid was transformed into P. pastoris GS115 at 1500 v for 5 ms. The electroporated cell suspension was coated on MD medium, and incubated upside down in a constant-temperature incubator at 30° C. until the transformants were grown out. Then the transformants were transferred to YPD solid medium with different concentrations of antibiotic G418 sulfate and cultured for additional three days, to screen out high-copy strains on the plate with high concentration of antibiotic. The recombinant strain was inoculated into 10 mL YPD medium and incubated 30° C. for 12 h and then inoculated into BMGY medium. After culturing for 24 h, the cells were harvested by centrifugation at 4500 rpm for 10 min and then resuspended in BMMY medium. Methanol with a final concentration of 5 g/L was added to the culture every 24 h for induction. After 96 h of induction, the fermentation broth was centrifuged for 10 min at 12000 rpm and 4° C. The fermentation supernatant containing phospholipase D was determined for enzyme activity. The enzyme activity of the recombinant P. pastoris GS115/pPIC9K-pld is 0.41 U/mL, which is lower than enzyme activity of phospholipase D expressed intracellularly.

Example 6

(61) This example provides the construction of the recombinant plasmid pDXW-pld and its expression method in Corynebacterium glutamicum. The specific steps are as follows:

(62) (1) Amplification of Phospholipase D Coding Sequence

(63) Using the recombinant plasmid pET-28a(+)-spld containing the phospholipase D coding gene as a template, the phospholipase D coding sequence was amplified with the designed primers (P11, P12):

(64) TABLE-US-00007 Primer P11: 5′-CGGAATTCATGGCACGTCATCCGCGCAAA-3′ (EcoRI) Primer P12: 5′-CCAAGCTTTTAATCCTGACAAATACCGCG-3′ (Hind III)

(65) The PCR amplification reaction was carried out in a 50 μL system, in which 25 μL of PrimeSTAR® (Premix), 20 μL of ddH.sub.2O, 2 μL of template DNA, and 1.5 μL of each of the upstream and downstream primers were added. The reaction conditions were as follows: pre-denaturation at 94° C. for 3 min; then 30 cycles of denaturation at 94° C. for 30 s, annealing at 57.2° C. for 30 s, and extension at 72° C. for 1.5 min; and final extension at 72° C. for 10 min. The PCR product was identified by electrophoresis and extracted for recovery and purification. The product recovered in Step (1) was cloned into pMD19-T vector to construct a recombinant cloning plasmid. The plasmid was transformed into E. coli JM 109. Multiple transformants were picked into LB liquid medium (Kan.sup.r) and incubated at 37° C. and 220 rpm for 10-12 h. The plasmid was extracted, and verified by PCR. The plasmid verified to be correct was sequenced.

(66) (2) Construction of Recombinant Plasmid pDXW-pld

(67) The pDXW-10 plasmid and the correct recombinant cloning plasmid containing the target gene were both cleaved with EcoR I and Hind III at 37° C. for 3 h. The electrophoresis was performed for verification and the target gene and target plasmid were extracted and recovered. The recovered products were ligated overnight at 16° C. with T4 DNA ligase, and the ligated product was transformed into E. coli JM 109 competent cells. The cells were cultured overnight on solid LB medium containing kanamycin (50 mg/L). Multiple transformants were picked into LB liquid medium containing kanamycin, and cultured at 37° C. and 220 rpm for 10-12 h. The plasmid was extracted.

(68) (3) Transformation of Recombinant Plasmid pDXW-pld into C. glutamicum ATCC 13032

(69) The recombinant plasmid obtained in Step (2) was shipped to Shanghai Ruidi Sequencing Company for sequencing. The recombinant plasmid with the correct sequence was designated as pDXW-pld. The recombinant plasmid was electroporated into C. glutamicum ATCC 13032 at 1900 V for 5 ms. The cells were coated on a solid seed medium containing kanamycin (50 mg/L), and incubated upside down in an incubator at 30° C. until the transformants were grown out. Positive transformants were screened out by PCR verification, and inoculated into a liquid seed medium containing kanamycin (50 mg/L), and incubated to OD562=25. The fermentation broth was centrifuged for 10 min at 12000 rpm and 4° C. The cells were re-suspended in 4 mL buffer (40 mM Tris-HCl, 0.1% (v/v) Triton X-100, 15 mM CaCl.sub.2). The cells were ultrasonically homogenized in an ice bath, and centrifuged. The supernatant was collected for activity determination. The enzyme activity of the recombinant C. glutamicum ATCC 13032/pDXW-pld is 0.25 U/mL.

