WO2020122430A1 - Système d'expression pour la production d'acide hyaluronique à l'aide de bactéries non pathogènes et procédé de production d'acide hyaluronique utilisant ledit système d'expression - Google Patents

Système d'expression pour la production d'acide hyaluronique à l'aide de bactéries non pathogènes et procédé de production d'acide hyaluronique utilisant ledit système d'expression Download PDF

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WO2020122430A1
WO2020122430A1 PCT/KR2019/015082 KR2019015082W WO2020122430A1 WO 2020122430 A1 WO2020122430 A1 WO 2020122430A1 KR 2019015082 W KR2019015082 W KR 2019015082W WO 2020122430 A1 WO2020122430 A1 WO 2020122430A1
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hyaluronic acid
expression system
promoter
strain
gene
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Korean (ko)
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고건
최영준
이인현
장준희
이한원
이한구
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대화제약 주식회사
(주)리독스바이오
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Priority claimed from KR1020190016267A external-priority patent/KR102152625B1/ko
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Priority to CN201980077146.7A priority Critical patent/CN113166735A/zh
Publication of WO2020122430A1 publication Critical patent/WO2020122430A1/fr

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins

Definitions

  • the present invention relates to an expression system using a constitutive expression promoter for producing hyaluronic acid using a non-pathogenic bacterium, a non-pathogenic bacterium comprising the expression system, and a method for producing hyaluronic acid using the same.
  • Hyaluronic acid is a biopolymer composed of disaccharide units of D-gluconic acid and N-acetyl-D-glucosamine, according to molecular weight, filler for molding, and treatment for arthritis And anti-adhesion agents.
  • the hyaluronic acid may be produced through fermentation of the Streptococcus spp. strain, and the Streptococcus strain is an infectious microorganism, and there is a possibility that pyrogens and the like are contaminated in the purification process.
  • a method of producing hyaluronic acid by transforming a GRAS (Generally Recognized As Safe) strain with a recombinant vector has been developed.
  • An object of the present invention is to provide an expression system or recombinant vector capable of synthesizing hyaluronic acid without a derivative in a non-pathogenic strain.
  • Another object of the present invention is to provide a method for producing hyaluronic acid using the strain and the strain transformed by the recombinant vector or the strain into which the expression system is introduced.
  • the present invention is an expression system for production of hyaluronic acid comprising a UDP-glucose 6-dehydrogenase gene and a hyaluronic acid synthase gene, preferably, a operably linked transcription promoter, a hyaluronic acid synthase gene UDP-glucose 6 -Dehydrogenase gene ribosome binding site (RBS), UDP-glucose 6- It provides an expression system for producing hyaluronic acid containing a 6-dehydrogenase gene.
  • the present inventors tried to make a system for constant expression with high production yield of hyaluronic acid (constitutive expression system), and selected optimal promoters through various promoter selection as described below, and also tuaD in an operon composed of hasA gene and tuaD gene
  • RBS ribosome binding sites
  • An example of the present invention relates to an expression system for producing hyaluronic acid, comprising a transcription promoter, a ribosome binding site, a UDP-glucose 6-dihydrogenase gene, and a hyaluronic acid synthase gene, which are operably linked.
  • the UDP-glucose 6-dehydrogenase gene and the hyaluronic acid synthase gene preferably constitute one operon, and more preferably, the hyaluronic acid synthase gene sequentially in the 5'to 3'direction, UDP-glucose RBS of the 6-dehydrogenase gene and UDP-glucose 6-dehydrogenase gene may be linked.
  • Another embodiment of the present invention relates to a transforming strain for producing hyaluronic acid, preferably a non-pathogenic bacterium, comprising the expression system for producing hyaluronic acid.
  • a further example relates to a hyaluronic acid-producing transforming strain comprising the expression system for producing hyaluronic acid, preferably a composition for producing hyaluronic acid comprising non-pathogenic bacteria.
