Transcription factor SugR coding gene, and use thereof in production of N-acetylglucosamine

Abstract

The present invention provides a recombinant Corynebacterium glutamicum producing N-acetylglucosamine and use thereof. The recombinant Corynebacterium glutamicum is obtained by overexpressing, in Corynebacterium glutamicum, the transcription factor SugR derived therefrom. The recombinant Corynebacterium glutamicum of the present invention increases the production of acetylglucosamine to up to 26 g/L, and lays a foundation for further metabolic engineering of Corynebacterium glutamicum to produce glucosamine.

Claims

1. A recombinant Corynebacterium glutamicum producing N-acetylglucosamine, wherein the recombinant Corynebacterium glutamicum expresses a transcription factor SugR, wherein said recombinant Corynebacterium glutamicum comprises the expression vector pJYW-4-ceN-C.glglmS-SugR, and wherein said transcription factor SugR is encoded by said expression vector.

2. The recombinant Corynebacterium glutamicum according to claim 1, wherein the acetylglucosamine deacetylase coding gene NagA, the acetylglucosamine deaminase coding gene GamA and the L-lactate dehydrogenase coding gene ldh in the Corynebacterium glutamicum are knocked out.

3. The recombinant Corynebacterium glutamicum according to claim 1, wherein the Corynebacterium glutamicum is C. glutamicum S9114 ΔnagA-ΔgamA-Δldh.

4. A method for constructing a recombinant Corynebacterium glutamicum according to claim 1, comprising a step of: transforming a host bacterium with the expression vector pJYW-4-ceN-C.glglmS-SugR to obtain the recombinant Corynebacterium glutamicum producing N-acetylglucosamine, wherein the host bacterium is Corynebacterium glutamicum in which the acetylglucosamine deacetylase coding gene NagA, the acetylglucosamine deaminase coding gene GamA, and the L-lactate dehydrogenase coding gene ldh are knocked out.

5. The method according to claim 4, wherein the host bacterium is C. glutamicum S9114 ΔnagA-ΔgamA-Δldh, which is constructed by the steps of: knocking out the acetylglucosamine deacetylase coding gene NagA, the acetylglucosamine deaminase coding gene GamA and the L-lactate dehydrogenase coding gene ldh in C. glutamicum S9114 sequentially by using a gene knockout frame of the acetylglucosamine deacetylase coding gene NagA, a gene knockout frame of the acetylglucosamine deaminase coding gene GamA, and a gene knockout frame of the L-lactate dehydrogenase coding gene ldh.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the map of pJYW-4-ceN-C.glglmS plasmid.

(2) FIG. 2 shows the map of the constructed recombinant pJYW-4-ceN-C.glglmS-SugR plasmid.

(3) FIG. 3 shows the GlcNAc production in supernatants of shake flask fermentation with different strains.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

(5) (1) In the following examples of the present invention, the determination method of N-acetylglucosamine is as follows:

(6) High performance liquid chromatography (HPLC): Agilent 1260, RID detector, HPX-87H column (Bio-Rad Hercules, Calif.), mobile phase: 5 mM H.sub.2SO.sub.4, flow rate 0.6 mL/min, column temperature 35° C., injection volume 10 μL.

(7) (2) In the following examples of the present invention, the culture media used are as follows:

(8) Liquid seed activation medium (LBG)(g/L): peptone 10.0, yeast extract 5.0, NaCl 10.0, glucose 5.0, where the filling volume is 20 ml per 250 ml Erlenmeyer flask.

(9) Solid seed activation medium (LBG solid) (g/L): peptone 10.0, yeast powder 5.0, NaCl 10.0, glucose 5.0, nutrient agar 15.0-20.0.

(10) Competent medium (g/L): peptone 10.0, yeast extract 5.0, NaCl 10.0, glycine 30.0, isoniazid 4.0, and 10 ml of Tween 80, where the filling volume is 50 ml per 500 ml Erlenmeyer flask.

(11) Recovery medium after electroporation LBHIS (g/L): peptone 5.0, yeast extract 2.5, NaCl 5.0, brain heart infusion 18.5, sorbitol 91.0.

(12) Solid medium for transformant detection (g/L): peptone 5.0, yeast extract 2.5, NaCl 5.0, brain heart infusion 18.5, sorbitol 91.0, nutrient agar 15.0-20.0.