Example 7

(70) This example provides the construction of the recombinant plasmid pDXW-pld4 and its expression method in Corynebacterium glutamicum. The specific steps are as follows:

(71) (1) Using the recombinant plasmid pET-28a(+)-spld containing the phospholipase D coding gene as a template, the phospholipase D coding sequence was amplified with the designed primers (P13, P14):

(72) TABLE-US-00008 Primer P13: 5′-AGCCGATGTACTAGCAGCTATGGCACGTCATCCGCGCA-3′  Primer P14: 5′-CCAAGCTTTTAATCCTGACAAATACCGCG-3′ (Hind III)

(73) The PCR amplification reaction was carried out in a 50 μL system, in which 25 μL of PrimeSTAR® (Premix), 20 μL of ddH.sub.2O, 2 μL of template DNA, and 1.5 μL of each of the upstream and downstream primers were added. The reaction conditions were as follows: pre-denaturation at 94° C. for 3 min; then 30 cycles of denaturation at 94° C. for 30 s, annealing at 57° C. for 30 s, and extension at 72° C. for 1.5 min; and final extension at 72° C. for 10 min. The PCR product was identified by electrophoresis and extracted for recovery and purification.

(74) (2) By using the genome of B. subtilis 168 as a template, the WapA signal peptide sequence was amplified with the designed primers (P15, P16):

(75) TABLE-US-00009 Primer P15: 5′-CGGAATTCTGAAAAAAAGAAAGAGG-3′ (EcoR I) Primer P16: 5′-TTGCGCGGATGACGTGCCATAGCAGCTGCTAGTACATCGGCT-3′ 

(76) The PCR amplification reaction was carried out in a 50 μL system, in which 25 μL of PrimeSTAR® (Premix), 20 μL of ddH.sub.2O, 2 μL of template DNA, and 1.5 μL of each of the upstream and downstream primers were added. The reaction conditions were as follows: pre-denaturation at 94° C. for 3 min; then 30 cycles of denaturation at 94° C. for 30 s, annealing at 53° C. for 30 s, and extension at 72° C. for 0.5 min; and final extension at 72° C. for 10 min. The PCR product was identified by electrophoresis and extracted for recovery and purification.

(77) (3) By using the extracted and purified phospholipase D sequence and WapA signal peptide sequence as templates, the two sequences were fused with Primers P14 and P15:

(78) TABLE-US-00010 Primer P14: 5′-CCAAGCTTTTAATCCTGACAAATACCGCG-3′ (Hind III) Primer P15: 5′-CGGAATTCAAAAAAAGAAAGAGG-3′ (EcoR I)

(79) The fusion process included two rounds of PCR reactions. First round: The PCR amplification reaction was carried out in a 47 μL system, in which 25 μL of PrimeSTAR® (Premix), 19 μL of ddH.sub.2O, and 1.5 μL of each of the phospholipase D gene and WapA templates were added. The reaction conditions were as follows: pre-denaturation at 94° C. for 3 min; then 8 cycles of denaturation at 94° C. for 30 s, annealing at 50° C. for 30 s, and extension at 72° C. for 2 min; and final extension at 72° C. for 10 min. Second round: The PCR amplification reaction was carried out in a 50 μL system where 1.5 μL of each of the primers were added to the reaction system of the first round. The reaction conditions were as follows: pre-denaturation at 94° C. for 3 min; then 25 cycles of denaturation at 94° C. for 30 s, annealing at 55° C. for 30 s, and extension at 72° C. for 2 min; and final extension at 72° C. for 10 min. The PCR product was identified by electrophoresis and extracted for recovery and purification. The product recovered in Step (3) was cloned into pMD19-T vector to construct a recombinant cloning plasmid. The plasmid was transformed into E. coli JM 109. Multiple transformants were picked into LB liquid medium (Kan.sup.r) and incubated at 37° C. and 220 rpm for 10-12 h. The plasmid was extracted, and verified by PCR. The plasmid verified to be correct was sequenced.

