Genetically Engineered Bacteria Producing Lacto-N-neotetraose and Production Method Thereof

Abstract

The disclosure discloses genetically engineered bacteria producing lacto-N-neotetraose and a production method thereof, and belongs to the fields of metabolic engineering and food biotechnology. To solve the problem of low yield of lacto-N-neotetraose produced by a microbial method in the prior art, through exogenous expression of lgtA and lgtB, reasonable combination and regulation of overexpression of lacY, pgm, galE, galT and galK in a lacto-N-neotetraose synthesis pathway, knockout of lacZ expression in an Escherichia coli host, and optimization of a carbon source in the culture process, the disclosure achieves the objectives of regulating the carbon flux of a metabolic pathway and improving the yield of lacto-N-neotetraose. In a shake flask experiment, the yield of lacto-N-neotetraose produced by E. coli increased from 304 mg/L to 1031 mg/L, laying a foundation for industrial production of the lacto-N-neotetraose.

Claims

1. Genetically engineered bacteria producing lacto-N-neotetraose, wherein in the genetically engineered bacteria, β-galactosidase gene lacZ is knocked out, and β-1,3-acetylglucosamine transferase gene lgtA, β-1,4-galactosyltransferase gene lgtB, phosphoglucomutase gene pgm, UDP-glucose-4-epimerase gene galE, galactose-1-phosphate uridyltransferase gene galT, galactokinase gene galK and β-galactoside permease gene lacY are overexpressed.

2. The genetically engineered bacteria according to claim 1, wherein the β-1,3-acetylglucosamine transferase gene lgtA and β-1,4-galactosyltransferase gene lgtB are both from Neisseria meningitidis; the nucleotide sequence of β-1,3-acetylglucosamine transferase gene lgtA is set forth in SEQ ID NO: 1; and the nucleotide sequence of β-1,4-galactosyltransferase gene lgtB is set forth in SEQ ID NO: 2.

3. The genetically engineered bacteria according to claim 1, wherein the phosphoglucomutase gene pgm, UDP-glucose-4-epimerase gene galE, galactose-1-phosphate uridyltransferase gene galT, galactokinase gene galK, β-galactoside permease gene lacY and β-galactosidase gene lacZ are all from Escherichia coli K-12; the nucleotide sequence of phosphoglucomutase gene pgm is set forth in SEQ ID NO: 24, the nucleotide sequence of UDP-glucose-4-epimerase gene galE is set forth in SEQ ID NO: 25, the nucleotide sequence of galactose-1-phosphate uridyltransferase gene galT is set forth in SEQ ID NO: 26, the nucleotide sequence of galactokinase gene galK is set forth in SEQ ID NO: 27, the nucleotide sequence of β-galactoside permease gene lacY is set forth in SEQ ID NO: 28, and the nucleotide sequence of β-galactosidase gene lacZ is set forth in SEQ ID NO: 23.

4. The genetically engineered bacteria according to claim 1, wherein the genetically engineered bacteria express the gene lacY with pETDuet-1 plasmid, express the genes pgm, galE, galT and galK sequentially with pRSFDuet-1 plasmid, and express the genes lgtA and lgtB sequentially with pCDFDuet-1 plasmid.

5. A method of use of the genetically engineered bacteria of claim 1 for producing lacto-N-neotetraose, comprising the following steps: (1) culturing the genetically engineered bacteria in a seed medium to obtain a seed solution; (2) inoculating the seed solution into a fermentation system and culturing the seed solution until the OD.sub.600 is 0.6-0.8; and (3) adding IPTG with a final concentration of 0.4 mM and lactose with a final concentration of 8 g/L-10 g/L for performing induction to obtain a fermentation broth containing lacto-N-neotetraose.

6. The method according to claim 5, wherein carbon source in the fermentation system is one or more of glucose, galactose and glycerin.

7. The method according to claim 6, wherein the carbon source is 20 g/L glycerin, or a mixture of 10 g/L glycerin and 10 g/L galactose; and the fermentation system further contains 13.5 g/L potassium dihydrogen phosphate, 4.0 g/L diammonium hydrogen phosphate, 1.7 g/L citric acid, 1.4 g/L magnesium sulfate heptahydrate and 10 ml/L trace metal elements, the trace metal elements comprising 10 g/L ferrous sulfate, 2.25 g/L zinc sulfate heptahydrate, 1.0 g/L anhydrous copper sulfate and 2.0 g/L calcium chloride dihydrate.

