Recombinant bacillus subtilis for synthesizing lacto-N-neotetraose and application thereof

Abstract

The disclosure discloses recombinant Bacillus subtilis for synthesizing e lacto-N-neotetraose yield. The recombinant Bacillus subtilis is obtained by integrating two -1,4-galactotransferase genes on a genome of a host bacterium Bacillus subtilis 168amyE:P.sub.43-lacY, P.sub.43-lgtB, P.sub.xylA-comK and exogenously expressing a -1,3-N-glucosaminotransferase gene. Compared with a strain before transformation, the recombinant Bacillus subtilis of the disclosure improves the yield of the synthesized lacto-N-neotetraose from 720 mg/L to 1300 mg/L, laying a foundation for further metabolic engineering transformation of Bacillus subtilis for producing the lacto-N-neotetraose.

Claims

1. Recombinant Bacillus subtilis for synthesizing lacto-N-neotetraose, wherein the recombinant Bacillus subtilis is obtained by the following steps: integrating two -1,4-galactotransferase genes on a genome of a host bacterium Bacillus subtilis 168amyE:P.sub.43-lacY, P.sub.43-lgtB, P.sub.xylA-comK; and exogenously expressing a -1,3-N-glucose aminotransferase gene.

2. The recombinant Bacillus subtilis of claim 1, wherein the host bacterium is obtained by the following steps: using Bacillus subtilis 168 as a starting strain, regulating the expression of a gene comK by a promoter P.sub.xylA, integrating a lactose permease-encoding gene onto an amyE site of the genome, and integrating a -1,4-galactotransferase-encoding gene between a ydeS site and a ydzO site of the genome.

3. A method for constructing the recombinant Bacillus subtilis of claim 1, comprising the following steps: (1) constructing a recombinant fragment containing homologous arms of ydaH and ydzA genes, a -1,4-galactotransferase gene, a P.sub.43 promoter, and a bleomycin resistance gene sequence by fusion PCR; (2) constructing a recombinant fragment containing homologous arms of yszA and ysxE genes, a -1,4-galactotransferase gene, a P.sub.43 promoter, and a chloramphenicol resistance gene sequence by fusion PCR; (3) transforming the recombinant fragment constructed in the step (1) into Bacillus subtilis 168amyE:P.sub.43-lacY, P.sub.43-lgtB, PxylA-comK, and verifying to obtain recombinant Bacillus subtilis BY03a; (4) transforming the recombinant fragment constructed in the step (2) into the recombinant Bacillus subtilis BY03a of the step (3), and verifying to obtain recombinant Bacillus subtilis BY03b; and (5) transforming pP43NMK-lgtA into the recombinant Bacillus subtilis BY03b to obtain recombinant Bacillus subtilis BY05 for synthesizing lacto-N-neotetraose.

4. The method of claim 3, wherein in the step (1), a left homologous arm of a ydaH gene and a right homologous arm of a ydzA gene are used.

5. The method of claim 3, wherein in the step (2), a left homologous arm of a yszA gene and a right homologous arm of a ysxE gene are used.

6. The method of claim 3, wherein a sequence of the recombinant fragment constructed in the step (1) is set forth in SEQ ID NO.47, and a sequence of the recombinant fragment constructed in the step (2) is set forth in SEQ ID NO.58.

7. The method of claim 3, wherein in the step (3), the -1,4-galactotransferase gene, the P.sub.43 promoter and the bleomycin resistance gene sequence in the recombinant fragment constructed in the step (1) are integrated between a ydaH site and a ydzA site of a genome by homologous recombination.

8. The method of claim 3, wherein in the step (4), the -1,4-galactotransferase gene, the P.sub.43 promoter and the chloramphenicol resistance gene sequence in the recombinant fragment constructed in the step (2) are integrated between a yszA site and a ysxE site of a genome by homologous recombination.

9. A method for producing lacto-N-neotetraose, comprising performing fermentation at 35-40 C. by using the recombinant Bacillus subtilis of claim 2.

10. The method of claim 9, wherein the recombinant Bacillus subtilis is fermented in a culture medium containing glucose and lactose.