(80) (4) Construction of Recombinant Plasmid pDXW-pld4

(81) The pDXW-10 plasmid and the correct recombinant cloning plasmid containing the target gene were both cleaved with EcoR I and Hind III at 37° C. for 3 h. The electrophoresis was performed for verification and the target gene and target plasmid were extracted and recovered. The recovered products were ligated overnight at 16° C. with T4 DNA ligase, and the ligated product was transformed into E. coli JM 109 competent cells. The cells were cultured overnight on solid LB medium containing kanamycin (50 mg/L). Multiple transformants were picked into LB liquid medium containing kanamycin, and cultured at 37° C. and 220 rpm for 10-12 h. The plasmid was extracted.

(82) (5) Transformation of Recombinant Plasmid pDXW-pld4 into C. Glutamicum ATCC 13032

(83) The recombinant plasmid obtained in Step (4) was shipped to Shanghai Ruidi Sequencing Company for sequencing. The recombinant plasmid with the correct sequence was designated as pDXW-pld. The recombinant plasmid was electroporated into C. glutamicum ATCC 13032 at 1900 v for 5 ms. The cells were coated on a solid seed medium containing kanamycin (50 mg/L), and incubated upside down in an incubator at 30° C. until the transformants were grown out. Positive transformants were screened out by PCR verification, and inoculated into a liquid seed medium containing kanamycin (50 mg/L), and incubated to OD562=25. The fermentation broth was centrifuged for 10 min at 12000 rpm and 4° C. The supernatant was collected for activity determination. The enzyme activity of the recombinant C. glutamicum ATCC 13032/pDXW-pld4 is 0.91 U/mL.

(84) In the above examples, the heterologous expression of phospholipase D in Bacillus subtilis, Pichia pastoris, and Corynebacterium glutamicum is achieved, and the enzyme activities of the recombinant strains are 0.14 U/mL, 0.22 U/mL, and 0.25 U/mL, respectively (FIG. 9). The use of signal peptides realizes the secretory expression of phospholipase D in Bacillus subtilis, Pichia pastoris, and Corynebacterium glutamicum. The enzyme activity of the recombinant strains is shown in FIG. 10. Because phospholipase D shows a highest hydrolysis activity of 0.91 U/mL in Corynebacterium glutamicum, Corynebacterium glutamicum is taken as the optimal expression host and WapA is the optimal signal peptide.

Example 8

(85) This example provides the construction of the recombinant plasmid pDXW-rpld4 added with RBS and its expression method in Corynebacterium glutamicum. The specific steps are as follows:

(86) (1) Using the recombinant plasmid pET-28a(+)-spld containing the phospholipase D coding gene as a template, the phospholipase D coding sequence was amplified with the designed primers (P13, P14):

(87) TABLE-US-00011 Primer P13: 5′-AGCCGATGTACTAGCAGCTATGGCACGTCATCCGCGCA-3′  Primer P14: 5′-CCAAGCTTTTAATCCTGACAAATACCGCG-3′ (Hind III)

(88) The PCR amplification reaction was carried out in a 50 μL system, in which 25 μL of PrimeSTAR® (Premix), 20 μL of ddH.sub.2O, 2 μL of template DNA, and 1.5 μL of each of the upstream and downstream primers were added. The reaction conditions were as follows: pre-denaturation at 94° C. for 3 min; then 30 cycles of denaturation at 94° C. for 30 s, annealing at 57° C. for 30 s, and extension at 72° C. for 1.5 min; and final extension at 72° C. for 10 min. The PCR product was identified by electrophoresis and extracted for recovery and purification.

