Method of constructing a recombinant Bacillus subtilis that can produce specific-molecular-weight hyaluronic acids

Abstract

The present invention relates to the field of biotechnology engineering. It provides a method of constructing a recombinant Bacillus subtilis that can produce specific-molecular-weight hyaluronic acids. By integranted expression of hasA from Streptococcus zooepidemicus and overexpression of genes of HA synthetic pathway, tuaD, glmU and glmS, high yield HA production was achieved in the recombinant strain. Additionally, introduction and functional expression of the leech hyaluronidase in the recombinant strain substantially increased the yield of HA to 19.38 g.Math.L.sup.1. Moreover, HAs with a broad range of molecular weights (10.sup.3 Da to 10.sup.6 MDa) were efficiently produced by controlling the expression level of hyaluronidase using RBS mutants with different translational strengths. The method of the present invention can be used to produce low molecular weight HAs at large scale in industrial applications.

Claims

1. A recombinant B. subtilis that can produce specific-molecular-weight hyaluronic acid (HA), wherein a HA biosynthetic pathway coupled with a secretory expression system of hyaluronidase is constructed in said recombinant B. subtilis; and wherein the secretory expression level of said hyaluronidase is regulated by ribosome binding sites with different translational strengths so as to produce said specific-molecular-weight HA.

2. The recombinant B. subtilis of claim 1, wherein a regulatory DNA fragment containing a constitutive promoter, a ribosome binding site sequence and a signal peptide, and a hyaluronidase gene are integrated into the genome of an engineered B. subtilis having a HA biosynthetic pathway.

3. The recombinant B. subtilis of claim 2, wherein the nucleotide sequence of said regulatory DNA fragment is set forth in SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 13.

4. The recombinant B. subtilis of claim 2, wherein said hyaluronidase gene is integrated at the glucosamine-6-phosphate deaminase 1 locus of B. subtilis chromosome by use of plasmid pBlueScript SK (+).

5. The recombinant B. subtilis of claim 1, wherein said HA biosynthetic pathway is to construct a biosynthetic pathway for HA precursors, UPD-N-acetylglucosamine and UDP-D-glucuronide, in a recombinant B. subtilis that expresses a hyaluronan synthase.

6. The recombinant B. subtilis of claim 5, wherein said hyaluronan synthase is encoded by hasA gene which is derived from Streptococcus zooepidemicus, Streptococcus equi or Streptococcus equissp.

7. The recombinant B. subtilis of claim 5, wherein genes for synthesizing UPD-N-acetylglucosamine and UDP-D-glucuronide are derived from Streptococcus species, Escherichia coli or Bacillus; wherein genes of said biosynthetic pathway includes tuaD which encodes a UDP-glucose dehydrogenase, glmU which encodes a UDP-N-acetylglucosamine pyrophosphorylase, gtaB which encodes a UDP-glucose pyrophosphorylase, glmM which encodes a mutase and glmS which encodes an amino transferase.

8. A method of constructing a recombinant B. subtilis of claim 1, comprising the steps of: 1) constructing a HA biosynthetic pathway: a hasA gene which encodes a hyaluronan synthase is integrated on the chromosome of B. subtilis by use of plasmid pAX01, resulting in a recombinant B. subtilis strain that contains said HA biosynthetic pathway; 2) over-expressing genes of biosynthetic pathway for UPD-N-acetylglucosamine and UDP-D-glucuronide: tuaD which encodes an UDP-glucose dehydrogenase, glmU which encodes a UDP-N-acetylglucosamine pyrophosphorylase, gtaB which encodes a UDP-glucose pyrophosphorylase, glmM which encodes a mutase and glmS which encodes an amino transferase are connected in series and inserted into vector pP43NMK; the resulting recombinant plasmid is transformed into the recombinant strain obtained in step 1), resulting in a recombinant B. subtilis strain with high yield of HA; 3) coexpressing HAase: a hyaluronidase gene fused with a regulatory DNA fragment containing a promoter, a ribosome binding site (RBS) sequence and a signal peptide is integrated onto the chromosome of the recombinant strain obtained in step 2), resulting in a recombinant B. subtilis which coexpresses a HAase with a HA synthetic pathway, wherein the resulting recombinant B. subtilis is said recombinant B. subtilis of claim 1.

9. The method of claim 8, further comprising regulating expression levels of HAase by using RBS mutants with different translational strengths to control the expression levels of HAase.