11. The method of claim 10, wherein the culture medium contains tryptone 6 g/L, yeast powder 12 g/L, (NH.sub.4)SO.sub.4 6 g/L, K.sub.2HPO.sub.4.3H.sub.2O 12.5 g/L, KH.sub.2PO.sub.4 2.5 g/L, MgSO.sub.4.7H.sub.2O 1.2 g/L, glucose 60 g/L, and lactose 5 g/L.

12. The method of claim 10, wherein the fermentation comprises inoculating the culture medium with 10% by volume of a recombinant Bacillus subtilis seed solution.

13. The method of claim 12, wherein the seed solution is obtained by culturing the recombinant Bacillus subtilis in a seed culture medium at 35-37 C. for 10-12 h.

14. The method of claim 13, wherein the seed culture medium is an LB culture medium and contains tryptone 10 g/L, yeast powder 5 g/L, and NaCl 10 g/L.

Description

BRIEF DESCRIPTION OF FIGURES

[0023] FIG. 1 is a PCR agarose gel electrophoresis diagram of recombinant BY03a strain according to the disclosure.

[0024] FIG. 2 is a PCR agarose gel electrophoresis diagram of the recombinant BY03b strain according to the disclosure.

[0025] FIG. 3 shows the effect of increasing the copy number of lgtB genes on the yield of lacto-N-neotetraose according to the disclosure.

DETAILED DESCRIPTION

[0026] The disclosure is explained in detail below with reference to the embodiments and the accompanying drawings.

[0027] Embodiment 1 Construction of Recombinant PZL(P.sub.43-lacY) Fragment

[0028] By using a genome of Bacillus subtilis 168 was used as a template, homologous arm primers on both sides were designed according to an amyE gene (Gene ID: 938356) published on NCBI, i.e., the left homologous arm primers of which the sequences are respectively SEQ ID NO:1 and SEQ ID NO:2:

TABLE-US-00001 amyE-1F: (SEQIDNO:1) 5-TATTCCGTATGTCAAGTGGCTGCGGTTTAT-3, amyE-1R: (SEQIDNO:2) 5-AATTGTTATCCGCTCTCTTGACACTCCTTATTTGATTTTTTGA AGACTTACTTCGG-3,

[0029] and the right homologous arm primers of which the sequences are respectively SEQ ID NO:3 and SEQ ID NO:4:

TABLE-US-00002 amyE-2F: (SEQIDNO:3) 5-CTTAAGGGCAAGGCTAGACGGGACTTA-3, amyE-2R: (SEQIDNO:4) 5-GGCACACCGATGTACACGTCATC-3.

[0030] The homologous arm gene sequences on both sides of amyE were amplified from the genome of Bacillus subtilis by using the above primers. By using a plasmid pP43NMK as a template, primers of which the sequences are SEQ ID NO:5 and SEQ ID NO:6 were designed respectively:

TABLE-US-00003 P.sub.43-F: (SEQIDNO:5) 5-CATTATACGAACGGTAAATCTGATAGGTGGTATGTTTTCGCTT GAACTTTTAAATACAG-3, P.sub.43-R: (SEQIDNO:6) 5-AGTTTGTGTTTTTTAAATAGTACATGTGTACATTCCTCTCTTA CCTATAATGGTACCGC-3.

[0031] An amplified fragment of P.sub.43 was obtained by using the above primers. By using a plasmid P7Z6 as a template, bleomycin resistance gene amplified primers of which the sequences are respectively SEQ ID NO:7 and SEQ ID NO:8 were designed:

TABLE-US-00004 zeo-F: (SEQIDNO:7) 5-TCAAATAAGGAGTGTCAAGAGAGCGGATAACAATTTCACACAG GAAACAG-3, zeo-R: (SEQIDNO:8) 5-AACATACCACCTATCAGATTTACCGTTCGTATAATGTATGCTA TACGAAGTTATTCAGT-3.

