# Patent

## Title
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·L−1. Moreover, HAs with a broad range of molecular weights (103 Da to 106 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.

## Background
CROSS-REFERENCES AND RELATED APPLICATIONS
This application claims the benefit of priority to Chinese Application No. 201510573851.X, entitled “A method of constructing a recombinant Bacillus subtilis that can produce specific-molecular-weight hyaluronic acids”, filed Sep. 10, 2015, which is herein incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to the field of biotechnology engineering, and more particularly relates to a method of constructing a recombinant Bacillus subtilis that can produce specific-molecular-weight hyaluronic acids.
Description of the Related Art
Hyaluronic Acid (HA or hyaluronan), a highly viscous polysaccharide, was first isolated from bovine vitreous and it is the best moisturizing substances found in nature. The unique rheological, the viscoelastic and hygroscopic properties along with the biocompatibility and non-immunogenicity has enabled HA to be widely used in cosmetics, food and pharmaceutics. Recent studies have found that HAs with low molecular weight (less than 1×104 Da) and HA oligosaccharides have unique biological functions, for example, HA oligosaccharides with molecular weight less than 1×104 Da are involved in wound healing and tumor cell apoptosis. In addition, the HA oligosaccharides can be easily absorbed by human body and act as precursors for the body's own synthesis of polysaccharides. Therefore, HA oligosaccharides have important applications in the areas of food, health care and medicine.
HA is widely distributed in animal tissues, such as comb, synovial fluid, cartilage and vitreous. And it is also distributed in bacteria, such as Bacillus aerogenes, Pseudomonas aeruginosa and Hemolytic streptococcus. Due to disadvantages (such as the risk of cross-species viral infection) of the traditional HA extraction methods from animal tissues, the commercial HA production is mainly relied on fermentation of certain attenuated strains of group C Streptococcus (such as S. equi and S. zooepidemicus). The HA produced by microbial fermentation was mostly high molecular weight HA. With increasing health and safety requirements, it becomes more and more urgent to find a safe and reliable microbial host for HA production.
Although de novo synthesis methods for preparing HA oligosaccharides have been reported, it is difficult to achieve large-scale production due to high cost of substrates, complex synthesis steps and low yields. Currently, physical and chemical degradation are the main methods used for low-molecular weight HA production, which have many disadvantages, such as generation of HA with a wide range of molecular weight distribution, poor product stability, high cost of purification, high energy consumption and high pollution. Compared to physical or chemical degradation, enzymatic catalytic synthesis of HA oligosaccharides is a promising approach that offers great industrial potential. However, the enzymatic method requires preparation of large amount of hyaluronidase (HAase) and precise control of the enzymatic catalytic reaction conditions. Therefore, constructing a single microbial strain that simultaneously produces both HA and HAase can offer great benefits in HA research and industrial applications.
HAase is a class of hyaluronic acid-degrading enzymes widely distributed in nature. According to sources, structures and function mechanisms of the enzyme, HAase are divided into three categories: endo-β-N-acetyl-glucosaminidase (EC 3.2.1.35, mainly exist in mammals and venom of bees, snakes and spiders), endo-β-glucuronidase enzyme (EC 3.2.1.36, mainly in the leech) and hyaluronic acid lyase (EC 4.2.2.1, mainly exist in bacteria, bacteriophages and fungi). As a “scatter factor”, HAase is widely used as auxiliaries for drug diffusion in clinical applications. However, the commercial HAase obtained from bovine testicular tissue is usually of poor quality and expensive to produce, and has the risk of Animal foci infection.
In the present invention, a HA biosynthetic pathway was constructed in Bacillus subtilis (B. subtilis) and high yield of HA was achieved by regulating the expression of important genes for synthesis of HA precursors, UDP-GlcNAc and UDP-GlcA, in the engineered strain. In addition, Leech HAase was co-expressed in the engineered strain with a HA biosynthetic pathway to achieve synchronous production of HA and HAase. Production of HA with specific molecular weights was achieved by precise regulation of the expression levels of HAase. The problem of fermentation stagnation caused by high viscosity of HA was solved by coupling the production of HA and HAase, thus greatly increasing the production efficiency. The present invention for the first time achieved efficient synthesis of HA with specific molecular weights, which has potential for bringing great economic gains in industrial applications.
DETAILED DESCRIPTION
The present invention provides a recombinant B. subtilis that can produce HA having molecular weights within specific ranges (specific-molecular-weight HA). A HA synthetic pathway is constructed and a HAase is coexpressed in the recombinant strain; and the RBS (ribosome binding sites) optimization strategy is performed to regulate the expression levels of HAase at the translational level, which in turn regulates the average molecular weight of HA products.
In one embodiment, the present invention provides a recombinant B. subtilis that the molecular weight of HA products in the recombinant strain is controlled by the expression level of HAase, wherein the higher is the expression of HAase, the lower is the molecular weight of the HA product and the higher is the yield of HA production.
In one embodiment of the present invention, a regulatory DNA fragment and a HAase gene are integrated into the genome of a B. subtilis containing a HA synthetic pathway. The said regulatory DNA fragment contains a constitutive promoter PlepA, a RBS sequence and a signal peptide. In one embodiment, the nucleotide sequences of the regulatory DNA fragment is set forth in SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 13. The DNA integration in the recombinant strain is mediated by plasmid pBlueScript SK (+).
In one embodiment of the present invention, the B. subtilis host is B. subtilis 168.
In one embodiment of the present invention, the nucleotide sequence of the HAase is SEQ ID NO: 7.
In one embodiment of the present invention, genes of the HA synthetic pathway contains hasA, which encodes a hyaluronan synthase. The hasA is derived from Streptococcus zooepidemicus, Streptococcus equi or Streptococcus equissp. 
In one embodiment of the present invention, the hasA is derived from Streptococcus zooepidemicus with a nucleotide sequence of SEQ ID NO: 1.
In one embodiment of the present invention, the HA biosynthetic pathway is obtained by further constructing a biosynthetic pathway for HA precursors, UPD-N-acetylglucosamine (UDP-GlcNAc) and UDP-D-glucuronide (UDP-GlcA), in a recombinant B. subtilis containing a hyaluronan synthase.
In one embodiment of the present invention, genes of UDP-GlcA and UDP-GlcNAc biosynthetic pathway are derived from Streptococcus species, Escherichia coli or Bacillus. In one embodiment of the present invention, the genes are derived from B. subtilis, containing tuaD (the nucleotide sequence is SEQ ID NO: 2) which encodes a UDP-glucose dehydrogenase, glmU (the nucleotide sequence is SEQ ID NO: 3) which encodes a UDP-N-acetylglucosamine pyrophosphorylase, gtaB (the nucleotide sequence is SEQ ID NO: 4) which encodes a UDP-glucose pyrophosphorylase, glmM (the nucleotide sequence is SEQ ID NO: 5) which encodes a mutase and glmS (the nucleotide sequence is SEQ ID NO: 6) which encodes an amino transferase.
In one embodiment of the present invention, the HAase gene deriving from leeches is fused with a signal peptide and a promoter, and then integrated into a recombinant B. subtilis with a HA biosynthetic pathway.
The present invention provides a method of constructing a recombinant B. subtilis that produces specific-molecular-weight HA. The method comprises the following steps:

    
    
        (1) construction of a HA biosynthetic pathway: a hyaluronan synthase hasA gene is inserted into plasmid pAX01, and the obtained recombinant plasmid is transformed into B. subtilis, resulting in the hasA gene integrated into the genome of B. subtilis under the control of Pxyl promoter. The recombinant B. subtilis strain is designated as E168T. The tuaD and glmU are respectively fused with strong ribosome binding site, P43. The gtaB was fused with promoter Pveg and P43 RBS, the glmM and glmS were respectively fused with P43 RBS. The five fused fragments are connected in series and inserted into pP43NMK vector. The resulting recombinant plasmid is transformed into E168T. A recombinant B. subtilis with a HA biosynthetic pathway is thus obtained.
        (2) The hyaluronidase gene is fused with PlepA promoter, a RBS and a signal peptide, and integrated into the genome of the recombinant B. subtilis obtained in step (1), resulting in a recombinant B. subtilis containing a HA biosynthetic pathway and HAase coexpression.
        (3) Expression levels of the HAase in step (2) are precisely controlled by the translational strength of different RBS sequences, the higher the translational strength of the RBS, the lower the molecular weight of HA produced by the recombinant B. subtilis. 
    
    


In one embodiment of the present invention, hasA gene is integrated at the lacA (β-galactosidase gene) locus of B. subtilis chromosome in the step (1).
In one embodiment of the present invention, hyaluronidase gene is integrated at the glucosamine-6-phosphate deaminase 1 (nagA-nagBA) locus of B. subtilis chromosome in the step (2).
In one embodiment of the present invention, the expression of HAase in step (2) is controlled by the wild-type RBS with a nucleotide sequence of aggaggaa (contained in the regulatory DNA fragment shown in SEQ ID NO:8). The average molecular weight of HA produced by the recombinant is 6628 dalton, the HAase activity reaches 1.62×106 U/mL and the yield of HA reaches 19.38 g/L.
In another embodiment of the present invention, the expression of HAase in step (2) is controlled by RBS mutant R1 with a nucleotide sequence of aagaggag (contained in the regulatory DNA fragment shown in SEQ ID NO:12). The average molecular weight of HA produced by the recombinant is 18,000 dalton, the HAase activity reaches 8.8×105 U/mL and the yield of HA reaches 9.18 g/L.
In another embodiment of the present invention, the expression of HAase in step (2) is controlled by RBS mutant R2 with a nucleotide sequence of acgtagac (contained in the regulatory DNA fragment shown in SEQ ID NO:13). The average molecular weight of HA produced by the recombinant is 49,600 dalton, the HAase activity reaches 6.4×104 U/mL and the yield of HA reaches 7.13 g/L.
In another embodiment of the present invention, the average molecular weight of HA produced by the recombinant strain obtained in step (1), which has no expression of HAse, is 1,420,000 dalton and the yield of HA is 5.96 g/L.
The present invention also provides a method of producing specific-molecular-weight HA (103 Da<Mr<106 Da) by using the recombinant B. subtilis strain. The method is performed by cultivating the recombinant in a 3 L fermenter with a fed-batch fermentation strategy at 37° C. for 56-96 hours, and specific-molecular-weight HA ranging from 6628 Da to 1420000 Da can be obtained. The medium for cultivation contains 5% yeast extract, 2% sucrose, 15.6 g/L sodium dihydrogen phosphate and 3.9 g/L potassium sulfate. In addition, the main products are low-molecular-weight HA (LMW-HA) or HA oligosaccharides (HA-4, HA-6, HA-8, HA-10, and so on) if secreted HAase is allowed to continue digesting the HA product after the end of fermentation.
In one embodiment, the carbon source in the cultivation medium is sucrose.
In one embodiment, the fermentation temperature is 37° C.
In one embodiment of the present invention, the fermentation time is set differently according to different recombinant strains.
After separation and purification, the HAase in the fermentation broth can be used in food, medical or clinical applications.
Compared with other engineered strains, the HA producing recombinant of the present invention has many advantages. Firstly, the present invention uses the food grade host of the recombinant strain that meets the requirements of health and food safety, and does not have any infection risk of endotoxins or pathogens. Secondly, the high molecular weight HA produced by the recombinant strain is degraded into LMW-HA by an extracellularly secreted HAase. The biosynthesis of HA is coupled with secretory expression of HAase in the present invention to reduce the viscosity of the fermentation broth, thus increasing the dissolved oxygen and enhance the yield of HA. Additionally, the molecular weight of HA in fermentation broth could be precisely controlled with a broad range from 103 to 106 Da, and purification and recovery of final products is very simple and easy to operate. Therefore, the method of the present invention has great value for large-scale production of specific low-molecular-weight hyaluronic acids.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1. Construction schematic of the regulatory DNA fragment for controlling HAase expression.
FIG. 2. High throughput screening of the different expression levels of HAase activity using a standard plate.
FIG. 3. The curve of dissolved oxygen (DO) in recombinant strains with different expression levels of HAase cultured in a 3 L fermentor.
FIG. 4. The HAase activity of recombinant strains with different expression levels of HAase cultured in a 3 L fermentor.
FIG. 5. The production of HA of recombinant strains with different expression levels of HAase cultured in a 3 L fermentor.
FIG. 6. The average molecular weight of HA produced in different recombinant strains.