  • the present invention relates to a method for producing hyaluronic acid comprising the step of culturing a transforming strain for producing hyaluronic acid, preferably a non-pathogenic bacterium, comprising the expression system for producing hyaluronic acid.
  • the expression system for producing hyaluronic acid according to the present invention improves the production yield of hyaluronic acid and the molecular weight of hyaluronic acid, increases the safety in synthesizing hyaluronic acid, and reduces production cost by not using expensive IPTG derivatives.
  • the hyaluronic acid produced according to the present invention has a merit of excellent moisturizing effect, viscosity increase, joint lubrication action, water absorption ability, elastic ability, etc. in a molecular weight range of 500 kDa to 10000 kDa.
  • An example of the present invention is an expression system for producing hyaluronic acid, preferably comprising a UDP-glucose 6-dehydrogenase gene and a hyaluronic acid synthase gene. It relates to an expression system for producing hyaluronic acid comprising a transcriptional promoter, a ribosome binding site (RBS), a UDP-glucose 6-dehydrogenase gene, and a hyaluronic acid synthase gene, which are operably linked.
  • RBS ribosome binding site
  • the expression system for producing hyaluronic acid includes both the UDP-glucose 6-dihydrogenase and hyaluronic acid synthase gene necessary for the synthesis of hyaluronic acid, and provides the RBS and constitutive expression promoters required for expression of the genes. By providing, it is possible to produce hyaluronic acid without a separate derivative.
  • the expression system for producing hyaluronic acid according to the present invention may be applied to a strain of the genus Bacillus, and may be, for example, Bacillus subtilis or Bacillus licheniformis, but is not limited thereto.
  • the transcription promoter applicable to the expression system of the present invention may be a constitutive expression promoter used in Bacillus strains, and the expression system produces hyaluronic acid synthase that is always expressed without an expression inducer.
  • the transcription promoter may be one having a high transcription level having a hyaluronic acid production amount of 1.1 to 10 times compared to a transformed strain having an expression system including the P43 promoter.
  • the constant expression promoter may be, for example, P43, Pmsm, Ppbp, Pylb, Pyob, Pyqe or Pyvl, preferably Psigx, Pyob, or Pyqe, but is not limited thereto, and compared to an inducible promoter. Any similar or high hyaluronic acid yield can be obtained and used without limitation.
  • the Psigx promoter may be obtained by PCR from the genome of Bacillus subtilis 168 strain (Bacillus Genetic Stock Center) with primers of SEQ ID NOs: 53 and 54.
  • the Psigx, Pyob, or Pyqe promoter may each include a nucleotide sequence of SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO: 64, respectively.
  • Specific examples of the promoters usable in the present invention are shown in Table 1 below, and specific primer sets used for the production of each promoter are shown in Table 2 below.
  • the hyaluronic acid yield was almost the same as when using an inducible promoter requiring an IPTG derivative (FIG. 4). Accordingly, it can be seen that the Psigx promoter is a promoter for constant expression suitable for hyaluronic acid production.
  • the transcription promoter is 1.1 to 10 times, 1.15 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times the hyaluronic acid production amount compared to a transformed strain having an expression system containing the P43 promoter.
  • Pear 5-10 times, 1.1-9 times, 1.15-9 times, 1.5-9 times, 2-9 times, 3-9 times, 4-9 times, 5-9 times, 1.1-8 times, 1.15-8 Pear, 1.5-8 times, 2-8 times, 3-8 times, 4-8 times, 5-8 times, 1.1-7 times, 1.15-7 times, 1.5-7 times, 2-7 times, 3-7 Pear, 4 to 7 times, 5 to 7 times, 1.1 to 6.5 times, 1.15 to 6.5 times, 1.5 to 6.5 times, 2 to 6.5 times, 3 to 6.5 times, 4 to 6.5 times, 5 to 6.5 times, 1.1 to 6 times Pear, 1.15 to 6 times, 1.5 to 6 times, 2 to 6 times, 3 to 6 times, 4 to 6 times, 5 to 6 times, 1.1 to 5.5 times, 1.15 to 5.5 times, 1.5 to 5.5 times, 2 to 5.5 times It may be a fold, 3 to 5.5 fold, 4 to 5.5 fold, or 5 to 5.5 fold promoter.