[0032] An amplified fragment of the bleomycin resistance gene was obtained by using the above primers. The amplified fragment of the P.sub.43 and the amplified fragment of the bleomycin resistance gene were verified to be correct by running the gel and then recovered by columns. The amplified fragment of the P.sub.43 and the amplified fragment of the bleomycin resistance gene were fused by a fusion PCR technology. The conditions of the first round of PCR were: the total amount of P.sub.43 and bleomycin resistant DNA recovered by equimolar columns was greater than 1000 ng, the amount of primer star enzyme was 25 L, the volume was adjusted to 50 L with ddH.sub.2O, and the PCR conditions were 55 C., 11 cycles. The conditions of the second round of PCR were: the above PCR product was used as a template, zeo-F and P43-R were used as upstream and downstream primers respectively, and a ZP43 fragment was obtained according to conventional PCR set conditions.

[0033] By using a genome of Escherichia coli K-12 as a template, primers of which the sequences are respectively SEQ ID NO:9 and SEQ ID NO:10 were designed according to lactose permease gene lacY (Gene ID: 949083) published on NCBI.

TABLE-US-00005 lacY-F: (SEQIDNO:9) 5-GTAAGAGAGGAATGTACACATGTACTATTTAAAAAACACAAAC TTTTGGATGTTCGGTT-3, lacY-R: (SEQIDNO:10) 5-TAAGTCCCGTCTAGCCTTGCCCTTAAGCGACTTCATTCACCTG ACGACG-3.

[0034] A lactose permease gene fragment was amplified by using the above primers. A left homologous arm of the amyE gene, a right homologous arm of the amyE gene, the ZP43 fragment and the lactose permease gene fragment were verified to be correct by running the gel and then recovered by columns. The left homologous arm of the amyE gene, the right homologous arm of the amyE gene, the ZP43 fragment and the lactose permease gene fragment were fused by a fusion PCR technology. The conditions of the first round of PCR were: the total amount of DNA recovered by equimolar columns was greater than 1000 ng, the amount of primer star enzyme was 25 L, the volume was adjusted to 50 L with ddH.sub.2O, and the PCR conditions were 55 C., 11 cycles. The conditions of the second round of PCR were: the above PCR product was used as a template, the sequences as shown in SEQ ID NO:11 and SEQ ID NO:12:

[0035] zong-F:5-CATGTAAGCCATAAGCCATTCGTAA-3 (SEQ ID NO:11),

[0036] zong-R:5-AGCAAGACTCATCGCAACCC-3 (SEQ ID NO:12) were respectively used as upstream and downstream primers respectively, and a PZL fragment was obtained according to conventional PCR set conditions. The sequence of the recombinant fragment PZL was shown in SEQ ID NO:13.

[0037] Embodiment 2 Construction of Recombinant P.sub.xylA-comk Fragment

[0038] By using a genome of Bacillus subtilis 168 as a template, homologous arm primers on both sides were designed, i.e., the left homologous arm primers of which the sequences are respectively SEQ ID NO:14 and SEQ ID NO:15:

TABLE-US-00006 yhzC-F: (SEQIDNO:14) 5-CATACATAGGAAGCAGGCATTGTTCATAAC-3, yhzC-R: (SEQIDNO:15) 5-atacgggatcaaatccgatgaaagagaaaaaatcgtacactga gctc-3,

[0039] and the right homologous arm primers of which the sequences are respectively SEQ ID NO:16 and SEQ ID NO:17:

TABLE-US-00007 comK-F: (SEQIDNO:16) 5-aagggggaaatgggatccatgagtcagaaaacagacgca cct-3, comK-R: (SEQIDNO:17) 5-ACTACCTCAGTTGAAGGCTATAATCCAAG-3.

[0040] The homologous arm gene sequences on both sides were amplified from the genome of the Bacillus subtilis by using the above primers. By using a plasmid pLCx-dcas9 as a template, primers of which the sequences are SEQ ID NO:18 and SEQ ID NO:19 were designed respectively:

TABLE-US-00008 P.sub.xylA-F: (SEQIDNO:18) 5-tttctctttcatcggatttgatcccgtataccgttcgtatagc atacattat-3, P.sub.xylA-R: (SEQIDNO:19) 5-gactcatggatcccatttccccctttgatttttagatatc ac-3.