(89) (2) By using the genome of B. subtilis 168 as a template, a WapA signal peptide sequence containing the RBS sequence (AGAAGGAGATATACC) was amplified with the designed primers (P16, P17):

(90) TABLE-US-00012 Primer P16: 5′-TTGCGCGGATGACGTGCCATAGCAGCTGCTAGTACATCGGCT-3′  Primer P17: 5′-CGGAATTCAGAAGGAGATATACCAAAAAAAGAAAGAGG-3′ (EcoR I)

(91) The PCR amplification reaction was carried out in a 50 μL system, in which 25 μL of PrimeSTAR® (Premix), 20 μL of ddH.sub.2O, 2 μL of template DNA, and 1.5 μL of each of the upstream and downstream primers were added. The reaction conditions were as follows: pre-denaturation at 94° C. for 3 min; then 30 cycles of denaturation at 94° C. for 30 s, annealing at 53.5° C. for 30 s, and extension at 72° C. for 0.5 min; and final extension at 72° C. for 10 min. The PCR product was identified by electrophoresis and extracted for recovery and purification.

(92) (3) By using the extracted and purified phospholipase D sequence and WapA signal peptide sequence containing the RBS sequence as templates, the two sequences were fused with Primers P14 and P17:

(93) TABLE-US-00013 Primer P14: 5′-CCAAGCTTTTAATCCTGACAAATACCGCG-3′ (Hind III) Primer P17: 5′-CGGAATTCAGAAGGAGATATACCAAAAAAAGAAAGAGG-3′ (EcoR I)

(94) The fusion process included two rounds of PCR reactions. First round: The PCR amplification reaction was carried out in a 47 μL system, in which 25 μL of PrimeSTAR® (Premix), 19 μL of ddH.sub.2O, and 1.5 μL of each of the phospholipase D gene and WapA templates were added. The reaction conditions were as follows: pre-denaturation at 94° C. for 3 min; then 8 cycles of denaturation at 94° C. for 30 s, annealing at 50° C. for 30 s, and extension at 72° C. for 2 min; and final extension at 72° C. for 10 min. Second round: The PCR amplification reaction was carried out in a 50 μL system where 1.5 μL of each of the primers were added to the reaction system of the first round. The reaction conditions were as follows: pre-denaturation at 94° C. for 3 min; then 25 cycles of denaturation at 94° C. for 30 s, annealing at 55.5° C. for 30 s, and extension at 72° C. for 2 min; and final extension at 72° C. for 10 min. The PCR product was identified by electrophoresis and extracted for recovery and purification. The product recovered in Step (3) was cloned into pMD19-T vector to construct a recombinant cloning plasmid. The plasmid was transformed into E. coli JM 109. Multiple transformants were picked into LB liquid medium (Kan.sup.r) and incubated at 37° C. and 220 rpm for 10-12 h. The plasmid was extracted, and verified by PCR. The plasmid verified to be correct was sequenced.

(95) (4) Construction of Recombinant Plasmid pDXW-rpld4

(96) The pDXW-10 plasmid and the correct recombinant cloning plasmid containing the target gene were both cleaved with EcoR I and Hind III at 37° C. for 3 h. The electrophoresis was performed for verification and the target gene and target plasmid were extracted and recovered. The recovered products were ligated overnight at 16° C. with T4 DNA ligase, and the ligated product was transformed into E. coli JM 109 competent cells. The cells were cultured overnight on solid LB medium containing kanamycin (50 mg/L). Multiple transformants were picked into LB liquid medium containing kanamycin, and cultured at 37° C. and 220 rpm for 10-12 h. The plasmid was extracted.

(97) (5) Transformation of Recombinant Plasmid pDXW-rpld4 into C. Glutamicum ATCC 13032

(98) The recombinant plasmid obtained in Step (4) was shipped to Shanghai Ruidi Sequencing Company for sequencing. The recombinant plasmid with the correct sequence was designated as pDXW-pld. The recombinant plasmid was electroporated into C. glutamicum ATCC 13032 at 1900 v for 5 ms. The cells were coated on a solid seed medium containing kanamycin (50 mg/L), and incubated upside down in an incubator at 30° C. until the transformants were grown out. Positive transformants were screened out by PCR verification, and inoculated into a liquid seed medium containing kanamycin (50 mg/L), and incubated to OD562=25. The fermentation broth was centrifuged for 10 min at 12000 rpm and 4° C. The supernatant was collected for activity determination. The enzyme activity of the recombinant C. glutamicum ATCC 13032/pDXW-rpld4 is 1.06 U/mL, which is increased by 16% compared with the recombinant strain without RBS.