  • the ribosome binding site (RBS) applicable to the expression system of the present invention is capable of producing hyaluronic acid in Bacillus by expressing the tuaD gene together with the hasA gene.
  • the RBS may be capable of translating the UDP-glucose 6-dehydrogenase coding gene to a high level, and the ribosome binding site is 1.1 to 3 times, 1.15 to 3 times, and 1.2 to 1 when compared with the tuaD RBS.
  • the RBS, BBa_B0030, BBa_B0031, BBa_B0032, BBa_B0033, BBa_B0034, BBa_B0035, RBS of the tuaD gene (tuaD RBS), or may be RBS of the pET plasmid, each comprising a nucleotide sequence of SEQ ID NOs: 65-72 It may be, and if specifically indicated, it is as shown in Table 3 below.
  • BBa_B0034 when BBa_B0034 was used as RBS, it exhibited a better yield than when using tuaD RBS, and unlike BBA_B0035 was previously known to exhibit superior expression efficiency compared to BBa_B0034, the BBa_B0034 RBS sequence was used. When shown, the highest yield of hyaluronic acid was produced (FIG. 5).
  • the expression system for hyaluronic acid production includes a UDP-glucose 6-dehydrogenase gene and a hyaluronic acid synthase gene, and the two The gene is preferably composed of one operon, more preferably 5'to 3'direction sequentially hyaluronic acid synthase gene, RBS and UDP-glucose 6-di of UDP-glucose 6-dehydrogenase gene It may be an operon to which the hydrogenase gene is linked.
  • the UDP-glucose 6-dehydrogenase gene according to the present invention may be, for example, a tuaD gene or a variant thereof.
  • the tuaD gene may be a tuaD gene derived from a species known to have a tuaD gene, without limitation, and may be, for example, a Bacillus strain, preferably a tuaD gene derived from Bacillus subtilis.
  • the tuaD gene may be a tuaD gene of Bacillus subtilis 2217 strain, but is not limited thereto, and may be a tuaD gene into which an appropriate mutation is introduced, if necessary, of UDP-glucose 6-dehydrogenase. It can be freely modified and used within a range that does not affect the activity.
  • the tuaD gene may include the nucleotide sequence of SEQ ID NO: 73.
  • the ribosomal binding site and tuaD were obtained from a Bacillus subtilis 2217 strain through a polymerase chain reaction (PCR) using primer pairs of SEQ ID NOs: 37 and 38.
  • the hyaluronic acid synthase gene may be, for example, a hasA gene or a variant gene thereof.
  • the hasA gene may use a hasA gene derived from a species known to have a hasA gene without limitation, for example, a strain of the genus Streptococcus, preferably a strain derived from Streptococcus juepidemicus.
  • the mutant gene of the hasA gene may include a mutation on all genes in a range in which hyaluronic acid synthesis activity is maintained.
  • the hasA gene may be a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 74 or 76, and preferably may include the nucleotide sequence of SEQ ID NO: 75 or 77.
  • a hasA gene is obtained through a PCR-based two-step DNA synthesis method using the primers of SEQ ID NOs: 1 to 36 (Table 4) from Streptococcus jupipidemicus. Did.
  • DNA fragments 1 were prepared using primers of SEQ ID NOs: 1 to 12, and DNA fragments 2 and 3 were produced using SEQ ID NOs: 12 to 24 and SEQ ID NOs: 25 to 36, respectively.
  • the obtained DNA fragment 1, fragment 2 and fragment 3 were mixed and PCR was performed using a primer pair consisting of SEQ ID NO: 1 and SEQ ID NO: 36.
  • An example of the present invention may be a transformation strain or a recombinant strain for producing hyaluronic acid, including the expression system for producing hyaluronic acid.