8. The method according to claim 5, wherein the seed medium in the method is an LB liquid medium, and the seeds are cultured at 35° C.-40° C. and 200 rpm-250 rpm for 10-14 hours in a shake flask.

9. The method according to claim 5, wherein the seed solution is inoculated into the fermentation system and is cultured at 35° C.-40° C. and 200 rpm-250 rpm until the OD.sub.600 is 0.6-0.8, and culture is induced at 22° C.-30° C. and 200 rpm-250 rpm for 42-48 hours.

Description

BRIEF DESCRIPTION OF FIGURES

[0021] FIG. 1 shows a diagram of the metabolic pathway of lacto-N-neotetraose.

[0022] FIG. 2A shows a secondary mass spectrogram of a lacto-N-neotetraose standard sample.

[0023] FIG. 2B shows a secondary mass spectrogram of a lacto-N-neotetraose product sample.

DETAILED DESCRIPTION

[0024] The specific implementation of the disclosure is further described in the following examples and figures. Plasmids, PCR reagents, restriction endonucleases, plasmid extraction kits, DNA gel extraction kits, and the like used in the following examples are commercial products, and are specifically operated according to the kit instructions. The embodiments of the disclosure are not limited thereto, and other unspecified experimental operations and process parameters are carried out according to conventional technology.

[0025] Plasmids and DNA products are sequenced by Talen Biotechnology (Shanghai) Co., Ltd.

[0026] E. coli competent cells are prepared using a kit of Shanghai Sangon Biotechnology Co., Ltd.

[0027] An LB liquid medium contains 10 g/L peptone, 5 g/L yeast extract, and 10 g/L sodium chloride.

[0028] An LB solid medium contains 10 g/L peptone, 5 g/L yeast extract powder, 10 g/L sodium chloride, and 15 g/L agar powder.

[0029] The lacto-N-neotetraose in the embodiments of the disclosure is measured by HPLC, specifically:

[0030] 1 mL of fermentation broth is boiled at 100° C. for 10 min and centrifuged at 13400 rpm for 10 min, the supernatant is filtered with a 0.22 μM membrane, and the yield of lacto-N-neotetraose is detected by HPLC. HPLC detection uses a differential refraction detector, the chromatographic column is Rezex ROA organic acid (Phenomenex, USA), the column temperature is 50° C., the mobile phase is a 5 mM H.sub.2SO.sub.4 aqueous solution, the flow rate is 0.6 mL/min, and the injection volume is 10 μL.

Example 1 Knockout of Gene lacZ from Genome of E. coli BL21 (DE3)

[0031] LacZ was knocked out from E. coli BL21 (lacZ has a gene ID of 945006 and is shown in SEQ ID NO: 23) using a CRISPR-Cas9 gene knockout system by the following specific steps (the primer sequences involved are shown in Table 1): [0032] (1) The upstream and downstream fragments of lacZ were amplified by PCR using lacZ-up-FIR and lacZ-down-F/R respectively and the genome of E. coli BL21 as a template, and a DNA fragment was extracted from gel. Then a complete lacZ template was obtained by overlapping PCR using a lacZ-up-F/lacZ-down-R primer and the lacZ upstream and downstream fragments as templates respectively, and a DNA fragment was extracted from gel. [0033] (2) An N20 sequence on an original plasmid was replaced with an N20 sequence complementary to the lacZ sequence by PCR amplification using the original pTargetF plasmid as a template and lacZ-sg-F/R as a primer, and a pTargetF plasmid targeting lacZ was obtained (the constructed plasmid was named pTargetF-lacZ, and the targeting plasmid information was a targeting plasmid pTargetF with the lacZ specific N20 sequence). The targeting plasmid was transformed into E. coli DH5a competent cells, spread on an LB plate (containing spectinomycin), cultured in a large scale at 37° C., extracted and sequenced. [0034] (3) PCas plasmids and E. coli BL21 competent cells were placed on ice for 5 min until the competent cells melted. 5 μL of plasmids were added to 100 μL of competent cells, gently mixed, placed in an ice bath for 30 min, thermally activated at 42° C. for 90 s, and immediately placed on ice for 5 min. The mixture was added to 1 mL of LB medium and cultured at 30° C. and 180 rpm for 1 h. 200 μL of concentrated bacterial solution was taken and evenly spread on an LB plate (containing kanamycin), and cultured upside down at 30° C. overnight until a single colony of E. coli BL21/pCas grows. [0035] (4) The single colony of E. coli BL21/pCas was picked and cultured in an LB medium at 30° C. for 1.0 h, and L-arabinose with a final concentration of 30 mM was added to induce expression of a pCas-λ-red system. When the OD.sub.600 reached 0.6-0.8, E. coli BL21/pCas competent cells were prepared. [0036] (5) 400 ng of pTargetF plasmids and 1000 ng of donor DNA fragments (i.e., lacZ template fragments obtained in step 1) were electro-transformed into the E. coli BL21/pCas competent cells obtained in step (4), spread on an LB plate (containing kanamycin and spectinomycin), and cultured at 30° C. for 24 h. Positive colonies on the plate were picked, cultured in an LB medium for 10 h, and sent to Talen Biotechnology (Shanghai) Co., Ltd. for sequencing verification. [0037] (6) The positive cloned colonies obtained in step (5) were picked into a 4 ml LB liquid test tube, IPTG with a final concentration of 1 mM and 30 mg/L kanamycin were added, and the cells were cultured at 30° C. for 8-16 h to remove the pTargetF plasmids, and then cultured at 42° C. for 12 h to remove the pCas plasmids.