(13) Seed culture medium (g/L): glucose 25.0, corn steep liquor 20.0, KH.sub.2PO.sub.4 1.0, (NH.sub.4).sub.2SO.sub.4 0.5, urea 1.25, pH 7.0.

(14) Fermentation medium (g/L): glucose 40.0, corn steep liquor 20.0, KH.sub.2PO.sub.4 1.0, (NH.sub.4).sub.2SO.sub.4 20.0, MgSO.sub.4 0.5, CaCO.sub.3 20.0, pH 7.0.

(15) Optimized fermentation medium (g/L): glucose 100.0, corn steep liquor 10.0, KH.sub.2PO.sub.4 1.0, (NH.sub.4).sub.2SO.sub.4 20.0, MgSO.sub.4 0.5, CaCO.sub.3 20.0, FeSO.sub.4 0.18, pH 7.0.

(16) Sterilization conditions: 115° C., 20 min. 25 mg/L Kanamycin sulfate is added to all media for transformant detection or recombinant culture.

Example 1: Knockout of L-Lactate Dehydrogenase Coding Gene (Ldh)

(17) According to the upstream and downstream sequences of L-lactate dehydrogenase coding gene (ldh) (having a nucleotide sequence as shown in SEQ ID NO:1) in Corynebacterium glutamicum ATCC 13032 published on NCBI, amplification primers for knocking out the homologous arms were designed. The upstream and downstream primers for the left arm were respectively LdhloxPUF (having a nucleotide sequence as shown in SEQ ID NO:2) and LdhloxPUR (having a nucleotide sequence as shown in SEQ ID NO:3). The upstream and downstream primers for the right arm were respectively LdhloxPDF (having a nucleotide sequence as shown in SEQ ID NO:4) and LdhloxPDR (having a nucleotide sequence as shown in SEQ ID NO:5). By using the genome DNA of Corynebacterium glutamicum strain 59114 as a template, the left and right arms were respectively amplified by PCR.

(18) The primers KanloxpldhF (having a nucleotide sequence as shown in SEQ ID NO:6) and KanloxpldhR (having a nucleotide sequence as shown in SEQ ID NO:7) were designed according to the nucleotide sequence of loxp-kana-loxp gene on the plasmid pDTW-202 (provided by Dr. Wang Xiaoyuan of Jiangnan University), and by using the plasmid pDTW-202 as a template, the loxp gene and kanamycin resistance gene were amplified for loxp-kana-loxp gene. By restriction enzyme cleavage and ligation, the left arm after enzymatic cleavage with the fast cleavage enzyme XhoI/XbaI for 2 hours, the loxp-kana-loxp gene fragment after enzymatic cleavage with the fast cleavage enzyme XbaI/BamHI for 2 hours, the right arm after enzymatic cleavage with the fast cleavage enzyme BamHI/EcoRI for 2 hours, and the plasmid pBluescriptIISK (+) after enzymatic cleavage with the fast cleavage enzyme XhoI/EcoRI for 2 hours (provided by Dr. Xiaoyuan Wang of Jiangnan University) were ligated overnight with T4 ligase at 16° C.

(19) The constructed pBluescriptIISK (+) ligated system with the ldh knockout frame was transformed into E. coli JM109 competent cells (see the instruction of Takara Preparation Kit for Competent Escherichia coli for the preparation method; article number: 9128). The transformant that was confirmed to be correct by colony PCR was sequenced for verification, to obtain the recombinant plasmid pBluescriptIISK (+)-ldh. The recombinant plasmid pBluescriptIISK (+)-ldh was extracted and electroporated into Corynebacterium glutamicum S9114-ΔNagA-GamA. The cells were screened in a plate for kanamycin resistance, and verified by colony PCR. It was confirmed that both the left and right arms of the knockout frame were bound to the 59114 genome, and the L-lactate dehydrogenase coding gene ldh was knocked out to obtain Corynebacterium glutamicum S9114-ΔNagA-GamA-Δldh. After 72 h, the GlcNAc production of this strain was 24.7 g/L.

Example 2: Construction of Recombinant Plasmid pJYW-4-ceN-C.glglmS-SugR and Construction of Recombinant Corynebacterium glutamicum

(20) (1) Amplification Primers were Designed According to the Genome of S9114 to Amplify SugR.