EXAMPLES
Materials and Methods:
Information of related nucleotide sequences:

    
    
        (1) SEQ ID NO: 1 is the nucleotide sequence of hyaluronic acid synthase gene hasA from Streptococcus pneumoniae. 
        (2) SEQ ID NO: 2 is the nucleotide sequence of UDP-glucose dehydrogenase gene tuaD from B. subtilis. 
        (3) SEQ ID NO: 3 is the nucleotide sequence of UDP-N-acetylglucosamine pyrophosphorylase gene glmU from B. subtilis. 
        (4) SEQ ID NO: 4 is the nucleotide sequence of UDP-glucose pyrophosphorylase gene gtaB from B. subtilis. 
        (5) SEQ ID NO: 5 is the nucleotide sequence of a mutase gene glmM from B. subtilis. 
        (6) SEQ ID NO: 6 is the nucleotide sequence of an amino transferase gene glmS from B. subtilis. 
        (7) SEQ ID NO: 7 is the nucleotide sequence of a Leech hyaluronidase gene.
        (8) SEQ ID NO: 8 is the nucleotide sequence of a regulatory DNA fragment PlepA-RBS-yewA.
        (9) SEQ ID NO: 9 is the nucleotide sequence of a bleomycin resistant gene.
        (10) SEQ ID NO: 10 is the nucleotide sequence of constitutive promoter P43.
        (11) SEQ ID NO: 11 is the nucleotide sequence of inducible promoter Pveg.
        (12) SEQ ID NO: 12 is the nucleotide sequence of a regulatory DNA fragment PlepA-RBS1-yewA.
        (13) SEQ ID NO:13 is the nucleotide sequence of a regulatory DNA fragment PlepA-RBS2-yewA.
    
    


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.
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·ml−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
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·L−1 NaNO3 and the temperature of the column was maintained at 40° C. The sample size was 40 μL 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
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.
The nucleotide sequences of primers hasA-F and hasA-R were as follows (from 5′ to 3′):









hasA-F:



(SEQ ID NO: 15)



CGCGGATCCATGAGAACATTAAAAAACCTCATAAC



 



hasA-R:



(SEQ ID NO: 16)



TGCATGCATTTATAATAATTTTTTACGTGTTCC






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
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.
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.
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.
The primers used were as follows:







tuaD-F:


(SEQ ID NO: 17)


CGGGGTACCAAGAGAGGAATGTACACATGAAAAAAATAGCTGTCATTGG


 


tuaD-R:


(SEQ ID NO: 18)


CCGGAGCTCTTATAAATTGACGCTTCCCAAG


 


glmU-F:


(SEQ ID NO: 19)


CGGGAGCTCAAGAGAGGAATGTACACATGGATAAGCGGTTTGCAGTTG


 


glmU-R:


(SEQ ID NO: 20)


CCGCTCGAGCGGACTCTAGTCTAGATTATTTTTTATGAATATTTTTCAC


 


Pveg-F:


(SEQ ID NO: 21)


GGACTAGTGGAGTTCTGAGAATTGGTATGC


 


Pveg-R:


(SEQ ID NO: 22)


ATGTAAATCGCTCCTTTTTAACTAC


 


Pveg-gtaB-F:


(SEQ ID NO: 23)


GTAGTTAAAAAGGAGCGATTTACATATGAAAAAAGTACGTAAAGC


 


glmM-F:


(SEQ ID NO: 24)


GGACTAGTAAGAGAGGAATGTACACATGGGCAAGTATTTTGGAACAG


ACGG


 


glmM-R:


(SEQ ID NO: 25)


CCGCTCGAGCGGACTCTAGTCTAGATTACTCTAATCCCATTTCTGAC


CGGAC


 


glmS-F:


(SEQ ID NO: 26)


GGACTAGTAAGAGAGGAATGTACACATGTGTGGAATCGTAGGTTATA


TCGG


 


glmS-R:


(SEQ ID NO: 27)


CCGCTCGAGCGGACTCTAGTCTAGATTACTCCACAGTAACACTCTTCGC






Example 3
Construction of the Integrated Gene Fragment of LHyal
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.
The primers used were as follows:









H6LHyal-F:



(SEQ ID NO: 28)



ATGCACAGTCTGCAGAATTCCACCACCACCACCACCACATG



 



H6LHyal-R:



(SEQ ID NO: 29)



TTACTTTTTGCACGCTTCAACAT



 



ZHLHPlepA-F:



(SEQ ID NO: 30)



CGCAGCCAAAGGAGTGGATTGCCTCAATCCTAGGAGAAACAG



 



ZHLHPlepA-R:



(SEQ ID NO: 31)



GAATTCTGCAGACTGTGCATGAGC



 



ZHLH-front-F:



(SEQ ID NO: 32)



TCAGCTGGTCTAGATCACTAGTC



 



ZHLH-front-R:



(SEQ ID NO: 33)



AATCCACTCCTTTGGCTGCGCTC



 



ZHLH-zeocin-F:



(SEQ ID NO: 34)



TTGAAGCGTGCAAAAAGTAAGAGCTCGGTACCCGGGGATCC



 



ZHLH-zeocin-R:



(SEQ ID NO: 35)



GCTTGCATGCCTGCAGGTCGAC



 



ZHLH-back-F:



(SEQ ID NO: 36)



CGACCTGCAGGCATGCAAGCCACTTCTTTCAGACGGAACCCTTGC



 



ZHLH-back-R:



(SEQ ID NO: 37)



CGGTCGTTCATATAGAAGTGATAG



 



ZHLH-pSK-F:



(SEQ ID NO: 38)



CACTTCTATATGAACGACCGCCTGTGTGAAATTGTTATCCGCTC



 



ZHLH-pSK-R:



(SEQ ID NO: 39)



TAGTGATCTAGACCAGCTGAGTGACTGGGAAAACCCTGGCGTTAC






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.
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
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:







JB/lepA-RBS-R:


(SEQ ID NO: 40)


ACGGGGTACCACTNTNYNHBYACTATTAAACGCAAAATACACTAGCTTAG


 


ZHLH-H6F:


(SEQ ID NO: 41)


ACGGGGTACCATGCTAAAAAGAACTTCATTCG






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.
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 μL 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
Recombinant strains E168T/pP43-DU-PBMS, E168TH/pP43-DU-PBMS, E168THR1/pP43-DU-PBMS and E168THR2/pP43-DU-PBMS4 were fermented, respectively.
The recombinant strains were grown in a LB medium with 50 μg/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.
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·h−·L−1 for the first 4 hours. The constant feed rate was maintained at 5 g·h−·L−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·L−1 NaCl solution. The suspension was used for further determination of yield and molecular weight.
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 1.62×106U/mL, 8.8×105U/mL and 6.4×104 U/mL, respectively. The HA yield of E168T/pP43-DU-PBMS reached the maximal HA titer of 5.96 g·L−1 due to viscous fermentation. However, the HA yield of the highest HAase expression strain, E168TH/pP43-DU-PBMS, reached 19.38 g·L−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·L−1 and 7.13 g·L−1, respectively. These results demonstrated that the higher is the HAase production, the higher is the HA yield.
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 1.42×106 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.
The results showed that the molecular weight of HA could be precisely controlled within a range from 103 to 106 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.
* * *
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.