Example 9

(99) This example provides the construction of the recombinant plasmid pDXW-wrpld4 in which the tac-M promoter is replaced by the pWapA promoter, and its expression method in Corynebacterium glutamicum. The specific steps are as follows:

(100) (1) By using the genome of B. subtilis 168 as a template, the pWapA promoter sequence was amplified with the designed primers (P18, P19):

(101) TABLE-US-00014 Primer P18: 5′-GGGGTACCATTTTTATCAACGAAATTTATTT-3′ (Kpn I) Primer P19: 5′-CGGAATTCTTCCTCTCTCCTTTTGTAATA-3′ (EcoRI)

(102) The PCR amplification reaction was carried out in a 50 μL system, in which 25 μL of PrimeSTAR® (Premix), 20 μL of ddH.sub.2O, 2 μL of template DNA, and 1.5 μL of each of the upstream and downstream primers were added. The reaction conditions were as follows: pre-denaturation at 94° C. for 3 min; then 30 cycles of denaturation at 94° C. for 30 s, annealing at 58° C. for 30 s, and extension at 72° C. for 0.5 min; and final extension at 72° C. for 10 min. The PCR product was identified by electrophoresis and extracted for recovery and purification.

(103) (2) By using the pDXW-10 plasmid as a template, the Kpn I restriction site GGTACC on the multiple cloning site in the plasmid was mutated to GGAACC with the designed primers (P20, P21, P22, P23), and a Kpn I restriction site was designed before the tac-M promoter. The engineered pDXW-10 plasmid was designated as pDXW-10a plasmid.

(104) TABLE-US-00015 Primer P20: 5′-GCCTCGAGGGAACCAGATCTCCGCGGCTTAA-3′  Primer P21: 5′-AGATCTGGTTCCCTCGAGGCGGCCGCCCAT-3′  Primer P22: 5′-TCATAACGGTACCGGCAAATATTCTGAAATGAGCTGTTG-3′  Primer P23: 5′-ATTTCAGAATATTTGCCGGTACCGTTATGATGTCGGCGCA-3′ 

(105) The PCR amplification reaction was carried out in a 50 μL system, in which 25 μL of PrimeSTAR® (Premix), 20 μL of ddH.sub.2O, 2 μL of template DNA, and 1.5 μL of each of the upstream and downstream primers were added. The reaction conditions were as follows: pre-denaturation at 94° C. for 3 min; then 30 cycles of denaturation at 94° C. for 30 s, annealing at 59° C. for 30 s, and extension at 72° C. for 10 min; and final extension at 72° C. for 10 min. The PCR product was identified by electrophoresis and extracted for recovery and purification.

(106) (3) Construction of Recombinant Plasmid pDXW-wrpld4

(107) The pDXW-10a plasmid (SEQ ID NO.6) was cleaved for 3 h with Kpn I and Hind at 37° C. Then electrophoresis was performed for verification and the target plasmid were extracted and recovered. The correct recombinant cloning plasmid containing the target gene in Example 9 was cleaved for 3 h with Kpn I and EcoRI at 37° C. Then electrophoresis was performed for verification and the target gene were extracted and recovered. The pWapA fragment obtained in Step (1) was cleaved for 3 h with EcoRI and Hind III at 37° C. Then electrophoresis was performed for verification and the pWapA gene were extracted and recovered. The three fragments of recovered products were ligated overnight at 16° C. with T4 DNA ligase, and the ligated product was transformed into E. coli JM 109 competent cells. The cells were cultured overnight on solid LB medium containing kanamycin (50 mg/L). Multiple transformants were picked into LB liquid medium containing kanamycin, and cultured at 37° C. and 220 rpm for 10-12 h. The plasmid was extracted.