TABLE-US-00001 TABLE 1 Primer sequences for lacZ knockout Primer name Sequence NO. Primer sequences (5′-3′) lacZ-sg-F SEQ ID NO: 3 TCGTTTTACAACGTCGTGACGTTTTAGAGCTAGAAATAGCAAGTT lacZ-sg-R SEQ ID NO: 4 GTCACGACGTTGTAAAACGAACTAGTATTATACCTAGGACTGAGC lacZ-up-F SEQ ID NO: 5 TGAATGAGGGCATCGTTCCCACTGCGA lacZ-up-R SEQ ID NO: 6 CTCGAGAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTTCA CACAAC lacZ-down-F SEQ ID NO: 7 CCATTACGGTCAATCCGCCGT lacZ-down-R SEQ ID NO: 8 ACGCTCATCGATAATTTCACCGCCGAAAGGCGCGG

Example 2 Construction of Recombinant Bacteria and Screening of Plasmid Combinations

[0038] Recombinant bacteria are constructed specifically by the following steps (the primer sequences involved are shown in Table 2): [0039] (1) Acquisition of lacY gene fragment (the Gene ID of lacY is 949083): A lacY gene fragment was amplified by PCR using the genome of E. coli K-12 as a template and lacY-F/lacY-R as a primer, and a DNA fragment was extracted from gel. The extracted DNA gene fragment was ligated to the BamHI and SalI restriction sites of the vector pETDuet-1 through a seamless cloning kit (Nanjing Vazyme Biotech Co., Ltd), and finally the plasmid pET-lacY was acquired. [0040] (2) Acquisition of pgm, galE, galE-galT and galE-galT-galK gene fragments (the Gene ID of pgm is 945271, the Gene ID of galE is 945354, the Gene ID of galT is 945357 and the Gene ID of galK is 945358): A pgm gene fragment was amplified by PCR using the genome of E. coli K-12 as a template and pgm-F/pgm-R as a primer, and a DNA fragment was extracted from gel; a galE gene cluster fragment was amplified by PCR using galE-F/galE-R as a primer, and a DNA fragment was extracted from gel; a galE-galT gene cluster fragment was amplified by PCR using galET-F/galET-R as a primer, and a DNA fragment was extracted from gel; and a galE-galT-galK gene cluster fragment was amplified by PCR using galETK-F/galETK-R as a primer, and a DNA fragment was extracted from gel. The extracted DNA gene fragments were ligated to the BamHI, SalI, BgiII and XhoI restriction sites of the vector pRSFDuet-1 respectively through a seamless cloning kit (Nanjing Vazyme Biotech Co., Ltd), and finally the plasmids pRSF-pgm, pRSF-pgm-galE, pRSF-pgm-galET and pRSF-pgm-galETK were acquired. [0041] (3) Acquisition of lgtA and lgtB gene fragments: The gene sequences of lgtA and lgtB (the nucleotide sequence of lgtA is shown in SEQ ID NO: 1, and the nucleotide sequence of lgtB is shown in SEQ ID NO: 2) of N. meningitidis were found out and synthesized by entrusting Talen Biotechnology (Shanghai) Co., Ltd. By means of the BamHI, SalI, BgiII and XhoI restriction sites, the synthesized gene fragment was ligated to the BamHI, SalI, BgiII and XhoI restriction sites of the vector pCDFDuet-1 through a seamless cloning kit (Nanjing Vazyme Biotech Co., Ltd), and finally the plasmid pCDF-lgtA-lgtB was acquired.