(21) Upstream primer FragmentSugR.FOR:

(22) TABLE-US-00005 5′--CCGTCGAATAAAAGAAATTCGGACATATTTAGTAAATTGGC TTTT--3′

(23) Downstream primer FragmentSugR.REV:

(24) TABLE-US-00006 5′--CTTTGCTAGTCGGACTTGCAGTGACTGTAAGAATCA--3′

(25) Primers for linearizion of the vector pJYW-4-ceN-C.glglmS was also designed. Upstream primer VectorSugR.FOR:

(26) TABLE-US-00007 5′--TGCAAGTCCGACTAGCAAAGGAGAAGAAAAGCCG--3′

(27) Downstream primer VectorSugR.REV:

(28) TABLE-US-00008 5′--TCCGAATTTCTTTTATTCGACGGTGACAGACTTTGC--3′

(29) Primers FragmentSugR.FOR and FragmentSugR.REV were used, and the laboratory-preserved Corynebacterium glutamicum S9114 was used as a template. PCR conditions: pre-denaturation at 95° C. for 10 min; 30 cycles of denaturation at 98° C. for 1 min, annealing at 55° C. for 1 min, and extension at 72° C. for 1 min; and final extension at 72° C. for 10 min. The PCR product was recovered with a DNA purification kit. The SugR gene was amplified from the genome of Corynebacterium glutamicum S9114, and the SugR gene was amplified by LA Taq HS DNA polymerase.

(30) The plasmid pJYW-4-ceN-C.glglmS previously constructed in the laboratory was used as an expression vector to express the SugR gene, and the specific construction process of pJYW-4-ceN-C.glglmS plasmid was as described in Chen Deng, XueqinLv, Yanfeng Liu, Long Liu. Metabolic engineering of Corynebacterium glutamicum S9114 based on whole-genome sequencing for efficient N-acetylglucosamine synthesis. Synthetic and Systems Biotechnology, 2019. 4: 120-129.

(31) Primers VectorSugR.FOR and VectorSugR.REV were used, and the extracted plasmid pJYW-4-ceN-C.glglmS was used as a template. PCR conditions: pre-denaturation at 95° C. for 3 min; 30 cycles of denaturation at 98° C. for 1 min, annealing at 55° C. for 1 min, and extension at 72° C. for 1 min; and final extension at 72° C. for 10 min. The PCR product was recovered with a DNA purification kit to obtain the linearized plasmid pJYW-4-ceN-C.glglmS.

(32) (2) The ClonExpress II One Step Cloning Kit from Vazyme Biotech Co., Ltd. Was used for ligation. The linearized vector obtained by PCR and the target gene fragment carrying a homologous end of the vector were mixed at a molar ratio of 3:1 after extraction, 4 μL of 5×CE II Buffer and 2 μL of Exnase II were added, and then ddH.sub.2O was added to give a total volume of the ligation system of 20 μL. The system was reacted at 37° C. for 30 min, and allowed to stand at 4° C. after cooling. Then 10 μL of the ligation system was taken to transform E. coli.BL21(DE3) competent cells (see the instruction of Takara Preparation Kit for Competent Escherichia coli). The transformant that was confirmed to be correct by colony PCR was selected, sent to GENEWIZ, Inc. and sequenced for verification, to obtain the recombinant expression vector pJYW-4-ceN-C.glglmS-SugR. The vector pJYW-4-ceN-C.glglmS-SugR was deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China 430072, on Aug. 4, 2022, with CCTCC No. M20221239.

(33) The plasmid pJYW-4-ceN-C.glglmS-SugR was transformed into Corynebacterium glutamicum strain S9114ΔnagA-ΔgamA-Δldh by electroporation.

(34) Preparation of Electrocompetent Corynebacterium glutamicum:

(35) (1) C. glutamicum was inoculated onto LBG medium (where the cells needed to be picked up from a fresh slant culture, otherwise the growth of the bacteria would be affected), placed on a traveling shaker (200 rpm), and incubated at 30° C. for 16 h until OD.sub.562 reached 3.0.