Sequence Listing















<210> 1


<211> 1254


<212> DNA


<213> Streptococcus zooepidemicus


<400> 1








atgagaacat taaaaaacct cataactgtt gtggccttta gtattttttg ggtactgttg
60


 


atttacgtca atgtttatct ctttggtgct aaaggaagct tgtcaattta tggctttttg
120


 


ctgatagctt acctattagt caaaatgtcc ttatcctttt tttacaagcc atttaaggga
180


 


agggctgggc aatataaggt tgcagccatt attccctctt ataacgaaga tgctgagtca
240


 


ttgctagaga ccttaaaaag tgttcagcag caaacctatc ccctagcaga aatttatgtt
300


 


gttgacgatg gaagtgctga tgagacaggt attaagcgca ttgaagacta tgtgcgtgac
360


 


actggtgacc tatcaagcaa tgtcattgtt caccggtcag aaaaaaatca aggaaagcgt
420


 


catgcacagg cctgggcctt tgaaagatca gacgctgatg tctttttgac cgttgactca
480


 


gatacttata tctaccctga tgctttagag gagttgttaa aaacctttaa tgacccaact
540


 


gtttttgctg cgacgggtca ccttaatgtc agaaatagac aaaccaatct cttaacacgc
600


 


ttgacagata ttcgctatga taatgctttt ggcgttgaac gagctgccca atccgttaca
660


 


ggtaatattc tcgtttgctc aggcccgctt agcgtttaca gacgcgaggt ggttgttcct
720


 


aacatagata gatacatcaa ccagaccttc ctgggtattc ctgtaagtat cggtgatgac
780


 


aggtgcttga ccaactatgc aactgattta ggaaagactg tttatcaatc cactgctaaa
840


 


tgtattacag atgttcctga caagatgtct acttacttga agcagcaaaa ccgctggaac
900


 


aagtccttct ttagagagtc cattatttct gttaagaaaa tcatgaacaa tccttttgta
960


 


gccctatgga ccatacttga ggtgtctatg tttatgatgc ttgtttattc tgtggtggat
1020


 


ttctttgtag gcaatgtcag agaatttgat tggctcaggg ttttggcctt tctggtgatt
1080


 


atcttcattg ttgctctttg tcgtaatatt cactatatgc ttaagcaccc gctgtccttc
1140


 


ttgttatctc cgttttatgg ggtactgcat ttgtttgtcc tacagccctt gaaattgtat
1200


 


tctcttttta ctattagaaa tgctgactgg ggaacacgta aaaaattatt ataa
1254


 


<211> 1386



<212> DNA



<213> Bacillus subtilis



<400> 2



atgaaaaaaa tagctgtcat tggaacaggt tatgtaggac tcgtatcagg cacttgcttt
60


 


gcggagatcg gcaataaagt tgtttgctgt gatatcgatg aatcaaaaat cagaagcctg
120


 


aaaaatgggg taatcccaat ctatgaacca gggcttgcag acttagttga aaaaaatgtg
180


 


ctggatcagc gcctgacctt tacgaacgat atcccgtctg ccattcgggc ctcagatatt
240


 


atttatattg cagtcggaac gcctatgtcc aaaacaggtg aagctgattt aacgtacgtc
300


 


aaagcggcgg cgaaaacaat cggtgagcat cttaacggct acaaagtgat cgtaaataaa
360


 


agcacagtcc cggttggaac agggaaactg gtgcaatcta tcgttcaaaa agcctcaaag
420


 


gggagatact catttgatgt tgtatctaac cctgaattcc ttcgggaagg gtcagcgatt
480


 


catgacacga tgaatatgga gcgtgccgtg attggttcaa caagtcataa agccgctgcc
540


 


atcattgagg aacttcatca gccattccat gctcctgtca ttaaaacaaa cctagaaagt
600


 


gcagaaatga ttaaatacgc cgcgaatgca tttctggcga caaagatttc ctttatcaac
660


 


gatatcgcaa acatttgtga gcgagtcggc gcagacgttt caaaagttgc tgatggtgtt
720


 


ggtcttgaca gccgtatcgg cagaaagttc cttaaagctg gtattggatt cggcggttca
780


 


tgttttccaa aggatacaac cgcgctgctt caaatcgcaa aatcggcagg ctatccattc
840


 


aagctcatcg aagctgtcat tgaaacgaac gaaaagcagc gtgttcatat tgtagataaa
900


 


cttttgactg ttatgggaag cgtcaaaggg agaaccattt cagtcctggg attagccttc
960


 


aaaccgaata cgaacgatgt gagatccgct ccagcgcttg atattatccc aatgctgcag
1020


 


cagctgggcg cccatgtaaa agcatacgat ccgattgcta ttcctgaagc ttcagcgatc
1080


 


cttggcgaac aggtcgagta ttacacagat gtgtatgctg cgatggaaga cactgatgca
1140


 


tgcctgattt taacggattg gccggaagtg aaagaaatgg agcttgtaaa agtgaaaacc
1200


 


ctcttaaaac agccagtcat cattgacggc agaaatttat tttcacttga agagatgcag
1260


 


gcagccggat acatttatca ctctatcggc cgtcccgctg ttcggggaac ggaaccctct
1320


 


gacaagtatt ttccgggctt gccgcttgaa gaattggcta aagacttggg aagcgtcaat
1380


 


ttataa
1386


 