[0041] An amplified fragment of a P.sub.xylA promoter with chloramphenicol resistance was obtained by using the above primers. The recombinant homologous arms and the promoter with the resistance gene were fused by fusion PCR. The conditions of the first round of PCR were: the total amount of DNA recovered by equimolar columns was greater than 1000 ng, the amount of primer star enzyme was 25 L, the volume was adjusted to 50 L with ddH.sub.2O, and the PCR conditions were 55 C., 11 cycles. The conditions of the second round of PCR were: the above PCR product was used as a template, the sequences as shown in SEQ ID NO:20 and SEQ ID NO:21:

TABLE-US-00009 zong-F: (SEQIDNO:20) 5-gacccgcgtccggtttttgataagccgaagaaag-3, zong-R: (SEQIDNO:21) 5-agtaaattcagttgcgcccggaccaatttggacac-3,

[0042] were respectively used as upstream and downstream primers respectively, and a P.sub.xylA-comk fragment was obtained according conventional PCR set conditions. The sequence of the fragment P.sub.xylA-comk was shown in SEQ ID NO:22.

[0043] Embodiment 3 Construction of p7S6P43-lgtB Fragment

[0044] By using a genome of Bacillus subtilis 168 as a template, homologous arm primers on both sides were designed, i.e., the left homologous arm primers of which the sequences are respectively SEQ ID NO:23 and SEQ ID NO:24:

TABLE-US-00010 ydeS-F: (SEQIDNO:23) 5-gggacaaggaatagtaagccggcaa-3, ydeS-R: (SEQIDNO:24) 5-tcctgtgtgaaattgttatccgctcctacatactctctgtagcagagg tagcttga,

[0045] and the right homologous arm primers of which the sequences are respectively SEQ ID NO:25 and SEQ ID NO:26:

TABLE-US-00011 ydzO-F: (SEQIDNO:25) 5-tgaagcccgcctaatgagcgggcttttttctgataagaactgcaaaag ctgcggattat-3, ydzO-R: (SEQIDNO:26) 5-ccaccctatagataaatttttcggctgccatat-3.

[0046] The homologous arm gene sequences on both sides were amplified from the genome of Bacillus subtilis by using the above primers.

[0047] By using a plasmid p7S6P43 as a template, primers of which the sequences are SEQ ID NO:27 and SEQ ID NO:28 were designed respectively:

TABLE-US-00012 p7S6P43-F: (SEQIDNO:27) 5-agagtatgtaggagcggataacaatttcacacagga-3, p7S6P43-R: (SEQIDNO:28) 5-AAAGAAATGACATGATTTTGCATgtgtacattcctctcttacctataa tggtaccgc-3.

[0048] By using a plasmid pP43NMK-lgtA-lgtB as a template, primers of which the sequences are SEQ ID NO:29 and SEQ ID NO:30 were designed respectively (disclosed in patent CN108410787A):

TABLE-US-00013 IgtB-F: (SEQIDNO:29) 5-aggtaagagaggaatgtacacATGCAAAATCATGTCATTTCTTTAGCA TCAGCAG-3, IgtB-R: (SEQIDNO:30) 5-cccgctcattaggcgggcttcatcaTTACTGAAACGGAACGATAAACT GTTCGCG-3.

[0049] By using the above primers, the obtained amplified fragments were verified to be correct by running the gel and then recovered by columns. Recombinant homologous arms, a -1,4-galactotransferase encoding gene and a promoter with the resistance gene were fused by fusion PCR. The conditions of the first round of PCR were: the total amount of DNA recovered by equimolar columns was greater than 1000 ng, the amount of primer star enzyme was 25 L, the volume was adjusted to 50 L with ddH.sub.2O, and the PCR conditions were 55 C., 11 cycles. The conditions of the second round of PCR were: the above PCR product was used as a template, the sequences as shown in SEQ ID NO:31 and SEQ ID NO:32:

[0050] zong-F:5-CGTATCCGATTTGGGTGAGTGTGA-3 (SEQ ID NO:31),

[0051] zong-R:5-TGATGTCAATCTAATGCCTCCTTACTGG-3 (SEQ ID NO:32) were respectively used as upstream and downstream primers respectively, and a p7S6P43-lgtB fragment was obtained according to conventional PCR set conditions. The sequence of the p7S6P43-lgtB fragment was shown in SEQ ID NO:33.

