High-efficiency synthesis and high-purity hyaluronic acid, and recombinant <i>Corynebacterium glutamicum </i>for oligosaccharide thereof

Abstract

The invention discloses a recombinant Corynebacterium glutamicum for efficient synthesis of highly pure hyaluronic acid and oligosaccharides thereof, belonging to the technical field of bioengineering. The recombinant Corynebacterium glutamicum constructed in the present invention can produce hyaluronic acid with a yield up to 40 g/L, and a crude product purity of 95%. Addition of exogenous hyaluronic acid hydrolase and optimization of the fermentation conditions results in hyaluronic acid oligosaccharides with specific molecular weight, and can further improve the yield of hyaluronic acid to 72 g/L. The invention lays a solid foundation for the efficient synthesis of highly pure hyaluronic acid by microorganisms, and the constructed recombinant Corynebacterium glutamicum is suitable for industrial production and application.

Claims

1. A recombinant Corynebacterium glutamicum, wherein exopolysaccharide genes cg0420 and/or cg0424 are/is silenced or knocked out, and hyaluronan synthase is expressed; wherein the hyaluronan synthase comprises the amino acid sequence of SEQ ID NO:3.

2. The recombinant Corynebacterium glutamicum according to claim 1, wherein an UDP-N-acetylglucosamine pathway and/or an UDP-glucuronic acid pathway are/is enhanced, wherein the UDP-N-acetylglucosamine pathway comprises: a glutamine-frutose-6-phosphate aminotransferase, a phosphoglucomutase, a UDP-N-acetylglucosamine pyrophosphorylase/glucose-1-phosphate acetyltransferase bifunctional enzyme; and the UDP-glucuronic acid pathway comprises: a phosphoglucomutase, a glucose-6-phosphate uramidotransferase, and a UDP-glucose dehydrogenase.

3. The recombinant Corynebacterium glutamicum according to claim 1, wherein the recombinant Corynebacterium glutamicum also expresses hemoglobin VHb derived from Vitreoscilla.

4. The recombinant Corynebacterium glutamicum according to claim 2, wherein genes encoding the UDP-N-acetylglucosamine pathway and/or UDP-glucuronic acid pathway and gene encoding a hemoglobin VHb are ligated to a vector to be expressed in the Corynebacterium glutamicum; the vector includes any of the following: pXMJ19, pECXK99E, pEC-XT99A, pEKEx1, pEKEx2, pVWEx1, pVWEx2, pZ8-1, pECTAC-K99 and pAPE12.

5. A method for constructing the recombinant Corynebacterium glutamicum according to claim 1, comprising the steps of: (1) knocking out exopolysaccharide synthesis genes cg0420 and cg0424 stepwise or simultaneously by constructing knockout box(es) and recombining the knockout box(es) with genomic genes cg0420 and cg0424 in the Corynebacterium glutamicum; and (2) ligating a hyaluronan synthase-encoding gene, a hemoglobin VHb-encoding gene, and at least one gene selected from pgM, ugd, galU, glmS, glmM and glmU to a vector, which is in turn transformed into the cell of the Corynebacterium glutamicum, wherein the hyaluronan synthase comprises the amino acid sequence of SEQ ID NO.3.

6. A method for producing hyaluronic acid, comprising steps of fermenting the recombinant Corynebacterium glutamicum according to claim 1 in culture media comprising carbon source, nitrogen source, inorganic salts, metal ions and oxygen, wherein the fermentation is performed at 25-35? C. for 24-72 h.

7. The method according to claim 6, wherein a hyaluronan hydrolase or hyaluronan lyase is added in the early stage of the fermentation; and the amount of the added hyaluronan hydrolase or hyaluronan lyase is 500-50000 U/mL.

8. The method according to claim 6, wherein glucose is supplemented during the fermentation process.

9. A method for the preparation of hyaluronic acid and derivative products, comprising steps of fermenting the recombinant Corynebacterium glutamicum according to claim 1 in culture media comprising carbon source, nitrogen source, inorganic salts, metal ions and oxygen, and collecting the produced hyaluronic acid and derivative products in the culture media, wherein the fermentation is performed at 25-35? C. for 24-72 h.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Metabolic pathways and related enzymes for the production of hyaluronic acid by the recombinant Corynebacterium glutamicum.