  • the strain may be a GRAS grade strain, and may be a Gram-positive bacterium, for example, Bacillus strain, preferably Bacillus subtilis or Bacillus licheniformis .
  • Bacillus strain preferably Bacillus subtilis or Bacillus licheniformis
  • the expression system for producing hyaluronic acid was introduced into the Bacillus subtilis 2217 strain to obtain a hyaluronic acid producing strain without a derivative such as IPTG.
  • the present invention relates to a method for producing hyaluronic acid using a non-pathogenic bacterium comprising culturing a transforming strain for producing hyaluronic acid containing the hyaluronic acid expression system. More specifically, the method for producing hyaluronic acid according to the present invention may further include the step of separating and/or purifying hyaluronic acid in addition to the step of culturing the transforming strain for producing hyaluronic acid, for example, in a culture medium. The method may include removing the strain and precipitating hyaluronic acid in the culture medium from which the strain is removed.
  • a transcription promoter In the transforming strain for producing hyaluronic acid and the method for producing hyaluronic acid, a transcription promoter, a hyaluronan synthase gene, a ribosome binding site for UDP-glucose 6-dehydrogenase gene expression, and UDP-glucose 6-di Hydrogenase genes and the like are as described above.
  • the method for producing hyaluronic acid using the recombinant strain according to the present invention may exhibit a hyaluronic acid yield equal to or higher than that of using an inducible promoter through an expression promoter at all times without a derivative such as IPTG.
  • the step of culturing the strain may use sucrose as a carbon source, but is not limited thereto.
  • the culture of the strain, the removal of the strain and the precipitation step of hyaluronic acid can be performed by methods known in the art, and can be used by appropriate modifications by a person skilled in the art as necessary.
  • the method of producing hyaluronic acid may further include a step of concentration, purification, or concentration and purification of hyaluronic acid after the precipitation step of hyaluronic acid.
  • the hyaluronic acid obtained using the above production method may have a molecular weight of 100 to 10,000 kDa, 500 to 10,000 kDa, 500 to 8,000 kDa, 3,000 to 8,000 kDa, or 5,000 to 6,000 kDa.
  • a hyaluronic acid having a maximum peak of 5,455 kDa from Bacillus bacteria incorporating the hyaluronic acid synthesis system.
  • the polymer hyaluronic acid has excellent properties such as moisturizing effect, viscosity increase, joint lubrication, water absorption ability, elasticity, etc., compared to low molecular hyaluronic acid. Therefore, high-molecular hyaluronic acid has high utility value as a medicine, such as injections for knee joints, eye drops, and fillers for molding.
  • ultra-high molecular weight hyaluronic acid of 3000 kDa or higher such as hyaluronic acid produced by using the expression system provided by the present invention, can be used as an anti-adhesion agent due to a slow decomposition rate in the body.
  • the strain for synthesizing hyaluronic acid of the present invention is a non-pathogenic strain, which increases safety during hyaluronic acid synthesis, and does not use expensive IPTG derivatives as an expression inducing agent, thereby reducing production costs.
  • 1 shows a vector map of the pHCMC02-hasA-RBS34-tuaD plasmid prepared according to an example of the present invention.
  • Figure 2 is a schematic diagram of the cloning process for the production of pHCMC02-hasA-RBS34-tuaD plasmid prepared according to an example of the present invention.
  • FIG. 3 is a graph showing the concentration of hyaluronic acid produced after introducing an expression system including various promoters into a Bacillus strain according to an embodiment of the present invention.
  • FIG. 4 is a graph showing the concentration of hyaluronic acid produced in the case of using the always-expressing promoter Psigx and the IPTC-inducing promoter Pgrac according to an embodiment of the present invention.
  • FIG. 5 is a graph showing the relative concentration of hyaluronic acid produced when various ribosome binding sites are used according to an embodiment of the present invention.
  • FIG. 6 is a diagram showing the results of infrared spectrum analysis of hyaluronic acid purified from a culture of a recombinant strain according to an example of the present invention and a commercially available hyaluronic acid standard.