Embodiment 4 Construction of Recombinant Plasmid pP43NMK-lgtA

[0052] By using a recombinant plasmid pP43NMK-lgtA-lgtB as a template, reverse recombinant primers were designed. The sequences of the reverse recombinant primers were respectively SEQ ID NO:34 and SEQ ID NO:35:

TABLE-US-00014 fz-IgtA-F: (SEQIDNO:34) 5-tgaagcccgcctaatgagcgggcttttttaagcttggcgtaatcatgg tcatagctgtt-3, fz-IgtA-R: (SEQIDNO:35) 5-CTGTGTGAAATTGTTATCCGCTCCCAGCCTTTCTTATTAAAACCACTT TGTCAGCC-3.

[0053] After using the above primers to verify that the obtained amplified fragment is correct by running the gel, 10 L of PCR reaction system was taken to transform an Escherichia coli JM109 competent cell. By sequencing, it was verified that the recombinant plasmid pP43NMK-lgtA was successfully constructed, and the gene sequence of the recombinant plasmid pP43NMK-lgtA was shown in SEQ ID NO:36.

Embodiment 5 Construction of Recombinant p7Z6P43-lgtB Fragment

[0054] By using a genome of Bacillus subtilis 168 as a template, homologous arm primers on both sides were designed, i.e., the left homologous arm primers of which the sequences are respectively SEQ ID NO:37 and SEQ ID NO:38:

TABLE-US-00015 ydaH-F: (SEQIDNO:37) 5-GGCTTTCGTTGTCTTGTGTTCAAGAAATTTCCA-3, ydaH-R: (SEQIDNO:38) 5-CTGTGTGAAATTGTTATCCGCTCCCAGCCTTTCTTATTAAAACCACTT TGTCAGCC-3,

[0055] and the right homologous arm primers of which the sequences are respectively SEQ ID NO:39 and SEQ ID NO:40:

TABLE-US-00016 ydZA-F: (SEQIDNO:39) 5-AAGCCCGCCTAATGAGCGGGCTTTTTTATCATAAAGATCCAGCCTTTT TGCGCT-3, ydZA-R: (SEQIDNO:40) 5-CTAAAGCCCAAGTCACAATATATTGATCGCCT-3.

[0056] By using a plasmid p7Z6P43 as a template, primers of which the sequences are SEQ ID NO:41 and SEQ ID NO:42 were designed respectively:

TABLE-US-00017 p7Z6P43-F: (SEQIDNO:41) 5-GTTTTAATAAGAAAGGCTGGGAGCGGATAACAATTTCACACAGGAAAC AGC-3, p7Z6P43-R: (SEQIDNO:42) 5-AAAGAAATGACATGATTTTGCATGTGTACATTCCTCTCTTACCTATAA TGGTACCGC-3.

[0057] By using a genome of the starting strain Bacillus subtilis 168amyE:P.sub.43-lacY, P.sub.43-lgtB, P.sub.xylA-comK as a template, primers of which the sequences are SEQ ID NO:43 and SEQ ID NO:44 were respectively designed:

TABLE-US-00018 IgtB-1F: (SEQIDNO:43) 5-AGGTAAGAGAGGAATGTACACATGCAAAATCATGTCATTTCTTTAGCA TCAGCAG-3, IgtB-1R: (SEQIDNO:44) 5-CCCGCTCATTAGGCGGGCTTCATCATTACTGAAACGGAACGATAAACT GTTCGCG-3.

[0058] By using the above primers, the obtained amplified fragments were verified to be correct by running the gel and then recovered by columns. Recombinant homologous arms, a -1,4-galactotransferase encoding gene and a promoter with the resistance gene were fused by fusion PCR. The conditions of the first round of PCR were: the total amount of DNA recovered by equimolar columns was greater than 1000 ng, the amount of primer star enzyme was 25 L, the volume was adjusted to 50 L with ddH.sub.2O, and the PCR conditions were 55 C., 11 cycles. The conditions of the second round of PCR were: the above PCR product was used as a template, primers for fusion PCR were respectively SEQ ID NO:45 and SEQ ID NO:46:

[0059] zong-1F:5-AACCTCCTTGTCTTCTTTCCAGTCTTATCTC-3 (SEQ ID NO:45),

[0060] zong-1R:5-ATCACTTCTCTTTCTTTCACGCTCATCCT-3 (SEQ ID NO:46), and a p7Z6P43-lgtB fragment was obtained according to the set conditions of conventional PCR. The sequence of the p7Z6P43-lgtB fragment was shown in SEQ ID NO:47.