<210> 3



<211> 1371



<212> DNA



<213> Bacillus subtilis



<400> 3



atggataagc ggtttgcagt tgttttagcg gctggacaag gaacgagaat gaaatcgaag
60


 


ctttataaag tccttcatcc agtttgcggt aagcctatgg tagagcacgt cgtggacgaa
120


 


gccttaaaat tatctttatc aaagcttgtc acgattgtcg gacatggtgc ggaagaagtg
180


 


aaaaagcagc ttggtgataa aagcgagtac gcgcttcaag caaaacagct tggcactgct
240


 


catgctgtaa aacaggcaca gccatttctt gctgacgaaa aaggcgtcac aattgtcatt
300


 


tgcggagata cgccgctttt gacagcagag acgatggaac agatgctgaa agaacataca
360


 


caaagagaag cgaaagctac gattttaact gcggttgcag aagatccaac tggatacggc
420


 


cgcattattc gcagcgaaaa cggagcggtt caaaaaatag ttgagcataa ggacgcctct
480


 


gaagaagaac gtcttgtaac tgagatcaac accggtacgt attgttttga caatgaagcg
540


 


ctatttcggg ctattgatca ggtgtctaat gataatgcac aaggcgagta ttatttgccg
600


 


gatgtcatag agattcttaa aaatgaaggc gaaactgttg ccgcttacca gactggtaat
660


 


ttccaagaaa cgctcggagt taatgataga gttgctcttt ctcaggcaga acaatttatg
720


 


aaagagcgca ttaataaacg gcatatgcaa aatggcgtga cgttgattga cccgatgaat
780


 


acgtatattt ctcctgacgc tgttatcgga agcgatactg tgatttaccc tggaactgtg
840


 


attaaaggtg aggtgcaaat cggagaagat acgattattg gccctcatac ggagattatg
900


 


aatagtgcca ttggcagccg tacggttatt aaacaatcgg tagtcaatca cagtaaagtg
960


 


gggaatgatg taaacatagg accttttgct cacatcagac ctgattctgt catcgggaat
1020


 


gaagtgaaga tcgggaattt tgtagaaatt aaaaagactc aattcggaga ccgaagcaag
1080


 


gcatctcatc taagctatgt cggcgatgct gaggtaggca ctgatgtaaa cctgggctgc
1140


 


ggttcaatta ctgtcaatta tgatggaaag aataagtatt tgacaaaaat tgaagatggc
1200


 


gcgtttatcg gctgcaattc caacttggtt gcccctgtca cagtcggaga aggcgcttat
1260


 


gtggcggcag gttcaactgt tacggaagat gtacctggaa aagcacttgc tattgccaga
1320


 


gcgagacaag taaataaaga cgattatgtg aaaaatattc ataaaaaata a
1371


 


<210> 4



<211> 879



<212> DNA



<213> Bacillus subtilis



<400> 4



atgaaaaaag tacgtaaagc cataattcca gcagcaggct taggaacacg ttttcttccg
60


 


gctacgaaag caatgccgaa agaaatgctt cctatcgttg ataaacctac cattcaatac
120


 


ataattgaag aagctgttga agccggtatt gaagatatta ttatcgtaac aggaaaaagc
180


 


aagcgtgcga ttgaggatca ttttgattac tctcctgagc ttgaaagaaa cctagaagaa
240


 


aaaggaaaaa ctgagctgct tgaaaaagtg aaaaaggctt ctaacctggc tgacattcac
300


 


tatatccgcc aaaaagaacc taaaggtctc ggacatgctg tctggtgcgc acgcaacttt
360


 


atcggcgatg agccgtttgc ggtactgctt ggtgacgata ttgttcaggc tgaaactcca
420


 


gggttgcgcc aattaatgga tgaatatgaa aaaacacttt cttctattat cggtgttcag
480


 


caggtgcccg aagaagaaac acaccgctac ggcattattg acccgctgac aagtgaaggc
540


 


cgccgttatc aggtgaaaaa cttcgttgaa aaaccgccta aaggcacagc accttctaat
600


 


cttgccatct taggccgtta cgtattcacg cctgagatct tcatgtattt agaagagcag
660


 


caggttggcg ccggcggaga aattcagctc acagacgcca ttcaaaagct gaatgaaatt
720


 


caaagagtgt ttgcttacga ttttgaaggc aagcgttatg atgttggtga aaagctcggc
780


 


tttatcacaa caactcttga atttgcgatg caggataaag agcttcgcga tcagctcgtt
840


 


ccatttatgg aaggtttact aaacaaagaa gaaatctaa
879


 


<210> 5



<211> 1347



<212> DNA



<213> Bacillus subtilis



<400> 5



atgggcaagt attttggaac agacggtgta agaggtgtcg ccaatagtga gcttacacct
60


 


gagctggcct ttaaagtcgg acgtttcggc ggttatgtgc tgacaaaaga caaacaacgt
120


 


ccaaaagtgc tgataggccg cgatacacgc atctccggcc atatgctgga gggagccctt
180


 


gtcgccggac ttttatccat tggcgcagaa gtcatgcgcc tgggtgtcat ttctacacca
240


 


ggtgtatctt atttgacaaa agcgatggat gcagaggcgg gcgtcatgat ttccgcttct
300


 


cataacccag tgcaggataa cggcatcaaa ttctttgggg gagatggatt taagctttct
360


 


gatgaacagg aggctgaaat tgagcgcctg atggacgaac ctgaggataa gctgccaaga
420


 


cctgtcggag cagaccttgg acttgtaaac gattattttg aaggcggaca aaaatatctg
480


 


caattcttaa aacagacagc tgatgaagat ttcacaggca ttcatgtggc attggactgt
540


 


gccaatggcg caacgtcatc cttggcgaca cacctgtttg ctgatttaga tgcagatgtt
600


 


tctacaatgg ggacttcccc gaacggatta aacattaatg acggcgtcgg ttcgactcat
660


 


cccgaagcgc tcagcgcgtt tgtcaaagag aaaaacgcgg atctcggtct tgcgttcgac
720


 


ggtgacggcg accgcctgat tgctgtcgat gaaaaaggaa atattgtaga cggcgaccaa
780


 


atcatgtaca tatgctcaaa acatctgaaa tcagagggcc gtttaaagga tgatacagtg
840


 


gtttcaaccg tgatgagcaa cctcggcttc tataaggcgc tcgaaaaaga aggcatcaaa
900


 


agcgtgcaga cagctgtcgg cgaccgctac gtagtagaag caatgaaaaa agacggctac
960


 


aacgtcggcg gagagcagtc aggacatctt attttccttg attacaacac gacaggggac
1020


 


ggattattgt ctgctattat gctgatgaac actttaaaag caacaggcaa gccgctgtca
1080


 


gagcttgcag ctgaaatgca gaagttcccg cagctgttag tcaatgtgag agtgactgat
1140


 


aaatataaag ttgaagaaaa tgaaaaagta aaagcagtta tttctgaagt tgaaaaagaa
1200


 


atgaacggcg acggccggat tttggtgcgc ccttcaggaa ctgaaccgct cgtccgtgtc
1260


 


atggctgaag cgaagacgaa agagctgtgc gatgagtatg tcaatcgcat tgttgaagtc
1320


 


gtccggtcag aaatgggatt agagtaa
1347


 