TABLE-US-00002 TABLE 2 Primers for plasmid construction Primer name Sequence NO. Primer sequences (5′-3′) lacY-F SEQ ID NO: 9 TCATCACCACAGCCAGGATCCAATGTACTATTTAAAAAACACAAACTTTTGG lacY-R SEQ ID NO: 10 TGCGGCCGCAAGCTTGTCGACTTAAGCGACTTCATTCACCTGACG pgm-F SEQ ID NO: 11 TCATCACCACAGCCAGGATCCAATGGCAATCCACAATCGTGCAGGC pgm-R SEQ ID NO: 12 TGCGGCCGCAAGCTTGTCGACTTACGCGTTTTTCAGAACTTCGC galE-F SEQ ID NO: 13 TCATCACCACAGCCAGGATCCAATGAGAGTTCTGGTTACCGGTGG galE-R SEQ ID NO: 14 TGCGGCCGCAAGCTTGTCGACTTAATCGGGATATCCCTGTGG galET-F SEQ ID NO: 15 TCATCACCACAGCCAGGATCCAATGAGAGTTCTGGTTACCGGTGG galET-R SEQ ID NO: 16 TGCGGCCGCAAGCTTGTCGACTTACACTCCGGATTCGCGAAAATGG galETK-F SEQ ID NO: 17 TCATCACCACAGCCAGGATCCAATGAGAGTTCTGGTTACCGGTGG galETK-R SEQ ID NO: 18 TGCGGCCGCAAGCTTGTCGACTCAGCACTGTCCTGCTCCTTGTGA lgtA-F SEQ ID NO: 19 TCATCACCACAGCCAGGATCCAATGGGCCAGCCGCTGGT lgtA-R SEQ ID NO: 20 TGCGGCCGCAAGCTTGTCGACTTAACGGTTTTTCAGCAGACGG lgtB-F SEQ ID NO: 21 AGATATACATATGGCAGATCTAATGCAAAATCACGTCATTAGTCTTG lgtB-R SEQ ID NO: 22 GGTTTCTTTACCAGACTCGAGTTAATGGTGGTGATGATGATGCTG [0042] (4) The plasmids pET-lacY, pRSF-pgm, pRSF-pgm-galE, pRSF-pgm-galET, pRSF-pgm-galETK and pCDF-lgtA-lgtB acquired by the above steps were combined with different number of key genes in the synthetic pathway of lacto-N-neotetraose, and 6 different engineered strains represented as A0, A1, A2, A3, A4 and A5 respectively were obtained (see Table 3).

[0043] After fermentation, the yields of lacto-N-neotetraose of different engineered strains were 304 mg/L, 508 mg/L, 595 mg/L, 654 mg/L, 749 mg/L and 837 mg/L respectively. Compared with A0, the yield of lacto-N-neotetraose of strain A5 increased by 175.3%. Therefore, the expression of the endogenous genes pgm and galE-galT-galK of E. coli related to UDP-galactose synthesis can increase the yield of lacto-N-neotetraose. The metabolic pathway of lacto-N-neotetraose of the strain A5 is shown in FIG. 1.

[0044] The fermentation method was as follows: the constructed genetically engineered strains A0-A5 were inoculated into LB liquid media, and the strains were cultured at 37° C. and 200 rpm for 12 h in a shake flask to obtain seed solutions; the seed solutions were inoculated into 50 ml of fermentation media at an inoculation volume of 2 mL/100 mL, and the seed solutions were cultured at 37° C. and 200 rpm in a shake flask until the OD.sub.600 was 0.6; and IPTG with a final concentration of 0.4 mM was added, lactose was added until the lactose concentration was 10 g/L, and culture was induced at 25° C. and 200 rpm for 48 h to obtain the fermentation broths.