Embodiment 6 Construction of Recombinant p7C6P43-lgtB Fragment

[0061] By using a genome of Bacillus subtilis 168 as a template, homologous arm primers on both sides were designed, i.e., the left homologous arm primers of which the sequences are respectively SEQ ID NO:48 and SEQ ID NO:49:

TABLE-US-00019 yszA-F: (SEQIDNO:48) 5-CATCCAGCGTAAAACGTTCACGGGAATAATCTAGG-3, yszA-R: (SEQIDNO:49) 5-TTATCCGCTCTGGCGCGGACTTGTTTGTTTATATCCATTCTAAATGAA GG-3,

[0062] and the right homologous arm primers of which the sequences are respectively SEQ ID NO:50 and SEQ ID NO:51:

TABLE-US-00020 ysxE-F: (SEQIDNO:50) 5-GCCCGCCTAATGAGCGGGCTTTTTTTTAAGACGTGGACTCGTTTTCAG CCTGAAATTTT-3, ysxE-R: (SEQIDNO:51) 5-CATCCCAGCAGCTGATCAGGATGAATTCT-3.

[0063] By using a plasmid p7C6P43 as a template, primers of which the sequences are SEQ ID NO:52 and SEQ ID NO:53 were designed respectively:

TABLE-US-00021 p7C6P43-F: (SEQIDNO:52) 5-ACAAGTCCGCGCCAGAGCGGATAACAATTTCACACAGGAAACAGCTAT G-3, p7C6P43-R: (SEQIDNO:53) 5-AGAAATGACATGATTTTGCATGTGTACATTCCTCTCTTACCTATAATG GTACCGCT-3.

[0064] By using the genome of the starting strain as a template, primers of which the sequences are SEQ ID NO:54 and SEQ ID NO:55 were designed respectively:

TABLE-US-00022 IgtB-2F: (SEQIDNO:54) 5-ATAGGTAAGAGAGGAATGTACACATGCAAAATCATGTCATTTCTTTAG CATCAGCAGCG-3, IgtB-2R: (SEQIDNO:55) 5-GCCCGCTCATTAGGCGGGCTTCATCATTACTGAAACGGAACGATAAAC TGTTCGCGT-3.

[0065] By using the above primers, the obtained amplified fragments were verified to be correct by running the gel and then recovered by columns. Recombinant homologous arms, a -1,4-galactotransferase encoding gene and a promoter with the resistance gene were fused by fusion PCR. The conditions of the first round of PCR were: the total amount of DNA recovered by equimolar columns was greater than 1000 ng, the amount of primer star enzyme was 25 L, the volume was adjusted to 50 L with ddH.sub.2O, and the PCR conditions were 55 C., 11 cycles. The conditions of the second round of PCR were: the above PCR product was used as a template, primers for fusion PCR were respectively SEQ ID NO:56 and SEQ ID NO:57:

TABLE-US-00023 zong-2F: (SEQIDNO:56) 5-GCCAGGAAGCCACAGCACATCATAGC-3, zong-2R: (SEQIDNO:57) 5-TTCGTTCATTCGTTCCCGTTTACAGAACAAATAGC-3,
and a p7C6P43-lgtB fragment was obtained according to conventional PCR set conditions. The sequence of the p7C6P43-lgtB fragment was shown in SEQ ID NO:58.

Embodiment 7 Construction of Starting Strain Bacillus subtilis

[0066] A constructed recombinant fragment PxylA-comK was transformed into Bacillus subtilis competent cells (Bacillus subtilis 168). The addition amount of the recombinant fragment is 100-300 ng. The conditions for electrotransformation were: the voltage was 2.5 kV, the electroshock reagent was 5 ms, after performing recovery at 37 C. for 5 h, a chloramphenicol-resistant plate was coated, and culture was carried out at 37 C. for 24 h to obtain recombinant Bacillus subtilis BS168comk.