10. A method of producing HA, comprising using the recombinant B. subtilis of claim 1 to produce HA.

11. A method of producing specific-molecular-weight HA or HA oligosaccharides, comprising using the recombinant B. subtilis of claim 1 as the fermentation strain, and the fermentation is conducted at 30-37 C. and pH 6.0-7.0 for 48-96 hours with glucose or sucrose as carbon source for fermentation.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0036] FIG. 1. Construction schematic of the regulatory DNA fragment for controlling HAase expression.

[0037] FIG. 2. High throughput screening of the different expression levels of HAase activity using a standard plate.

[0038] FIG. 3. The curve of dissolved oxygen (DO) in recombinant strains with different expression levels of HAase cultured in a 3 L fermentor.

[0039] FIG. 4. The HAase activity of recombinant strains with different expression levels of HAase cultured in a 3 L fermentor.

[0040] FIG. 5. The production of HA of recombinant strains with different expression levels of HAase cultured in a 3 L fermentor.

[0041] FIG. 6. The average molecular weight of HA produced in different recombinant strains.

EXAMPLES

Materials and Methods:

[0042] Information of related nucleotide sequences:

[0043] (1) SEQ ID NO: 1 is the nucleotide sequence of hyaluronic acid synthase gene hasA from Streptococcus pneumoniae.

[0044] (2) SEQ ID NO: 2 is the nucleotide sequence of UDP-glucose dehydrogenase gene tuaD from B. subtilis.

[0045] (3) SEQ ID NO: 3 is the nucleotide sequence of UDP-N-acetylglucosamine pyrophosphorylase gene glmU from B. subtilis.

[0046] (4) SEQ ID NO: 4 is the nucleotide sequence of UDP-glucose pyrophosphorylase gene gtaB from B. subtilis.

[0047] (5) SEQ ID NO: 5 is the nucleotide sequence of a mutase gene glmM from B. subtilis.

[0048] (6) SEQ ID NO: 6 is the nucleotide sequence of an amino transferase gene glmS from B. subtilis.

[0049] (7) SEQ ID NO: 7 is the nucleotide sequence of a Leech hyaluronidase gene.

[0050] (8) SEQ ID NO: 8 is the nucleotide sequence of a regulatory DNA fragment P.sub.lepA-RBS-yewA.

[0051] (9) SEQ ID NO: 9 is the nucleotide sequence of a bleomycin resistant gene.

[0052] (10) SEQ ID NO: 10 is the nucleotide sequence of constitutive promoter P43.

[0053] (11) SEQ ID NO: 11 is the nucleotide sequence of inducible promoter Pveg.

[0054] (12) SEQ ID NO: 12 is the nucleotide sequence of a regulatory DNA fragment P.sub.lepA-RBS1-yewA.

[0055] (13) SEQ ID NO:13 is the nucleotide sequence of a regulatory DNA fragment P.sub.lepA-RBS2-yewA.

[0056] The HA titers were routinely estimated by the modified carbazole assay. The HA titer is assumed to be 2.067 times the glucuronic acid titer.

[0057] Leech hyaluronidase (LHAse or LHyal) activity was quantified by measuring the amount of reducing sugar liberated from HA using the 3,5-dinitrosalicylic acid (DNS) colorimetric spectrophotometric method. One unit of enzymatic activity is defined as equal to the reducing power of glucuronic acid (glucose equivalents in micrograms) liberated per hour from HA at 38 C., pH 5.5. Specific activity is defined as units of enzyme per ml of culture supernatant. The standard enzymatic reaction contained appropriate volumes of fermentation supernatant and 1.6 mg.Math.ml.sup.1 of HA as the substrate was incubated in 50 mM citrate-disodium hydrogen phosphate buffer at 38 C., pH 5.5 for 10 min in a total volume of 1 ml. The reaction was stopped by immersing in boiling water for 2 min and the enzyme activity was examined using the DNS method. Controls with fermentation supernatant of B. subtilis 168 were prepared and analyzed in the same manner

[0058] The average molecular weight of HA was measured by high performance gel filtration chromatography (HPGFC) with a multi-angle laser light scattering detector (MALLS). The mobile phase was 0.1 mol.Math.L.sup.1 NaNO.sub.3 and the temperature of the column was maintained at 40 C. The sample size was 40 L and elution time for each sample was 25 min. Dextran produced from Chinese Institute of food and drug testing was used as a standard and GPC software was used to calculate the average molecular weight.

Example 1

Construction of the Recombinant Plasmid pAX01-hasA

[0059] Hyaluronan synthase hasA was cloned from S. zooepidemicus ATCC 35246 with primers hasA-F/hasA-R to amplify the hasA gene by polymerase chain reaction (PCR). The S. zooepidemicus strain was incubated in 5 mL M17 media at 37 C., 200 rpm for 16 hours, and the chromosome of S. zooepidemicus was extracted by a bacterial genome extraction kit.