  • Streptococcus zooepidemicus-derived hyaluronic acid synthase gene (hasA, Genbank No. AY173078 base sequences 1 to 1254) (SEQ ID NO: 75) shows the primers from SEQ ID NOS: 1 to 36 shown in Table 4 It was synthesized by PCR-based two-step DNA synthesis (PTDS; Xiong, 2004, Nucleic Acids Research 32:e98). Specifically, DNA fragments 1 were prepared using SEQ ID NOs: 1 to 12, and DNA fragments 2 and 3 were prepared using SEQ ID NOs: 13 to 24 and 25 to 36, respectively.
  • the obtained DNA fragment 1, fragment 2 and fragment 3 were mixed and PCR was performed using a primer pair consisting of SEQ ID NO: 1 and SEQ ID NO: 36 to obtain the hasA gene.
  • PCR conditions were performed 25 times in total for 15 seconds at 94°C, 15 seconds at 55°C, 15 minutes at 55°C, and stretching at 72°C for 1 minute and 30 seconds using Veriti® Thermal Cycler (applied biosystem).
  • Veriti® Thermal Cycler applied biosystem.
  • As the hyaluronic acid synthase gene a hasA gene derived from Streptococcus jupie epidemius was used, and the full length hasA gene was obtained using primers (SEQ ID NO: 4) of SEQ ID NOS: 1 to 36 in the manner described above.
  • the obtained full-length hasA gene was digested with restriction enzymes BamHI and XbaI, and linked to a pHCMC02 (Bacillus Genetic Stock Center) plasmid digested with BamHI and XbaI using T4 DNA ligase (NEB).
  • the vector was introduced into E. coli DH5alpha (Enzynomics), and the plasmid pHCMC02-hasA was isolated from the ampicillin-resistant transformant obtained by plating on a plate medium containing ampicillin.
  • the obtained plasmid pHCMC02-hasA was confirmed that the normal hasA gene was cloned through sequencing.
  • the DNA of Bacillus subtilis 2217 strain (Bioresource Center (KCTC)) is used as a template for the RBS_tuaD_forward primer of SEQ ID NO: 37 and SEQ ID NO: 38.
  • RBS_tuaD_reverse primer the tuaD gene was amplified to include RBS34 (BioBrick BBa_B0034) at the 5'-end of the tuaD gene. PCR was performed 30 times in total by denaturing at 94°C for 15 seconds, binding at 55°C for 15 seconds, and stretching at 72°C for 1 minute and 30 seconds using a Veriti® Thermal Cycler (applied biosystem).
  • SEQ ID NO: 37 5'-aatctagaaagaggagaaatactagatgaaaaaatagctgtcattgg-3'
  • SEQ ID NO: 38 5'-gggttataaattgacgcttcccaagtctttagccaatt-3'
  • the amplified RBS34-tuaD gene was digested with restriction enzymes XbaI, and linked to pBluescriptII SK+ (Stratagene) plasmids cut with XbaI and SmaI using T4 DNA ligase (NEB). This was introduced into E. coli DH5alpha (Enzynomics), and the plasmid pBSIISK-RBS34-tuaD was isolated from the ampicillin-resistant transformant obtained by plating on a plate medium containing ampicillin. The obtained plasmid pBSIISK-RBS34-tuaD was analyzed by sequencing to Genbank No. It was confirmed that the base sequences of AF015609 3599 to 4984 bp (protein coding region of the tuaD gene, SEQ ID NO: 73) were cloned normally.
  • the pBSIISK-RBS34-tuaD obtained in Example 1-2 was digested with restriction enzymes XbaI and SmaI, and the truncated RBS34-tuaD gene was treated with the same restriction enzyme.
  • the pHCMC02-hasA plasmid obtained in 1-1 was ligated using T4 DNA ligase (NEB). This was introduced into E. coli DH5alpha (Enzynomics), and the plasmid pHCMC02-hasA-RBS34-tuaD plasmid was isolated from the ampicillin resistant transformant obtained by plating on a plate medium containing ampicillin.