[0067] Competent cells were prepared from BS168comk recombinant Bacillus subtilis by xylose induction. After performing culture in an LB liquid medium at 37 C. and 220 rpm for 12 h, the OD600 was diluted to 1 with the LB liquid medium, xylose with a final concentration of 2% was added, and culture was continued to be carried out for 2 h to obtain the BS168comk competent cells. The addition amount of a recombinant fragment PZL is 500 ng. The cells were cultured at 37 C. for 1 h, and a bleomycin resistant plate was coated with the cells and cultured over night at 37 C. to obtain recombinant Bacillus subtilis BY00.

[0068] Competent cells were prepared from The BY00 by xylose induction. The addition amount of a recombinant fragment p7S6P43-lgtB was 500 ng. After performing culture at 37 C. for 1 h, a spectinomycin resistant plate was coated, and culture was carried out at 37 C. overnight to obtain recombinant Bacillus subtilis BY01. A resistance gene was knocked out by a Cre/lox recombination system to obtain BY01.2 bacteria.

[0069] Competent cells were prepared from the BY01.2 by xylose induction. The addition amount of the recombinant plasmid pP43NMK-lgtA was 500 ng. After performing culture at 37 C. for 1 h, a kanamycin resistant plate was coated, and culture was carried out at 37 C. overnight to obtain a strain BY03.

Embodiment 8

[0070] A constructed recombinant p7Z6P43-lgtB fragment was transformed into competent cells BY01.2. Transformants were selected for colony PCR verification, and primers for verification of primer sequences SEQ ID NO:59 and SEQ ID NO:60 were designed respectively:

[0071] YZ-1F:5-CGAAGGAATGCCGGTGTGCCAACTGAATC-3 (SEQ ID NO:59),

[0072] YZ-1R:5-CGACCTCCAGCGTGTATTGCTTGATGATGC-3 (SEQ ID NO:60), and recombinant Bacillus subtilis BY03a was obtained after verifying the primer sequences to be correct. The colony PCR agarose gel electrophoresis diagram is shown in FIG. 1.

[0073] A recombinant plasmid pP43NMK-lgtA was transformed into the recombinant Bacillus subtilis BY03a. After performing culture at 37 C. for 1 h, a kanamycin resistant plate was coated, and culture was carried out at 37 C. overnight to obtain recombinant Bacillus subtilis BY04.

[0074] A constructed recombinant p7C6P43-lgtB fragment was transformed into the recombinant Bacillus subtilis BY03a competent cells. Transformants were selected for colony PCR verification, and primers for verification of primer sequences SEQ ID NO:61 and SEQ ID NO:62 were designed respectively:

TABLE-US-00024 YZ-2F: (SEQIDNO:61) 5-GGGCACAGCGGGATACATCATCTCAAGAAA-3, YZ-2R: (SEQIDNO:62) 5-TGAACCACGGCAGCCTGTCTATACACCAT-3.

[0075] Recombinant Bacillus subtilis BY03b was obtained after verifying the primer sequences to be correct, and the obtained colony PCR agarose gel electrophoresis diagram is shown in FIG. 2.

[0076] A recombinant plasmid pP43NMK-lgtA was transformed into the recombinant Bacillus subtilis BY03b. After performing culture at 37 C. for 1 h, a kanamycin resistant plate was coated, and culture was carried out at 37 C. overnight to obtain recombinant Bacillus subtilis BY05 for synthesizing lacto-N-neotetraose.