<210> 6



<211> 1803



<212> DNA



<213> Bacillus subtilis



<400> 6



atgtgtggaa tcgtaggtta tatcggtcag cttgatgcga aggaaatttt attaaaaggg
60


 


ttagagaagc ttgagtatcg cggttatgac tctgctggta ttgctgttgc caacgaacag
120


 


ggaatccatg tgttcaaaga aaaaggacgc attgcagatc ttcgtgaagt tgtggatgcc
180


 


aatgtagaag cgaaagccgg aattgggcat actcgctggg cgacacacgg cgaaccaagc
240


 


tatctgaacg ctcacccgca tcaaagcgca ctgggccgct ttacacttgt tcacaacggc
300


 


gtgatcgaga actatgttca gctgaagcaa gagtatttgc aagatgtaga gctcaaaagt
360


 


gacaccgata cagaagtagt cgttcaagta atcgagcaat tcgtcaatgg aggacttgag
420


 


acagaagaag cgttccgcaa aacacttaca ctgttaaaag gctcttatgc aattgcttta
480


 


ttcgataacg acaacagaga aacgattttt gtagcgaaaa acaaaagccc tctattagta
540


 


ggtcttggag atacattcaa cgtcgtagca tctgatgcga tggcgatgct tcaagtaacc
600


 


aacgaatacg tagagctgat ggataaagaa atggttatcg tcactgatga ccaagttgtc
660


 


atcaaaaacc ttgatggtga cgtgattaca cgtgcgtctt atattgctga gcttgatgcc
720


 


agtgatatcg aaaaaggcac gtaccctcac tacatgttga aagaaacgga tgagcagcct
780


 


gttgttatgc gcaaaatcat ccaaacgtat caagatgaaa acggcaagct gtctgtgcct
840


 


ggcgatatcg ctgccgctgt agcggaagcg gaccgcatct atatcattgg ctgcggaaca
900


 


agctaccatg caggacttgt cggtaaacaa tatattgaaa tgtgggcaaa cgtgccggtt
960


 


gaagtgcatg tagcgagtga attctcctac aacatgccgc ttctgtctaa gaaaccgctc
1020


 


ttcattttcc tttctcaaag cggagaaaca gcagacagcc gcgcggtact cgttcaagtc
1080


 


aaagcgctcg gacacaaagc cctgacaatc acaaacgtac ctggatcaac gctttctcgt
1140


 


gaagctgact atacattgct gcttcatgca ggccctgaga tcgctgttgc gtcaacgaaa
1200


 


gcatacactg cacaaatcgc agttctggcg gttcttgctt ctgtggctgc tgacaaaaat
1260


 


ggcatcaata tcggatttga cctcgtcaaa gaactcggta tcgctgcaaa cgcaatggaa
1320


 


gctctatgcg accagaaaga cgaaatggaa atgatcgctc gtgaatacct gactgtatcc
1380


 


agaaatgctt tcttcatcgg acgcggcctt gactacttcg tatgtgtcga aggcgcactg
1440


 


aagctgaaag agatttctta catccaggca gaaggttttg ccggcggtga gctaaagcac
1500


 


ggaacgattg ccttgatcga acaaggaaca ccagtattcg cactggcaac tcaagagcat
1560


 


gtaaacctaa gcatccgcgg aaacgtcaaa gaagttgctg ctcgcggagc aaacacatgc
1620


 


atcatctcac tgaaaggcct agacgatgcg gatgacagat tcgtattgcc ggaagtaaac
1680


 


ccagcgcttg ctccgttggt atctgttgtt ccattgcagc tgatcgctta ctatgctgca
1740


 


ctgcatcgcg gctgtgatgt ggataaacct cgtaaccttg cgaagagtgt tactgtggag
1800


 


taa
1803


 


<210> 7



<211> 1470



<212> DNA



<213> Leech



<400> 7



atgaaagaga tcgcggtgac aattgacgat aagaacgtta ttgcctctgt cagcgagtca
60


 


ttccatggtg ttgcctttga tgcgtcgtta ttttcaccga aggggttgtg gagctttgtt
120


 


gacattacct caccgaaatt gtttaaactc ttggagggtc tctctcctgg ttacttcagg
180


 


gttggaggaa cgtttgctaa ctggctgttc tttgacttag atgaaaataa taagtggaaa
240


 


gactattggg cttttaaaga taaaacaccc gagactgcaa caatcacaag gaggtggctg
300


 


tttcgaaaac aaaacaacct gaaaaaagag acttttgacg acttagtcaa actaaccaaa
360


 


ggaagcaaaa tgagactgtt atttgattta aacgctgaag tgagaactgg ttatgaaatt
420


 


ggaaagaaaa tgacatccac ttgggatagc tcggaagctg aaaaattatt caaatactgt
480


 


gtgtcaaaag gttatggaga taatattgat tgggaacttg gtaatgaacc ggaccatacc
540


 


tccgcacaca atcttactga aaagcaagtt ggagaggact ttaaagccct gcataaagtg
600


 


ctagagaaat atccgacgtt gaataaagga tcgcttgttg gacctgacgt tggatggatg
660


 


ggagtctctt atgtgaaagg attagcagac ggggctggtg atcacgtaac cgcttttact
720


 


cttcatcagt attattttga cggcaatacc tcagatgtgt caacatacct tgacgctact
780


 


tattttaaaa aacttcaaca gctgtttgac aaagttaagg atgtcttgaa aaattctcca
840


 


cataaagata aaccgctctg gcttggagaa acaagttctg gatacaacag cggcacaaaa
900


 


gatgtatccg atcgatatgt tagcggattt ctaacattgg acaagttggg actcagtgca
960


 


gcgaacaatg tgaaagttgt gataagacaa acgatctata atggatacta cggacttctt
1020


 


gataaaaata ctctagagcc aaatccggat tattggctaa tgcatgttca caattctctg
1080


 


gttggaaata cggtttttaa agttgacgtt agtgacccta caaataaagc tagagtttat
1140


 


gcacagtgca ccaaaacaaa tagcaaacat actcagagta gatactacaa gggctcattg
1200


 


acgatctttg ctcttaatgt tggagatgaa gatgtgacgt tgaagattga tcaatacagt
1260


 


ggaaaaaaga tttattcata tattctgacc ccagaaggcg gccaacttac atcacaaaaa
1320


 


gttcttttga atggaaaaga attaaaatta gtgtcggatc aattgccaga actgaatgca
1380


 


gacgagtcga aaacctcttt cactctgtct ccaaagacat ttggattttt tgttgttagc
1440


 


gatgctaacg ttgaagcctg caaaaaataa
1470


 