  • 1 and 2 show schematic diagrams of the vector map and cloning process for the pHCMC02-hasA-RBS34-tuaD, respectively.
  • Example 2 Selection of a promoter for the expression of hasA-tuaD operon
  • the expression of the hasA-tuaD operon is regulated by the PlepA promoter, which is known to have weak activity. Accordingly, the promoter having high hasA-tuaD operon expression activity was selected by replacing the PlepA promoter with various promoters.
  • the candidate promoters were selected as those having higher expression activity than P43, which is a constitutive expression promoter used in Bacillus strains (Yu, 2015, Scientific Reports, 5:18405; Song, 2016, PLoS One. 11:e0158447).
  • each promoter was amplified by PCR using the primers shown in Table 2 above as a template for Bacillus subtilis 168 strain DNA (Bacillus Genetic Stock Center). Specifically, forward and reverse primers of the PP43 promoter (SEQ ID NOs: 39 and 40), forward and reverse primers of the Pmsm promoter (SEQ ID NOs: 41 and 42), forward and reverse primers of the Ppbp promoter (SEQ ID NOs: 43 and 44), Forward and reverse primers of the Pylb promoter (SEQ ID NOs: 45 and 46), forward and reverse primers of the pyob promoter (SEQ ID NOs: 47 and 48), forward and reverse primers of the Pyqe promoter (SEQ ID NOs: 49 and 50), and Pyvl promoter Forward and reverse primers (SEQ ID NOs: 51 and 52), forward and reverse primers (SEQ ID NOs: 53 and 54) of the Psigx promoter were used.
  • Each promoter amplified through PCR was digested with restriction enzymes NheI and BamHI, and linked to pHCMC02-hasA-RBS34-tuaD of Example 1 digested with the same restriction enzyme using T4 DNA ligase (NEB). This was introduced into E. coli DH5alpha (Enzynomics), and each plasmid was isolated from the ampicillin-resistant transformant obtained by plating on a plate medium containing ampicillin. Through sequencing, it was confirmed that each promoter was cloned normally into each isolated plasmid.
  • Plasmids having different promoters were introduced into the Bacillus 2217 strain by electroporation (Sun, 2015, Applied Microbiology and Biotechnology, 99:5151-5162) to prepare transforming strains having chloroamphenicol resistance.
  • each transformed strain was inoculated into LB medium and cultured overnight. 50 mL of 50 mM potassium phosphate (pH7.) containing 20 mL sucrose medium (50 g sucrose per 1 L, 20 g yeast extract, 1.5 g magnesium sulfate (MgSO4)) in a 250 mL Erlenmeyer flask containing 0.2 mL of overnight cultured strain. After inoculation at 0)), the cells were shaken and cultured at 180 rpm at a temperature of 37° C. Each culture was taken at 65 hours after the start of culture, and centrifuged at 10,000 rpm for 1 minute, and then passed through a 0.45 ⁇ m filter to remove the strain.
  • 50 mM potassium phosphate pH7.
  • sucrose medium 50 g sucrose per 1 L, 20 g yeast extract, 1.5 g magnesium sulfate (MgSO4)
  • MgSO4 magnesium sulfate
  • hyaluronic acid was precipitated by centrifugation at 15,000 rpm for 10 minutes at a temperature of 4°C. After drying the precipitated hyaluronic acid and dissolving it in water, the hyaluronic acid content was measured using a HA quantitative Test Kit (Corgenix, Riverside, CO, USA), and containing P43, which is a constant expression promoter.
  • the content of hyaluronic acid produced by the transformed strain (g/L) is set to 100, and the content of hyaluronic acid produced by the transformed strain containing the test promoter is relatively displayed, and the percentages thereof are shown in Table 5 and FIG. 3. Showed.
  • the Psigx promoter of the pSigx-hasA-RBS34-tuaD plasmid was used as the IPTG-derived Pgrac Pgrac-hasA-RBS34-tuaD was produced by replacing with a promoter.