[0077] Plasmids pLCx-dcas9, p7S6P43, p7Z6P43 and p7C6P43 used were from the Institute of Biological Engineering, Lab of Biosystem and Bioprocessing Engineering, No. 1800, Lihu Avenue, Binhu District, Wuxi, Jiangsu Province. The plasmids p7S6P43, p7Z6P43 and p7C6P43 have been disclosed in Modular pathway engineering of key carbon-precursor supply-pathways for improved N-acetylnuraminic acid production in Bacillus subtilis (Zhang X L, Liu Y F, Liu L, Wang M, Li J H, Du G C, Chen J. 2018). The plasmid pLCx-dcas9 has been disclosed in CRISPRi allows optimal temporal control of Nacetylglucosamine bioproduction by a dynamic coordination of glucose and xylose metabolism in Bacillus subtilis (Wu Y K, Chen T C, Liu Y F, Lv X Q, Li J H, Du G C, Amaro R L, Liu L. 2018).

Embodiment 9 Production of Lacto-N-Neotetraose from Recombinant Bacillus subtilis by Fermentation

[0078] The recombinant Bacillus subtilis BY05 in Embodiment 8 was prepared into a seed solution. A preparation method of the seed solution was: a single colony on the plate was selected and placed in a seed culture medium (the seed culture medium contains tryptone 10 g/L, yeast powder 5 g/L, and NaCl 10 g/L). Meanwhile, kanamycin with a final concentration of 30 g/mL was added, and culture was carried out at 37 C. and 220 rpm for 10-12 h.

[0079] The seed solution was transferred into a fermentation medium according to an inoculation amount of 10% (the fermentation medium contains tryptone 6 g/L, yeast powder 12 g/L, (NH.sub.4)SO.sub.4 6 g/L, K.sub.2HPO.sub.4.3H.sub.2O 12.5 g/L, KH.sub.2PO.sub.4 2.5 g/L, MgSO.sub.4.7H.sub.2O 1.2 g/L, glucose 60 g/L, and lactose 5 g/L), and culture was carried out at 37 C. and 220 rpm for 48 h. Lacto-N-neotetraose was detected in the supernatant of a fermentation broth, and the content was 1300 mg/L. The yield was improved by 80.6% compared with the strain (Bacillus subtilis 168amyE:P.sub.43-lacY, P.sub.43-lgtB, PxylA-comK, pP43NMK-lgtA), efficient synthesis of lacto-N-neotetraose from recombinant Bacillus subtilis was realized, and a foundation was laid for further metabolic engineering transformation of Bacillus subtilis to produce lacto-N-neotetraose and complicated oligosaccharides with the lacto-N-neotetraose as a core element.

Comparative Embodiment 1 Production of Lacto-N-Neotetraose from BY03 and BY04 Strains by Fermentation

[0080] Bacillus subtilis 168amyE:P.sub.43-lacY, P.sub.43-lgtB, P.sub.xylA-comK, pP43NMK-lgtA and recombinant Bacillus subtilis BY04 were prepared into a seed solution. The preparation method of the seed solution was: a single colony on the plate was selected and placed in a seed culture medium. Meanwhile, kanamycin with a final concentration of 30 g/mL was added, and culture was carried out at 37 C. and 220 rpm for 10-12 h.

[0081] The seed solution was transferred into a fermentation medium according to an inoculation amount of 10% and culture was carried out at 37 C. and 220 rpm for 48 h. Lacto-N-neotetraose was detected in the supernatant of a fermentation broth, the content of BY03 in the supernatant was about 720 mg/L, and the content of the recombinant Bacillus subtilis BY04 in the supernatant was about 1090 mg/L. The yield of recombinant Bacillus subtilis BY04 was 51.3% higher than that of the starting strain (Bacillus subtilis 168amyE:P.sub.43-lacY, P43-lgtB, P.sub.xylA-comK, pP43NMK-lgtA), and was 16% lower than the yield of lacto-N-neotetraose in BY05 which was 1300 mg/L, as shown in FIG. 3. It is known that a genome of BY05 bacteria has 2 copy numbers of -1,4-galactotransferase encoding genes, and the genome of the BY05 bacteria having 3 copy numbers of -1,4-galactotransferase encoding genes indicates that increasing the copy number of key genes effectively improves the synthesis efficiency of lacto-N-neotetraose from recombinant Bacillus subtilis.

[0082] Although the disclosure has been disclosed above with the preferred embodiments, it is not intended to limit the disclosure. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the disclosure. Therefore, the scope of the disclosure should be determined by the scope of the claims.