(2) FIG. 2: A graph indicating the yield and product purity of hyaluronic acid by the recombinant Corynebacterium glutamicum.

(3) FIG. 3: A graph indicating the yield of hyaluronic acid by the recombinant Corynebacterium glutamicum in 5 L fermentation tank.

(4) FIG. 4: A graph indicating the yield of hyaluronic acid by the recombinant Corynebacterium glutamicum with exogenous addition of hyaluronan hydrolase in 5 L fermentation tank.

(5) FIG. 5: A mass spectrum of the disaccharide unit of hyaluronic acid produced by the recombinant Corynebacterium glutamicum.

DETAILED DESCRIPTION OF THE INVENTION

(6) Strain: Corynebacterium glutamicum ATCC 13032, plasmids: pXMJ19, pEC-XK99E, pK18mobSacB

(7) LB medium: Yeast powder 5 g/L, peptone 10 g/L, and sodium chloride 10 g/L

(8) BHI: Brain heart extract 17 g/L, sorbitol 21 g/L

(9) Fermentation Medium: Glucose: 40 g/L, corn steep powder: 20 g/L, KH.sub.2PO.sub.4: 15 g/L, K.sub.2HPO.sub.4: 5 g/L, MgSO.sub.4: 1 g/L.

(10) Determination of the hyaluronic acid yield: The fermentation broth was taken appropriately and 10-fold diluted, centrifuged at 10,000 rpm for 10 minutes, the supernatant was taken and added with 4? volume of pre-cooled ethanol, placed at ?20 for 6h-ethanol precipitation, centrifuged at 10,000 rpm for 10 minutes, the supernatant was discarded, resuspended with water up to the original volume, centrifuged at 10,000 rpm for 5 minutes, the precipitation was discarded and the supernatant was taken and added with 4? volume of pre-cooled ethanol, placed at ?20 for 6h-ethanol precipitation, centrifuged at 10,000 rpm for 10 minutes, the supernatant was discarded, resuspended with water up to the original volume, centrifuged at 10,000 rpm for 5 minutes, the supernatant was taken and the precipitation was discarded. The supernatant was taken and 50-fold diluted, up to the final dilution of 500-fold. For the measurement of the sample, the sample was diluted according to the linear effective range, and then measured by the sulfuric acid carbazole method.

(11) Determination by sulfuric acid carbazole method: 1 ml sample was added to a glass tube containing 5 mL of borax sulfuric acid (4.77 g borax dissolved in 500 mL of concentrated H2504), mixed well, boiled in a boiling water bath for 15 min, and cooled on ice. 250 ?L of carbazole reagent (0.125 g carbazole dissolved in 100 mL absolute ethanol) was added, mixed well, and boiled in a boiling water bath for 15 min. The blank control was prepared in the same condition, except the sample was replaced with an equal volume of distilled water, and the absorbance value at the wavelength of 530 nm was measured. Different concentrations (10, 20, 30, 40, 50 ?g mL.sup.?1) of D-glucuronic acid were used as standard samples to plot a standard curve, and then the content of hyaluronic acid was calculated by fitting the absorbance value thereof to the standard curve. Standard curve equation: y=126.88x?9.2639, R.sup.2=0.9991 (x, A530 absorbance; y, glucuronic acid content in the sample (?g mL.sup.?1)). Calculation formula for the yield of chondroitin: hyaluronic acid content (g/L)=(concentration determined by the standard curve*dilution factor*2.067)/1000. For the measurement of the sample, the sample was diluted according to the linear effective range.

(12) Determination of the molecular weight of hyaluronic acid: The supernatant of the fermentation was removed impurities by repeated alcohol precipitation to obtain high-purity hyaluronic acid, which was measured by HPLC for the molecular weight.

(13) LC-MS Determination of the Structure of Hyaluronic Acid. The supernatant of the fermentation was removed impurities by repeated alcohol precipitation to obtain high-purity hyaluronic acid, and then the sample was treated overnight at 37? C. by hyaluronidase to cleave hyaluronic acid into disaccharide units. Then, nine times the volume of anhydrous methanol was added to the sample. The impurities and unhydrolyzed hyaluronic acid were removed by centrifugation and the insoluble impurities were filtered out through an organic membrane prior to analyzing the structure of the disaccharide units by LC-MS.