[0045] The above fermentation medium contains 20 g/L glycerin, 13.5 g/L potassium dihydrogen phosphate, 4.0 g/L diammonium hydrogen phosphate, 1.7 g/L citric acid, 1.4 g/L magnesium sulfate heptahydrate and 10 ml/L trace metal elements, the trace metal elements including 10 g/L ferrous sulfate, 2.25 g/L zinc sulfate heptahydrate, 1.0 g/L anhydrous copper sulfate and 2.0 g/L calcium chloride dihydrate, and the pH of the fermentation medium being 6.8.

TABLE-US-00003 TABLE 3 Details of engineered strains Yield of lacto-N- Strain neotetraose name Plasmids and genotypes contained in the host (mg/L) A0 E. coli BL21 (DE3) with lacZ gene knocked out, 304 containing plasmid pCDF-IgtA-IgtB A1 E. coli BL21 (DE3) with lacZ gene knocked out, 508 containing plasmids pCDF-IgtA-IgtB and pET-lacY A2 E. coli BL21 (DE3) with lacZ gene knocked out, 595 containing plasmids pCDF-IgtA-IgtB, pET-lacY and pRSF-pgm A3 E. coli BL21 (DE3) with lacZ gene knocked out, 654 containing plasmids pCDF-IgtA-IgtB, pET-lacY and pRSF-pgm-galE A4 E. coli BL21 (DE3) with lacZ gene knocked out, 749 containing plasmids pCDF-IgtA-IgtB, pET-lacY and pRSF-pgm-galE-galT A5 E. coli BL21 (DE3) with lacZ gene knocked out, 837 containing plasmids pCDF-IgtA-IgtB, pET-lacY and pRSF-pgm-galE-galT-galK

Example 3: Screening of Different Combinations of Carbon Sources for Producing Lacto-N-Neotetraose

[0046] The engineered strain A5 with high lacto-N-neotetraose yield obtained in Example 2 was fermented in fermentation media with different combinations of carbon sources by the following specific fermentation method:

[0047] The constructed genetically engineered strain A5 was inoculated into an LB liquid medium, and the strain was cultured at 37° C. and 200 rpm for 12 h in a shake flask to obtain a seed solution; the seed solution were inoculated into 50 ml of fermentation media at an inoculation volume of 2 mL/100 mL, and the seed solution was cultured at 37° C. and 200 rpm in a shake flask until the OD.sub.600 was 0.6; and IPTG with a final concentration of 0.4 mM was added, lactose was added until the lactose concentration was 10 g/L, and culture was induced at 25° C. and 200 rpm for 48 h to obtain the fermentation broths.

[0048] The above fermentation media contain carbon sources (see Table 4 for combinations and contents of the carbon sources), 13.5 g/L potassium dihydrogen phosphate, 4.0 g/L diammonium hydrogen phosphate, 1.7 g/L citric acid, 1.4 g/L magnesium sulfate heptahydrate and 10 ml/L trace metal elements, the trace metal elements including 10 g/L ferrous sulfate, 2.25 g/L zinc sulfate heptahydrate, 1.0 g/L anhydrous copper sulfate and 2.0 g/L calcium chloride dihydrate, and the pH of the fermentation media being 6.8.

[0049] Table 4 shows the results of the yields of lacto-N-neotetraose of the strain A5 in the fermentation media of different combinations of carbon sources. From the table, when the combination of carbon sources in the medium was 10 g/L galactose+10 g/L glycerin, the yield of lacto-N-neotetraose was increased by 23.2% compared to that fermented by using 20 g/L glycerin as the carbon source at the very start.

TABLE-US-00004 TABLE 4 Details of combinations of carbon sources in the media Yield of lacto-N- neotetraose Carbon source composition in the media (mg/L) 20 g/L Glycerin 837 20 g/L Galactose 642 20 g/L Glucose 402 10 g/L Glycerin 701 10 g/L Galactose 794 10 g/L Glucose 540 10 g/L Glycerin + 10 g/L Glucose 217 10 g/L Galactose + 10 g/L Glycerin 1031 10 g/L Glucose + 10 g/L Galactose 161

[0050] Although the disclosure has been disclosed as above in preferred embodiments, it is not intended to limit the disclosure. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the disclosure. Therefore, the protection scope of the disclosure should be defined by the claims.