<210> 8



<211> 403



<212> DNA



<213> Artificial sequence



<400> 8



gcctcaatcc taggagaaac agtcacggca aaagatttag tagaaaaaca aaaagagctg
60


 


gaaaaggtgg agacattcaa tatgttttca aaagccggaa aagcgctttc ggacaccgta
120


 


accaatactg cccagtcaat gtatgaatgg atacgggata tgaatcaata agtacgtgaa
180


 


agagaaaagc aacccagata tgatagggaa cttttctctt tcttgtttta cattgaatct
240


 


ttacaatcct attgatataa tctaagctag tgtattttgc gtttaatagt aggaggaaag
300


 


tggtaccatg ctaaaaagaa cttcattcgt atcttcatta ttcatcagtt cagctgtttt
360


 


actatcaatc ttacttcctt cgggccaagc tcatgcagaa ttc
403


 


<210> 9



<211> 556



<212> DNA



<213> Artificial sequence



<400> 9



gagctcggta cccggggatc ctctagagat tctaccgttc gtatagcata cattatacga
60


 


agttatcttg atatggcttt ttatatgtgt tactctacat acagaaagga ggaactaaat
120


 


atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc
180


 


gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt
240


 


gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac
300


 


aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag
360


 


gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag
420


 


ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc
480


 


gaggagcagg actgaataac ttcgtatagc atacattata cgaacggtag aatcgtcgac
540


 


ctgcaggcat gcaagc
556


 


<210> 10



<211> 268



<212> DNA



<213> Bacillus subtilis



<400> 10



tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat acggttgatt
60


 


taataactga caaacatcac cctcttgcta aagcggccaa ggacgctgcc gccggggctg
120


 


tttgcgtttt tgccgtgatt tcgtgtatca ttggtttact tatttttttg ccaaagctgt
180


 


aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga aaaaaagcgc
240


 


gcgattatgt aaaatataaa gtgatagc
268


 


<210> 11



<211> 257



<212> DNA



<213> Bacillus subtilis



<400> 11



ggagttctga gaattggtat gccttataag tccaattaac agttgaaaac ctgcatagga
60


 


gagctatgcg ggttttttat tttacataat gatacataat ttaccgaaac ttgcggaaca
120


 


taattgagga atcatagaat tttgtcaaaa taattttatt gacaacgtct tattaacgtt
180


 


gatataattt aaattttatt tgacaaaaat gggctcgtgt tgtacaataa atgtagttaa
240


 


aaaggagcga tttacat
257


 


<210> 12



<211> 403



<212> DNA



<213> Artificial sequence



<400> 12



gcctcaatcc taggagaaac agtcacggca aaagatttag tagaaaaaca aaaagagctg
60


 


gaaaaggtgg agacattcaa tatgttttca aaagccggaa aagcgctttc ggacaccgta
120


 


accaatactg cccagtcaat gtatgaatgg atacgggata tgaatcaata agtacgtgaa
180


 


agagaaaagc aacccagata tgatagggaa cttttctctt tcttgtttta cattgaatct
240


 


ttacaatcct attgatataa tctaagctag tgtattttgc gtttaatagt aagaggagag
300


 


tggtaccatg ctaaaaagaa cttcattcgt atcttcatta ttcatcagtt cagctgtttt
360


 


actatcaatc ttacttcctt cgggccaagc tcatgcagaa ttc
403


 


<210> 13



<211> 403



<212> DNA



<213> Artificial sequence



<400> 13



gcctcaatcc taggagaaac agtcacggca aaagatttag tagaaaaaca aaaagagctg
60


 


gaaaaggtgg agacattcaa tatgttttca aaagccggaa aagcgctttc ggacaccgta
120


 


accaatactg cccagtcaat gtatgaatgg atacgggata tgaatcaata agtacgtgaa
180


 


agagaaaagc aacccagata tgatagggaa cttttctctt tcttgtttta cattgaatct
240


 


ttacaatcct attgatataa tctaagctag tgtattttgc gtttaatagt acgtagacag
300


 


tggtaccatg ctaaaaagaa cttcattcgt atcttcatta ttcatcagtt cagctgtttt
360


 


actatcaatc ttacttcctt cgggccaagc tcatgcagaa ttc
403


 


<210> 14



<211> 17



<212> DNA



<213> Artificial sequence



<400> 14



aagagaggaa tgtacac
17


 


<210> 15



<211> 35



<212> DNA



<213> DNA



<400> 15



cgcggatcca tgagaacatt aaaaaacctc ataac
35


 


<210> 16



<211> 33



<212> DNA



<213> Artificial sequence



<400> 16



tgcatgcatt tataataatt ttttacgtgt tcc
33


 


<210> 17



<211> 49



<212> DNA



<213> Artificial sequence



<400> 17



cggggtacca agagaggaat gtacacatga aaaaaatagc tgtcattgg
49


 


<210> 18



<211> 31



<212> DNA



<213> Artificial sequence



<400> 18



ccggagctct tataaattga cgcttcccaa g
31


 


<210> 19



<211> 48



<212> DNA



<213> Artificial sequence



<400> 19



cgggagctca agagaggaat gtacacatgg ataagcggtt tgcagttg
48


 


<210> 20



<211> 49



<212> DNA



<213> Artificial sequence



<400> 20



ccgctcgagc ggactctagt ctagattatt ttttatgaat atttttcac
49


 


<210> 21



<211> 30



<212> DNA



<213> Artificial sequence



<400> 21



ggactagtgg agttctgaga attggtatgc
30


 


<210> 22



<211> 25



<212> DNA



<213> Artificial sequence



<400> 22



atgtaaatcg ctccttttta actac
25


 


<210> 23



<211> 45



<212> DNA



<213> Artificial sequence



<400> 23



gtagttaaaa aggagcgatt tacatatgaa aaaagtacgt aaagc
45


 