(36) (2) 10% was inoculated into a competent medium to allow for an OD.sub.562 of 0.3, placed on a traveling shaker (200 rpm), and incubated at 30° C. until OD.sub.562 reached 0.9 (where the incubation time was about 3-5 h, and the cells were in a logarithmic growth phase at this time; and the subsequent operations could also be performed if the OD.sub.562 was persistently to be low and at about 0.6). The concentration of the cells needed to be ensured to be as high as possible, and the concentration factor was generally 100 times (where 50 mL competent medium was concentrated to 0.5 mL to prepare 5 tubes of competent cells).

(37) (3) The cell suspension was allowed to stand in an ice water bath for 15 min and then centrifuged at 4,000 rpm and 4° C. for 10 min, and the supernatant was discarded carefully.

(38) (4) The cells were fully suspended in 30 mL of pre-cooled 10% glycerol and then centrifuged at 4,000 rpm and 4° C. for 10 min, and the supernatant was discarded carefully. The cells were repeatedly washed 4 times.

(39) (5) The cells (100 times concentrated) were re-suspended in 500 μL of pre-cooled 10% glycerol, and filled in 1.5 mL sterile centrifuge tubes in an amount of 100 μL per tube.

(40) (6) The cells were stored at −80° C. for later use. To ensure the transformation efficiency of competent cells, the cells are preferred to be used immediately after preparation. The cells should not be left for more than 1 week, otherwise the cell content will be released due to the lysis of competent cells, which causes the breakdown of electroporation cuvette during the subsequent electroporation and affects the transformation efficiency.

(41) Electroporation of Corynebacterium glutamicum

(42) (1) Competent Corynebacterium glutamicum stored at −80° C. were thawed in an ice bath.

(43) (2) 1-5.0 μL of plasmid was added and mixed well (where the total amount of DNA was about 1.0 μg), and allowed to stand in an ice bath for 5-10 min.

(44) (3) The system was added into a pre-cooled 0.1 cm electroporation cuvette and received 2 electric shocks at 1.8 KV for 5 ms each.

(45) (4) 1.0 mL of preheated recovery medium (LBWS) was quickly added, mixed well and transferred to a new 1.5 mL sterile centrifuge tube. The system was allowed to stand in a water bath at 46° C. for 6 min, and then stand in an ice bath.

(46) (5) The cells were placed on a travelling shaker (100 rpm) and incubated at 30° C. for 2 h.

(47) (6) The cells were centrifuged for 1 min at 6,000 rpm and normal temperature, coated onto a detection plate of corresponding resistant transformant, and incubated in an incubator at a constant temperature of 30° C. for 2-3 days.

(48) (7) Efficiency verification of competent cells: 5.0 μL of sterile ddH.sub.2O was added as a negative control, no colonies were grown. For the positive control, 1-5 μL of the plasmid pXMJl9 (where the total DNA content was about 1.0 μg) was added, a large number of colonies were grown. The correctly sequenced colonies were the recombinant Corynebacterium glutamicum.

Example 3. Effect of Over-Expression of SugR Gene on N-Acetylglucosamine Production in Recombinant Corynebacterium glutamicum

(49) The correctly sequenced recombinant Corynebacterium glutamicum strain containing the plasmid pJYW-4-ceN-C.glglmS-SugR was inoculated from a glycerol tube into an LBG plate (added with 25 mg/L kanamycin sulfate), and cultured at 220 rpm and 30° C. for 18 h. Then single colonies were picked up and inoculated into an LBG plate until a large number of colonies were grown.

(50) A loop of single colonies was inoculated to the seed culture medium, and cultured at 220 rpm and 30° C. for 16 to 18 h until the cells were grown to the early logarithmic phase.

(51) 10% of the seed culture was inoculated into the fermentation medium and incubated at 30° C. and 220 rpm for 72 h. The amount of GlcNAc produced was determined.

(52) The recombinant strain containing the plasmid pJYW-4-ceN-C.glglmS was used as a control, and cultured and fermented under the same conditions. After 72 h, the amount of GlcNAc is 24.7 g/L (CK as shown in FIG. 3), and the amount of GlcNAc produced by the SugR gene-over-expressing strain containing the plasmid pJYW-4-ceN-C.glglmS-SugR over 72 h was 26 g/L (SugR as shown in FIG. 3).

(53) 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.