[0060] The nucleotide sequences of primers hasA-F and hasA-R were as follows (from 5 to 3):

TABLE-US-00001 hasA-F: (SEQIDNO:15) CGCGGATCCATGAGAACATTAAAAAACCTCATAAC hasA-R: (SEQIDNO:16) TGCATGCATTTATAATAATTTTTTACGTGTTCC

[0061] Gene fragment of hasA amplified by PCR and pAX01 plasmid were digested with restriction enzymes BamHI and SacII, respectively. The digested fragments were recovered for ligation. Then the ligation products were used to transform to JM109 competent cells and positive recombinant plasmid pAX01-hasA was verified by sequencing. Then, the pAX01-hasA was transformed into B. subtilis 168, resulting in the hasA gene integrated into the genome of B. subtilis 168 under the control of Pxyl promoter. The recombinant strain was designated as E168T.

Example 2

Construction of the Recombinant Plasmid pP43NMK/pP43-DU-PBMS

[0062] tuaD gene and glmU gene were amplified from B. subtilis 168 by PCR using primers tuaD-F/tuaD-R and glmU-F/glmU-R, respectively. KpnI restriction site and P43 RBS sequence (shown in SEQ ID NO:14) were introduced to the 5 of tuaD-F. SacI restriction site was introduced to the 5 of tuaD-R. SacI restriction site and P43 RBS sequence were introduced to the 5 of glmU-F. XhoI and XbaI restriction sites were introduced to the 5 of glmU-R. The resulting tuaD fragment and glmU fragment were digested with KpnI/SacI and SacI/XhoI, respectively. The digested fragments were purified and ligated together with digested pP43NMK (KpnI/XhoI) fragment. Then, the obtained ligation product was transformed into JM109 competent cells. The positive recombinant cells was verified by sequencing and the recombinant plasmid was designated as pP43-DU.

[0063] The Pveg promoter fragment amplified with the primer pair Pveg-F/Pveg-R was fused with the gtaB gene amplified with the primer pair Pveg-gtaB-F(containing a P43 RBS) and gtaB-R. SpeI and XbaI-XhoI restriction sites were introduced to the 5 and the 3 of the fusion fragment, respectively. The fusion product was digested with SpeI and XhoI, and ligated with digested pP43-DU fragment (XbaI and XhoI), resulting in a recombinant plasmid designated as pP43 -DU-PB.

[0064] By use of the same isocaudarner SpeI/XbaI, glmM and glmS genes were amplified with primers glmM-F/R and glmS-F/R, respectively. The glmM and glmS fragments were inserted into plasmid pP43-DU-PB in order, generating the recombinant plasmid pP43-DU-PBMS. pP43-DU-PBMS was transformed into E168T competent cells and a recombinant strain E168T/pP43-DU-PBMS with high yield of HA was obtained.

[0065] The primers used were as follows:

TABLE-US-00002 tuaD-F: (SEQIDNO:17) CGGGGTACCAAGAGAGGAATGTACACATGAAAAAAATAGCTGTCATTGG tuaD-R: (SEQIDNO:18) CCGGAGCTCTTATAAATTGACGCTTCCCAAG glmU-F: (SEQIDNO:19) CGGGAGCTCAAGAGAGGAATGTACACATGGATAAGCGGTTTGCAGTTG glmU-R: (SEQIDNO:20) CCGCTCGAGCGGACTCTAGTCTAGATTATTTTTTATGAATATTTTTCAC Pveg-F: (SEQIDNO:21) GGACTAGTGGAGTTCTGAGAATTGGTATGC Pveg-R: (SEQIDNO:22) ATGTAAATCGCTCCTTTTTAACTAC Pveg-gtaB-F: (SEQIDNO:23) GTAGTTAAAAAGGAGCGATTTACATATGAAAAAAGTACGTAAAGC glmM-F: (SEQIDNO:24) GGACTAGTAAGAGAGGAATGTACACATGGGCAAGTATTTTGGAACAG ACGG glmM-R: (SEQIDNO:25) CCGCTCGAGCGGACTCTAGTCTAGATTACTCTAATCCCATTTCTGAC CGGAC glmS-F: (SEQIDNO:26) GGACTAGTAAGAGAGGAATGTACACATGTGTGGAATCGTAGGTTATA TCGG glmS-R: (SEQIDNO:27) CCGCTCGAGCGGACTCTAGTCTAGATTACTCCACAGTAACACTCTTCGC

Example 3

Construction of the Integrated Gene Fragment of LHyal

[0066] The gene encoding hyaluronidase was integrated at the glucosamine-6-phosphate deaminase 1 (nagA-nagBA) locus of B. subtilis 168 using Zeocin gene as the selection marker. The integrated fragment (shown in FIG. 1) was obtained by homologous recombination technique.