(108) (4) Transformation of Recombinant Plasmid pDXW-wrpld4 into C. Glutamicum ATCC 13032

(109) The recombinant plasmid obtained in Step (3) was shipped to Shanghai Ruidi Sequencing Company for sequencing. The recombinant plasmid with the correct sequence was designated as pDXW-wrpld4. The recombinant plasmid was electroporated into C. glutamicum ATCC 13032 at 1900 v for 5 ms. The cells were coated on a solid seed medium containing kanamycin (50 mg/L), and incubated upside down in an incubator at 30° C. until the transformants were grown out. Positive transformants were screened out by PCR verification, and inoculated into a liquid seed medium containing kanamycin (50 mg/L), and incubated to OD562=25. The fermentation broth was centrifuged for 10 min at 12000 rpm and 4° C. The supernatant was collected for activity determination. The enzyme activity of the recombinant C. glutamicum ATCC 13032/pDXW-wrpld4 is 1.3 U/mL.

(110) In this example, by means of heterologous expression, addition of signal peptide and RBS, and replacement of promoter, high-efficiency expression of phospholipase D is achieved in Corynebacterium glutamicum. Recombinant C. glutamicum ATCC 13032/pDXW-wrpld4 in the optimized fermentation medium has an enzyme activity 1.9 U/mL, which is 7.6 times that of the unengineered recombinant C. glutamicum ATCC 13032/pDXW-pld.

(111) In the above examples of the present invention, the process for determining the hydrolysis activity of phospholipase D was as follows:

(112) A 100 μL reaction system containing 60 μL substrate lecithin solution (preheated for 5 min) and 40 μL enzyme solution was thoroughly mixed and reacted at 60° C. for 20 min. 50 μL of a stop solution was added to terminate the reaction. The system was placed in boiling water for 5 min and then immediately cooled on ice. The system was centrifuged at 6000 rpm for 5 min. All the supernatant was pipetted and added with 60 μL choline oxidase, 200 μL phenol solution, 200 μL 4-aminoantipyrine solution and 40 μL peroxidase, fully mixed and reacted at 37° C. for 20 min. After reaction, the absorbency at OD505 nm was determined, and the enzyme activity was calculated according to the standard curve.

(113) The molecular weight of phospholipase D protein was also tested in the present invention, and the process was as follows.

(114) The separation gel and concentration gel were prepared according to the composition of 10% separation gel and concentration gel. Then 80 μL sample was added to 20 μL 5× loading buffer, mixed well and stood in a boiling water bath for 5 min to denature the protein. The sample was loaded according to the protein concentration and a protein marker was added. The upper concentration gel was applied with a voltage of 80 V (about 30 min) and the lower separation gel was applied with a voltage of 100 V. When the sample reached to about 1 cm from the bottom of the separation gel, the power was turned off. Subsequently, the sample was rinsed with distilled water, dried and decolored, and the molecular weight of phospholipase D protein was determined. The SDS-PAGE results show (FIG. 11) that the molecular weight of the phospholipase D expressed in Example 9 of the present invention is 60 kDa.

Example 10

(115) This example provides a method for producing phosphatidylserine (PS).

(116) The initial conversion process was as follows. Soybean lecithin (PC50) was dissolved in 8 mL ethyl acetate to give a concentration of 8 mg/mL and used as the organic phase, and 160 mg L-serine was dissolved in 4 mL phospholipase D crude enzyme solution and used as the aqueous phase. After the organic phase and the aqueous phase were ultrasonically mixed fully, and then reacted for 12 h with shaking at 120 rpm at 40° C.

(117) 20 mL mixed solution of chloroform/methanol (volume ratio 2/1) and 3 mL ultrapure water were added to the reaction solution, and centrifuged at 2500 rpm for 5 min. The lower solution was removed, and the remainder was concentrated by a vacuum centrifugal concentrator, dissolved in 2 mL n-hexane/isopropanol (volume ratio 1/1), and filtered through a 0.22 μm organic membrane. The product phosphatidylserine was detected and analyzed by HPLC.