  • the pHT01 plasmid (Mobitec) was cut with restriction enzymes NheI and BamHI to separate the Laci and Pgrac promoters, and the T4 DNA was cut into pSigx-hasA-RBS34-tuaD with the same restriction enzyme removed to remove the Psigx promoter. Connection was made using ligase (NEB). This was introduced into E. coli DH5alpha (Enzynomics), and the Pgrac-hasA-RBS34-tuaD plasmid was isolated from an ampicillin-resistant transformant obtained by plating on a plate medium containing ampicillin. The isolated plasmid was introduced into the Bacillus 2217 strain by electroporation, and a transformant strain having chloramphenicol resistance was completed.
  • Each strain was cultured in substantially the same manner as in Example 2, and the culture solution was taken at 65 hours of culture. However, in the case of the IPTG-derived type, the strain cultured overnight was inoculated into sucrose medium to induce the expression of syntheses, and IPTG was added so that IPTG became 0.5 mM after 2 hours, and in the case of the induced type, the culture solution was taken at 72 hours. Did.
  • the results of measuring the hyaluronic acid production of the two strains in substantially the same manner as in Example 2 are shown in FIG. 4.
  • Example 4 RBS screening for overexpression of tuaD gene
  • D-glucuronic acid is a component of hyaluronic acid and can be produced by the tuaD gene originally possessed by Bacillus, but over-expression of the tuaD gene is required for efficient hyaluronic acid production.
  • the hasA gene and the tuaD gene are produced in the form of an operon to induce overexpression of the tuaD gene together with the hasA gene.
  • a highly active RBS sequence exists at the 5'end of the tuaD gene, and thus the translation of the tuaD gene must be controlled.
  • RBS Since the activity of RBS is sequence context-dependent to the surrounding sequence, it may vary according to the sequence of the gene to be regulated (Mutalik, 2013, Nature Methods, 10:347-353). For this reason, it is advantageous for the translation of the actual tuaD, and as a result, an RBS selection process suitable for hyaluronic acid production was performed.
  • RBS RBS screening
  • 6 synthetic RBSs BBa_B0030, BBa_B0031, BBa_B0032, BBa_B0033, BBa_B0034, BBa_B0035
  • native RBS tuaD RBS
  • RBS plasmids commonly used as pET RBS
  • Table 8 shows the 8 RBS sequences tested.
  • PCR was performed using the DNA of Bacillus subtilis 168 strain (Bacillus Genetic Stock Center) as a template using the primers shown in Table 4 and SEQ ID NO: 38. .
  • the tuaD gene containing each amplified RBS was digested with XbaI, and then cut with XbaI and SmaI to connect pSigx-hasA-RBS34-tuaD plasmid with RBS34-tuaD removed using T4 DNA ligase (NEB).
  • NEB T4 DNA ligase
  • Plasmids having different RBSs obtained above were introduced into Bacillus subtilis 2217 strain by electroporation, and transformed strains having chloroamphenicol resistance were prepared. Next, the method and practical method of Example 2 were prepared. In the same way, each transformed strain was cultured and the culture solution was taken to measure the hyaluronic acid content, and the content (g/L) of the hyaluronic acid produced by the transformed strain using RBS of the pET plasmid was set to 100, The hyaluronic acid content produced by the transformed strain containing the test RBS sequence is relatively displayed, and the results are shown in Table 7 and FIG. 5.
  • BBa_B0035 has more expression efficiency compared to BBa_B0034 (corresponding to RBS of Example 1-2). It is known to be excellent.
  • the expression system according to the present invention confirmed the highest yield of hyaluronic acid production when using the BBa_B0034 RBS sequence.
  • the hyaluronic acid produced in the same manner as the method described in Example 2 was purified through ultrafiltration, and then the molecular weight was measured.
  • the process of purifying the produced hyaluronic acid is as follows.
  • the culture solution was centrifuged at 10,000 rpm for 10 minutes and then passed through a 0.45 ⁇ m filter to remove the strain.