[0067] The primers used were as follows:

TABLE-US-00003 H6LHyal-F: (SEQIDNO:28) ATGCACAGTCTGCAGAATTCCACCACCACCACCACCACATG H6LHyal-R: (SEQIDNO:29) TTACTTTTTGCACGCTTCAACAT ZHLHPlepA-F: (SEQIDNO:30) CGCAGCCAAAGGAGTGGATTGCCTCAATCCTAGGAGAAACAG ZHLHPlepA-R: (SEQIDNO:31) GAATTCTGCAGACTGTGCATGAGC ZHLH-front-F: (SEQIDNO:32) TCAGCTGGTCTAGATCACTAGTC ZHLH-front-R: (SEQIDNO:33) AATCCACTCCTTTGGCTGCGCTC ZHLH-zeocin-F: (SEQIDNO:34) TTGAAGCGTGCAAAAAGTAAGAGCTCGGTACCCGGGGATCC ZHLH-zeocin-R: (SEQIDNO:35) GCTTGCATGCCTGCAGGTCGAC ZHLH-back-F: (SEQIDNO:36) CGACCTGCAGGCATGCAAGCCACTTCTTTCAGACGGAACCCTTGC ZHLH-back-R: (SEQIDNO:37) CGGTCGTTCATATAGAAGTGATAG ZHLH-pSK-F: (SEQIDNO:38) CACTTCTATATGAACGACCGCCTGTGTGAAATTGTTATCCGCTC ZHLH-pSK-R: (SEQIDNO:39) TAGTGATCTAGACCAGCTGAGTGACTGGGAAAACCCTGGCGTTAC

[0068] The LHyal gene encoding a leech hyaluronidase (LHyal) was amplified with primers H6LHyal-F/H6LHyal-R and the Zeocin gene was amplified with primers ZHLH-zeocin-F/ZHLH-zeocin-R. The regulatory DNA fragment containing the promoter PlepA, the RBS P43 and the signal peptide yweA was amplified with primers ZHLHPlepA-F/R. The front and back flanking fragments of the target for integration were amplified with primers ZHLH-front-F/R and ZHLH-back-F/R, respectively. A recombinant vector was amplified with primers ZHLH-pSK-F/ZHLH-pSK-R using the plasmid pBlueScript SK(+) as template. The five DNA fragments and the recombinant vector described above were assembled using homologous recombination technology, and the assembled products were transformed into E. coli JM109 competent cells. The recombinant plasmid containing the regulatory DNA fragment and leech hyaluronidase gene was designated as pSKZHLH.

[0069] pSKZHLH was transformed into the competent cells of HA producing strain E168T/pP43-DU-PBMS and the recombinant strain was screened with 25 ug/ml Zeocin. The positive recombinant strain expressing HAase was designated as E168TH/pP43-DU-PBMS.

Example 4

Construction of RBS Mutant Library for Controlling the Expression of HAase

[0070] A RBS mutant library with a wide range of translational strength was constructed by genetic engineering at the ribosome regulation level. The degenerate primer JB/lepA-RBS-R, which includes the RBS region, and reverse primer ZHLH-H6F were used to amplify the RBS mutant library using the pSKZHLH as the template. KpnI restriction site was added to the 5 of both primers. The primers used were as follows:

TABLE-US-00004 JB/lepA-RBS-R: (SEQIDNO:40) ACGGGGTACCACTNTNYNHBYACTATTAAACGCAAAATACACTAGCTTAG ZHLH-H6F: (SEQIDNO:41) ACGGGGTACCATGCTAAAAAGAACTTCATTCG

[0071] The PCR product was first digested with DpnI, and then further digested with the restriction endonuclease KpnI, which was used for ligation. The ligation products were transformed into E168T/pP43-DU-PBMS competent cells. Five hundred transformants were picked from LB agar plates with 25 ug/ml Zeocin and then grown in 96-well microtiter at 37 C., 200 rpm for 60 hours. The culture medium contains 2% yeast powder, 7% sucrose, 15.6 g/L sodium dihydrogen phosphate, 3.9 g/L potassium sulfate.