Example 1

Construction of the Recombinant Corynebacterium glutamicum

(14) (1) Genes cg0420 and cg0424 Knockout

(15) A fragment about 500 bp upstream to cg0420, 0420-up, was obtained by PCR, with the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, and using 0420-up-F and 0420-up-R as primers, and the PCR product was purified;

(16) A fragment about 500 bp downstream to cg0420, 0420-down, was obtained by PCR, with the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, and using 0420-down-F and 0420-down-R as primers, and the PCR product was purified;

(17) The Corynebacterium glutamicum suicide plasmid pK18mobsacB was double digested with EcoRI/BamhI, and the 0420-up and 0420-down fragments were ligated to the digested pK18mobsacB in one step with the Gibson Assembly kit, and the obtained recombinant plasmid was named pK18-0420;

(18) Corynebacterium glutamicum ATCC13032 was transfected with the plasmid pK18-0420 by electroporation, and the electric shock condition was: voltage 1.5 KV, 5 ms, (the width of the electroporation vessel was 1 mm), and the electric shock was performed twice. The first screening of recombinant bacteria was performed on BHI plates containing 25 mg/L kanamycin. The positive recombinants were picked up and further cultured in liquid LB medium overnight, and then the second screening was performed on BHI plates containing 100 g/L sucrose. PCR was performed on the colony by using primers 0420-up-F and 0420-down-R, and a fragment of 1 Kb could be amplified from the 0420 gene knockout recombinant, and the recombinant strain was named as C. glutamicum ?0420. The gene cg0424 was also knocked out by the above method, resulting in a cg0424 single-knockout strain and a cg0420 and cg0424 double-knockout strain, named as C. glutamicum ?0420 and C. glutamicum ?0420 & ?0424, respectively.

(19) Among them, the primer sequences used were as follows:

(20) TABLE-US-00001 0420-UP-F: (SEQIDNO.11) GCAGGTCGACTCTAGAGGATCCAAGTTTCGAACCATGCTTGAAC 0420-UP-R: (SEQIDNO.12) GATCTGATTCTTCGCACCAATAGGCGACATACCGTTTCTAACTGCTCAG 0420-down-F: (SEQIDNO.13) CTGAGCAGTTAGAAACGGTATGTCGCCTATTGGTGCGAAGAATCAGATC 0420-down-R: (SEQIDNO.14) CTATGACCATGATTACGAATTCTGGACCCTAAACTGAGCAGTGA

(21) (2) Integration of Genes HasA and VHb

(22) A 500 bp fragment U-up was amplified by PCR, with the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, and using U-up-F and U-up-R as primers, and the PCR product was purified;

(23) A fragment of about 500 bp D-down was amplified by PCR, with the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, and using D-down-F and D-down-R as primers, and the PCR product was purified;

(24) A fragment as shown in SEQ ID NO.10 was amplified by PCR, with the genomic DNA of Vitreoscilla (Vitreoscilla stercoraria DSM 513) as a template, and using HasA-F and HasA-R as primers, and the PCR product was purified;

(25) The Corynebacterium glutamicum suicide plasmid pK18mobsacB was double digested with EcoRI/BamhI, and the fragments U-up, D-down and VHb were ligated to the digested pK18mobsacB in one step with the Gibson Assembly kit, and the obtained recombinant plasmid was named pK18-VHB;

(26) C. glutamicum ?0420 strain, C. glutamicum ?0424 strain and C. glutamicum?0420 ?0424 strain constructed in step (I), and wild-type strain were transfected with the plasmid pK18-VHB by electroporation, and the electric shock condition was: voltage 1.5 KV, 5 ms, (the width of the electroporation vessel was 1 mm), and the electric shock was performed twice. The first screening of recombinant bacteria was performed on BHI plates containing 25 mg/L kanamycin. The positive recombinants were picked up and further cultured in liquid LB medium overnight, and then the second screening was performed on BHI plates containing 100 g/L sucrose. PCR was performed on the colony by using primers U-up-F and D-down-R, and a fragment of 1.3 Kb could be amplified from the recombinant integrated with HasA gene, and the obtained recombinant strain was named as C. glutamicum-HasA. The above method was also used for the integration of Gene VHb, and the finally obtained strains were C. glutamicum ?0420-HasA-VHB, ?0424-HasA-VHB, 40420&40424-HasA-VHB and WT-HasA-VHB, respectively.