(118) In order to obtain the most desirable conversion rate, the conversion conditions were optimized in this example. The conversion process was optimized from the selection of organic solvent, the volume ratio of organic phase to aqueous phase, the ratio of substrate concentration, the conversion temperature, the conversion time and the concentration of calcium ions.

(119) (1) Selection of Organic Solvent

(120) Ethyl acetate in the initial conversion conditions was replaced by the same volume of n-hexane, chloroform, ether or toluene. Through detection and analysis by HPLC, when the organic phase is toluene, the rate of conversion to phosphatidylserine is the highest and is 43.3% (FIG. 12a), so toluene can be used as the optimal organic phase.

(121) (2) Volume Ratio of Organic Phase to Aqueous Phase

(122) The organic phase in the initial conversion conditions was replaced by toluene, and the volume ratio of the organic phase to the aqueous phase was set to 4:1, 4:2, 4:3 or 4:4 respectively, and the other conversion conditions remained unchanged. The influence of the volume ratio of the two phases on the conversion rate was investigated. When the volume ratio of the organic phase to the aqueous phase is 4:2, the rate of conversion to phosphatidylserine is the highest and is 44.5% (FIG. 12b), so the volume ratio of the two phases of 4:2 is the optimal ratio.

(123) (3) Ratio of Substrate Concentration

(124) PC50 was dissolved in 8 mL toluene to give a concentration of 8 mg/mL and used as the organic phase, and L-serine was dissolved in 4 mL crude enzyme solution to give a concentration of 8, 16, 24, 32, 40, or 48 mg/mL respectively and used as the aqueous phase. The organic phase and the aqueous phase were ultrasonically mixed thoroughly, and reacted for 12 h with shaking at 120 rpm at 40° C. The influence of different ratios of substrate concentration on the conversion rate was investigated. When the PC50 concentration is 8 mg/mL and the L-serine concentration is 40 mg/mL, the rate of conversion to phosphatidylserine is the highest, reaching 43.2% (FIG. 12c), so 1:5 is the optimal ratio of substrate concentration.

(125) (4) Conversion Temperature

(126) PC50 was dissolved in 8 mL toluene to give a concentration of 8 mg/mL and used as the organic phase, and L-serine was dissolved in 4 mL phospholipase D crude enzyme solution to give a concentration of 40 mg/mL and used as the aqueous phase. The organic phase and the aqueous phase were ultrasonically mixed thoroughly, and reacted for 12 h with shaking at 120 rpm at 25, 30, 35, 40, 45, and 50° C. respectively. The influence of different conversion temperatures on the conversion rate was investigated. According to the detection results by HPLC, it can be seen that when the conversion temperature is most preferably 40° C., the conversion rate is 44.6% (FIG. 12e), so 40° C. can be used as the optimal conversion temperature.

(127) (5) Conversion Time

(128) The time in the optimized conversion conditions obtained in Step (4) was set to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 h, and the influence of different conversion time on the conversion rate was investigated. According to the detection results by HPLC, it can be seen that when the conversion time is most preferably 10 h, the conversion rate is 45.8% (FIG. 12d), so a conversion time of 10 h can be used as the optimal conversion time.

(129) (6) The Concentration of Calcium Ions

(130) 8 mL toluene with 8 mg/mL PC50 was taken as the organic phase for reaction and 4 mL crude enzyme solution with 40 mg/mL L-serine was taken as the aqueous phase for reaction. Calcium chloride having a final concentration of 0 mM, 5 mM, 10 mM, 15 mM, or 20 mM was added to the aqueous phase respectively. The organic phase and the aqueous phase were mixed thoroughly, and then reacted for 10 h with shaking at 120 rpm at 40° C. The production of PS was detected. When the concentration of calcium chloride is 15 mM, the conversion rate is the highest and is 48.6% (FIG. 12f), so 15 mM CaCl.sub.2 can be added to the conversion system to obtain the highest conversion rate.

(131) While preferred embodiments of the present invention have been described above, the present invention is not limited thereto. It should be appreciated that some improvements and variations can be made by those skilled in the art without departing from the technical principles of the present invention, which are also contemplated to be within the scope of the present invention.