  • the culture solution from which the strain was removed was filtered through an ultrafiltration membrane having a cut-off value of 100 kDa to obtain a product.
  • Cetyl trimethyl ammonium bromide was added to a concentration of 1% (v/v) in the obtained total product, followed by stirring and centrifuging for 1 hour (7,000 rpm, 30 minutes) to obtain a precipitate.
  • the precipitate was dissolved in a 0.25M sodium iodide solution for 10 minutes and dissolved to allow cetyl trimethyl ammonium bromide to react with iodine and sodium.
  • the reaction solution was centrifuged (7,000 rpm, 30 minutes) and the supernatant was taken to remove the reactant of cetyl trimethyl ammonium bromide and sodium iodide.
  • Purified samples were obtained by adding 2% activated carbon to the supernatant and stirring for 1 hour to adsorb impurities and passing through a 0.22 ⁇ m filter.
  • the purified sample was confirmed to be consistent with the hyaluronic acid standard (sigma) through an infrared spectrum, and the spectrum analysis results are shown in FIG. 6.
  • the molecular weight of the purified hyaluronic acid showed a peak in the range of 1,000 to 7,000 kDa belonging to the ultra-high molecular range, and the main peak was measured at 5,455 kDa.
  • the polymer hyaluronic acid has excellent properties such as moisturizing effect, viscosity increase, joint lubrication, water absorption ability, elasticity, etc., compared to low molecular hyaluronic acid.
  • the decomposition rate in the body is slow and may be used as an anti-adhesion agent.

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Abstract

La présente invention concerne un système d'expression de la synthase d'acide hyaluronique permettant la synthèse d'acide hyaluronique dans des souches non pathogènes et une expression constitutive même en l'absence d'un agent inducteur, une souche transformée contenant le système d'expression, et un procédé de production d'acide hyaluronique utilisant la souche transformée.
PCT/KR2019/015082 2018-12-10 2019-11-07 Système d'expression pour la production d'acide hyaluronique à l'aide de bactéries non pathogènes et procédé de production d'acide hyaluronique utilisant ledit système d'expression WO2020122430A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030175902A1 (en) * 2001-12-21 2003-09-18 Novozymes Biotech, Inc. Methods for producing hyaluronan in a recombinant host cell
US20100136630A1 (en) * 2006-02-15 2010-06-03 Novozymes Biopolymer A/S Production of low molecular weight hyaluronic acid
CN104293726A (zh) * 2014-10-17 2015-01-21 江南大学 一种产小分子透明质酸的重组枯草芽孢杆菌
US20170073719A1 (en) * 2015-09-10 2017-03-16 Jiangnan University Method of constructing a recombinant Bacillus subtilis that can produce specific-molecular-weight hyaluronic acids
WO2017136795A1 (fr) * 2016-02-04 2017-08-10 Synlogic, Inc. Bactéries modifiées pour traiter des maladies associées au metabolisme du tryptophane

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Publication number Priority date Publication date Assignee Title
US20030175902A1 (en) * 2001-12-21 2003-09-18 Novozymes Biotech, Inc. Methods for producing hyaluronan in a recombinant host cell
US20100136630A1 (en) * 2006-02-15 2010-06-03 Novozymes Biopolymer A/S Production of low molecular weight hyaluronic acid
CN104293726A (zh) * 2014-10-17 2015-01-21 江南大学 一种产小分子透明质酸的重组枯草芽孢杆菌
US20170073719A1 (en) * 2015-09-10 2017-03-16 Jiangnan University Method of constructing a recombinant Bacillus subtilis that can produce specific-molecular-weight hyaluronic acids
WO2017136795A1 (fr) * 2016-02-04 2017-08-10 Synlogic, Inc. Bactéries modifiées pour traiter des maladies associées au metabolisme du tryptophane

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YU , XIAOXIA ET AL.: "Identification of a highly efficient stationary phase promoter in Bacillus subtilis", SCIENTIFIC REPORTS, vol. 5, no. 18405, 2015, pages 1 - 9, XP055660977 *

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