(27) TABLE-US-00002 U-up-F: (SEQIDNO.15) GCAGGTCGACTCTAGAGGATCCTTAGAAGAACTGCTTCTGAAT U-up-R: (SEQIDNO.16) AATAGGCATGATATACGCTCCTTCGAACACGGCGACACTGAAC D-down-F: (SEQIDNO.17) GTTACCGACGGTTTCTTTCATATTCCAAGCCGGAGAATTTCC D-down-R: (SEQIDNO.18) CTATGACCATGATTACGAAATGAAAGAAACCGTCGGTAAC HasA-F: (SEQIDNO.19) AAGGAGCGTATATCATGCCTATTTTCAAGAAGACT HasA-R: (SEQIDNO.20) AATAGGCATGATATACGCTCCTTTTATTTAAAAATAGTAACTTTTTTT CTAG

(28) (3) Construction of Recombinant Plasmids pXMJ19 Pgm-galU-Ugd and pECXK99E-glmS-glmM-glmU

(29) Pseudomonas putida KT2440 was inoculated in 3 ml LB liquid medium and cultured at 30? C. 220 rpm for 24 hours. The bacteria were collected and the genomic DNA was extracted with the cell genome extraction kit. Primers pgm-F/pgm-R, galU-F/galU-R, ugd-F/ugd-R, glmS-F/glmS-R, glmM-F/glmM-R and glmU-F/glmU-R were designed, and the extracted Pseudomonas putida genomic DNA was used as a template to amplify and obtain genes pgm, ugd, galU, glmU, glmS and glmM with the PCR amplification system and procedure. The plasmids pXMJ19 and pECXK99E were enzymatically digested at the selected restriction sites to obtain linear plasmids pXMJ19 and pECXK99E, and Gibson assembly reactions were performed on the amplified fragments pgm, ugd, galU and the linear plasmid pXMJ19, and on glmU, glmS, glmM and the linear plasmid pECXK99E. JM109 competent cells were transformed with the Gibson assembly reaction system. The transformants were selected for plasmid sequencing reaction and sequence alignment, and the recombinant plasmids pXMJ19-pgm-ugd-galU and pECX99E-glmU-glmM-glmS were successfully constructed. The recombinant plasmids were electro-transformed into Corynebacterium glutamicum ATCC13032, C. glutamicum?0420-HasA-VHB, ?0424-HasA-VHB, ?0420&40424-HasA-VHB and WT-HasA-VHB, strain and the obtained recombinant strain were named as WT, ?0420, ?0424 and ?0420&?0424, respectively.

(30) TABLE-US-00003 TABLE1 Primersusedforconstructionofrecombinant plasmidpXMJ19-pgm-ugd-galU Primer name Sequence(5-3) SEQIDNO: pgm-F GCATGCCTGCAGGTCGACTCTAGAGGATC SEQIDNO: CAAGGAGCGTATATCATGACGCTCAGTCC 21 TTTGGC pgm-R CTTCATGATATACGCTCCTTTCAGGCAAT SEQIDNO: GGCTTCATCGAC 22 ugd-F TCGATGAAGCCATTGCCTGAAAGGAGCGT SEQIDNO: ATATCATGAAGGTCACGGTTTTCGGAAC ugd-R GATCATGATATACGCTCCTTTCAAGCTGG SEQIDNO: CGCAATCTTGC 24 galU-R GCAAGATTGCGCCAGCTTGAAAGGAGCGT SEQIDNO: ATATCATGATCAAAAAATGCTTGTTCCCG 25 GCAG galU-R CTCATCCGCCAAAACAGCCAAGCTGAATT SEQIDNO: CTCAGTAAGCCTTGCCAGTCTTG 26

(31) TABLE-US-00004 TABLE2 Primersusedforconstructionofrecombinant plasmidpXMJ19-glmU-glmM-glmS Primer name Sequence(5-3) SEQIDNO: glmU-F CAATTTCACACAGGAAACAGACCATGGAA SEQIDNO: TTCAAGGAGCGTATATCATGTCACTCGAT 27 ATCGTTATTCTCGCC glmU-R CGTCGGTACCAAAGTATTTTCTGCTCATT SEQIDNO: CAGCTCTTCTTGATCTTCTCCG 28 glmM-P CGGAGAAGATCAAGAAGAGCTGAAAGGAG SEQIDNO: CGTATATCATGAGCAGAAAATACTTTGGT 29 ACCGACG glmM-R GACAGCACCAACGATTCCACACATTCAGA SEQIDNO: CACAAACTTCGCCGACC 30 glmS-F CTGGTCGGCGAAGTTTGTGTCTGAAGGAG SEQIDNO: CGTATATCATGTGTGGAATCGTTGGTGCT 31 G glmS-R GCAGGTCGACTCTAGAGGATCCCCGGGTA SEQIDNO: CCTTACTCGACAGTCACCGACTTG 32