[0072] The quantitation of hyaluronidase activity of culture supernatants was performed with high throughput screening by transparent ring colorimetric plate assay. 2 mg/ml HA was dissolved in citric acid buffer (pH 5.5) to make a HA buffer and 1.5% agarose was melted by heat in the same citric buffer. Equal volume of HA buffer and heated agarose buffer was mixed and poured into a plastic plate to allow solidification. Multiple holes were drilled in the agarose plate as shown in FIG. 2. After centrifuged at 4000 rpm for 5 min, 150 L supernatant of the fermentation broth of RBS mutant strains was added to the holes in the agarose plate and cultivated at 37 C. for 10 hours. After that, 2.5 g/L cetyltrimethyl ammonium bromide was added and incubated for 30 min. Results (FIG. 2) demonstrated that the mutant strains with RBS modifications exhibit significantly different levels of HAse expression. E168THR1/pP43-DU-PBMS and E168THR2/pP43-DU-PBMS were two mutant strains with different RBS translational strengths.

Example 5

Fed-Batch Fermentation of the Recombinant Strains in a 3-L Fermentor

[0073] Recombinant strains E168T/pP43-DU-PBMS, E168TH/pP43 -DU-PBMS, E168THR1/pP43 -DU-PBMS and E168THR2/pP43-DU-PBMS4 were fermented, respectively.

[0074] The recombinant strains were grown in a LB medium with 50 g/ml kanamycin at 37 C. and 200 rpm for 12 hours. The 3-L fermentor contained an initial 1.35 L of fermentation medium (2% Yeast extract, 1.5% sucrose, 15.6 g/L sodium dihydrogen phosphate and 3.9 g/L potassium sulfate, pH 7.0). The seed cultures were transferred into the fermentor with a 10% inoculation volume. Xylose with a final concentration of 20 g/L was used to induce the expression of hasA at 2 hours after the inoculation.

[0075] Feed started at about 8 hours after inoculation with a simple sucrose solution at index-fed-batch feed rates of 7.5, 7.5, 15.0, 10.0 g.Math.h.sup..Math.L.sup.1 for the first 4 hours. The constant feed rate was maintained at 5 g.Math.h.sup..Math.L.sup.1 until the end of fermentation. Samples were periodically withdrawn to determine the HA production and HAase activity of the fermentation. After centrifugation at 10000 rpm for 10 min, the fermentation supernatant was transferred to another tube, and 2 volumes ethanol was added to precipitate HA and incubated for 1 hour. The precipitate was collected by centrifugation (10000 rpm for 20 min) and redissolved in equal volume 1 mol.Math.L.sup.1 NaCl solution. The suspension was used for further determination of yield and molecular weight.

[0076] Due to the viscoelastic properties of HA, the fermentation of engineered strain E168T/pP43-DU-PBMS became very viscous after 15 h and concomitantly resulting in the dramatic decline of dissolved oxygen (DO), which seriously affected the growth of cells and the accumulation of HA. FIG. 3 showed that the fermentation DO of E168T/pP43-DU-PBMS was almost reduced to 0 at 40 hours, while the fermentation DO of other engineered strains which had different expression levels of HAase were maintained at a higher level. The HAase activities of E168TH/pP43-DU-PBMS, E168THR1/pP43-DU-PBMS and E168THR2/pP43-DU-PBMS4 reached high values of 1.6210.sup.6U/mL, 8.810.sup.5U/mL and 6.410.sup.4 U/mL, respectively. The HA yield of E168T/pP43-DU-PBMS reached the maximal HA titer of 5.96 g.Math.L.sup.1 due to viscous fermentation. However, the HA yield of the highest HAase expression strain, E168TH/pP43-DU-PBMS, reached 19.38 g.Math.L.sup.1, and the HA yield of the other two strains, E168THR1/pP43-DU-PBMS and E168THR2/pP43-DU-PBMS4, with lower HAase expression reached 9.18 g.Math.L.sup.1 and 7.13 g.Math.L.sup.1, respectively. These results demonstrated that the higher is the HAase production, the higher is the HA yield.

[0077] There was a significant difference between the average molecular weight of HA of engineered strains with different HAase expression levels (shown in FIG. 6). The average molecular weight of HA from strain E168T/pP43-DU-PBMS which did not express the HAase was 1.4210.sup.6 Da, while those of strains E168TH/pP43-DU-PBMS, E168THR1/pP43-DU-PBMS and E168THR2/pP43-DU-PBMS4 were 6628 Da, 18000 Da and 49600 Da, respectively.

[0078] The results showed that the molecular weight of HA could be precisely controlled within a range from 10.sup.3 to 10.sup.6 Da through controlling the expression level of HAase. Additionally, HA10, HA8, HA6, HA4 and other oligosaccharides could be obtained by allowing the supernatant of the fermentation broth to incubate at room temperature for additional 1-3 hours.

[0079] While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, appendices, patents, patent applications and publications, referred to above, are hereby incorporated by reference.