<210> 24



<211> 51



<212> DNA



<213> Artificial sequence



<400> 24



ggactagtaa gagaggaatg tacacatggg caagtatttt ggaacagacg g
51


 


<210> 25



<211> 52



<212> DNA



<213> Artificial sequence



<400> 25



ccgctcgagc ggactctagt ctagattact ctaatcccat ttctgaccgg ac
52


 


<210> 26



<211> 51



<212> DNA



<213> Artificial sequence



<400> 26



ggactagtaa gagaggaatg tacacatgtg tggaatcgta ggttatatcg g
51


 


<210> 27



<211> 49



<212> DNA



<213> Artificial sequence



<400> 27



ccgctcgagc ggactctagt ctagattact ccacagtaac actcttcgc
49


 


<210> 28



<211> 41



<212> DNA



<213> Artificial sequence



<400> 28



atgcacagtc tgcagaattc caccaccacc accaccacat g
41


 


<210> 29



<211> 23



<212> DNA



<213> Artificial sequence



<400> 29



ttactttttg cacgcttcaa cat
23


 


<210> 30



<211> 42



<212> DNA



<213> Artificial sequence



<400> 30



cgcagccaaa ggagtggatt gcctcaatcc taggagaaac ag
42


 


<210> 31



<211> 24



<212> DNA



<213> Artificial sequence



<400> 31



gaattctgca gactgtgcat gagc
24


 


<210> 32



<211> 23



<212> DNA



<213> Artificial sequence



<400> 32



tcagctggtc tagatcacta gtc
23


 


<210> 33



<211> 23



<212> DNA



<213> Artificial sequence



<400> 33



aatccactcc tttggctgcg ctc
23


 


<210> 34



<211> 41



<212> DNA



<213> Artificial sequence



<400> 34



ttgaagcgtg caaaaagtaa gagctcggta cccggggatc c
41


 


<210> 35



<211> 22



<212> DNA



<213> Artificial sequence



<400> 35



gcttgcatgc ctgcaggtcg ac
22


 


<210> 36



<211> 45



<212> DNA



<213> Artificial sequence



<400> 36



cgacctgcag gcatgcaagc cacttctttc agacggaacc cttgc
45


 


<210> 37



<211> 24



<212> DNA



<213> Artificial sequence



<400> 37



cggtcgttca tatagaagtg atag
24


<210> 38



<211> 44



<212> DNA



<213> Artificial sequence



<400> 38



cacttctata tgaacgaccg cctgtgtgaa attgttatcc gctc
44


 


<210> 39



<211> 45



<212> DNA



<213> Artificial sequence



<400> 39



tagtgatcta gaccagctga gtgactggga aaaccctggc gttac
45


 


<210> 40



<211> 50



<212> DNA



<213> Artificial sequence



<221> misc_feature



<222> (14)..(14)



<223> n is a, c, g, t or u



 


<221> misc_feature



<222> (16)..(16)



<223> n is a, c, g, t or u



 


<221> misc_feature



<222> (18)..(18)



<223> n is a, c, g, t or u



 


<400> 40



acggggtacc actntnynhb yactattaaa cgcaaaatac actagcttag
50


 


<210> 41



<211> 32



<212> DNA



<213> Artificial sequence



<400> 41



acggggtacc atgctaaaaa gaacttcatt cg
32

## Claims

1. A recombinant Bacillus subtilis bacterium having a hyaluronic acid (HA) biosynthetic pathway, which is further transformed to express and secrete hyaluronidase.

2. The Bacillus subtilis bacterium of claim 1, wherein the bacterium is transformed with a DNA fragment having a constitutive promoter and a ribosome binding site sequence and encoding a signal peptide and hyaluronidase.

3. The Bacillus subtilis bacterium of claim 2, wherein the expression of hyaluronidase is regulated by ribosomal binding sites having different translational strengths.

4. The Bacillus subtilis bacterium of claim 2, wherein the DNA fragment comprises the nucleic acid sequence of SEQ ID NO: 8, SEQ ID NO: 12, or SEQ ID NO: 13.

5. The Bacillus subtilis bacterium of claim 1, wherein said HA biosynthetic pathway comprises a heterologous hyaluronan synthase hasA gene derived from Streptococcus zooepidemicus, Streptococcus equi, or Streptococcus equissp.

6. The Bacillus subtilus bacterium of claim 1, wherein said HA biosynthetic pathway comprises a UDP-glucose dehydrogenase gene tauD, UDP-N-acetylglucosamine pyrophosphorylase gene glmU, UDP-glucose pyrophosphorylase gene gtaB, mutase gene glmM, and amino transferase gene glmS, said genes derived from Steptococcus species, Escherichia coli, and/or Bacillus species.

7. A method for making a Bacillus subtilis bacterium cell culture capable of providing specific molecular weight hyaluronic acid, said method comprising the steps of:
1) transforming said bacterium with a hasA gene which encodes a hyaluronan synthase, said gene is integrated into the chromosome of said bacterium by use of plasmid pAX01:
2) transforming said bacterium with genes 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, said genes are connected in series and inserted into vector pP43NMK, said vector is transformed into the bacterium;
3) coexpressing in said bacterium a hyaluronidase gene fused with a regulatory DNA fragment containing a promoter, a ribosome binding site (RBS) sequence and encoding a signal peptide, said DNA fragment is integrated onto the chromosome of the bacterium;
wherein said bacterium expresses hyaluronic acid that is acted on by the hyaluronidase to provide specific molecular weight hyaluronic acid in said cell culture.

8. The method of claim 7 further comprising regulating expression levels of hyaluronidase by using ribosomal binding site mutants with different translational strengths to control the expression levels of hyaluronidase.

9. The method of claim 7, wherein said Bacillus subtilis bacterium cell culture is fermented at 30-37° C. and pH 6.0-7.0 for 48-96 hours with glucose or sucrose as the carbon source for fermentation.

10. A method of producing specific-molecular-weight HA or HA oligosaccharides using a recombinant Bacillus subtilis bacterium of claim 1, comprising the steps of:
a) culturing said recombinant Bacillus subtilis bacterium at 30-37° C. and pH 6.0-7.0 for 48-96 hours with glucose or sucrose as the carbon source; and
b) purifying specific-molecular-weight HA or HA oligosaccharides from the culture of said recombinant Bacillus subtilis bacterium.