Example 2

Production of Hyaluronic Acid by Recombinant Corynebacterium glutamicum

(32) A single clone of each of the constructed recombinant Corynebacterium glutamicum strains WT, ?0420, ?0424 and ?0420&?0424 was inoculated in 5 ml BHI medium, at 200 rpm, 30? C. overnight. 10 hours later, 1% of the inoculum was transferred to a 250 ml Erlenmeyer flask (containing 25 ml fermentation medium). 3 hours after incubation at 200 rpm, 28? C., IPTG was added at a final concentration of 0.25 Mm to induce gene expression. The fermentation period was 48 hours. After the fermentation was ended, the supernatant was taken and four times volume of ethanol was added for alcohol precipitation to remove some impurities. After the alcohol precipitation was repeated twice, the content of hyaluronic acid was determined by the sulfuric acid carbazole method. It can be seen from FIG. 2 that both the yield and purity of hyaluronic acid were improved to a certain extent by knocking out cg0420 and cg0420. For the double knockout strain, the yield and purity of hyaluronic acid by shaking bottle were up to 6.9 g/L and 95%, respectively.

Example 3

Fermentation Culture of Recombinant Corynebacterium glutamicum in 5 L Fermentation Tank

(33) A single clone of each of the constructed recombinant Corynebacterium glutamicum strains Corynebacterium glutamicum HasA-VGB/pXMJ19-pgm-ugd-galU and pECX99E-glmU-glmM-glmS (cg0420 and cg0424 knockout) were inoculated in 5 ml BHI medium, at 200 rpm, at 30? C. overnight. 10 hours later, 1% of the inoculum was transferred to a 250 ml Erlenmeyer flask (containing 25 ml fermentation medium). Cultivated at 200 rpm, at 28? C. for 10 hours and 10% of the inoculum was inoculated into the fermentation tank. During the fermentation process, the glucose content in the tank was maintained at about 10 g/L by feeding glucose and pH was controlled to be neutral by feeding NaOH. 20 hours after fermentation, hyaluronic acid hydrolase was added exogenously at a final concentration of 6000 U/mL. The fermentation period was 72 h. It can be seen from FIG. 3 that the OD reached the highest level at 48 h without the addition of hyaluronic acid hydrolase. Hyaluronic acid accumulated rapidly from 24 to 48 hours, and the yield was up to 40 g/L at 60 hours. It can be seen from FIG. 4 that the bacteria were in the logarithmic growth phase from 4 to 36 hours, the bacteria grew rapidly, and entered the stationary phase at 36 hours. Further, hyaluronic acid accumulated rapidly from 24 hours to 60 hours. Compared to FIG. 3, the fermentation period was 12 hours longer, and the yield of hyaluronic acid was also increased significantly. At 72 hours, the yield of hyaluronic acid was up to 72 g/L, which was 32 g/L higher than that without hydrolase, and the OD was also increased to a certain extent.

Comparative Example

(34) Following the same strategy as in Example 1, encoding genes Cgl1118 (NC_003450.3) and Cgl0452 (NC_003450.3) in other pathway competing precursor were knocked out. The recombinant plasmids constructed according to steps (2) and (3) in Example 1 were transformed into the Cgl1118 (NC_003450.3) and Cgl0452 (NC_003450.3) knockout cells. Fermentation was carried out according to the method of Example 2 or 3. The results show that the yield of hyaluronic acid was greatly reduced in these genes knockout strains. The yield by shaking bottle was only 3.1 g/L. The main reason is that knocking out these genes affects the growth of bacteria, resulting in slow growth of the strain. The OD value of fermentation for 48 h was 34, which was only the half of that of the wild type strain cultured under the same conditions.

(35) Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Various changes and modifications can be made by those familiar with this technology, without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.