Recombinant strain for high production of chondroitin sulfate, application thereof and method for preparing chondroitin sulfate

Abstract

The present invention belongs to the technical field of chondroitin sulfate biosynthesis, and discloses a recombinant strain of high-yield chondroitin sulfate and its application and a method for preparing chondroitin sulfate. The recombinant strain provided by the present invention can simultaneously express UDP-glucosamine isomerase, UDP-glucose dehydrogenase, chondroitin polymerase, glucosamine synthetase, phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, glucose-6-phosphate isomerase, glucose phosphomutase, glucose-1-phosphate uridyl transferase, ATP sulfonase, sulfotransferase, pyruvate kinase and APS kinase, and the above enzyme molecules are combined with the original carbon metabolism pathway in the recombinant strain cell to form an enzyme cascade path with efficient CSA synthesis ability, which can achieve high yield and high purity preparation of CSA, and has the advantages of simple operation, high biological safety of the obtained product and easy large-scale production, and good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of chondroitin sulfate biosynthesis, and particularly relates to a recombinant strain with high chondroitin sulfate production, an application thereof and a method for preparing chondroitin sulfate. Background Art

[0002] Chondroitin sulfate (CS) refers to a class of acidic glycosaminoglycan macromolecular mucopolysaccharides, mostly in the form of proteoglycan side chains. CS is widely present in animal connective tissues, especially cartilage, skin, blood vessels, ligaments and tendons. CS plays an important role in the treatment of rheumatism, osteoporosis, arthritis, lumbar disc herniation, etc. In Europe, America, Japan and other countries, CS is widely used to prevent and treat cardiovascular and cerebrovascular diseases, atherosclerosis, osteoarthritis, etc. CS is often used in combination with glucosamine to stimulate the proliferation of chondrocytes and the biosynthesis of PGs, type II collagen, and hyaluronic acid, thereby relieving pain in patients with osteoarthritis, reducing joint swelling and effusion, and has a good effect on improving joint function and treating osteoarthritis.

[0003] CS is formed by alternating β-1,3 and β-1,4 glycosidic bonds between glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc), and then sulfonated at different sites by sulfotransferases. Sulfonation modification refers to the process of transferring the sulfonic acid group to the C-2 hydroxyl group of GlcA or the C-4 / C-6 hydroxyl group of GalNAc by the corresponding sulfotransferase. According to the different sites of sulfonation modification in the structure, CS can be divided into CSA (GlcA-GalNAc(4S)), CSC (GlcA-GalNAc(6S)), CSD (GlcA(2S)-GalNAc(6S)) and CSE (GlcA-GalNAc(4,6S)).

[0004] At present, commercial CS is mainly prepared by animal tissue extraction. The animal tissue extraction method mainly uses cartilage from animals such as cattle, pigs, chickens, and sharks as raw materials to extract CS. However, the CS extracted by this method needs to undergo complex and tedious purification treatment, and the resulting product may also induce an immune response, and has low biosafety. Compared with the traditional animal tissue extraction method, the CS prepared by microbial fermentation has better biosafety, which can well solve the many shortcomings of the animal tissue extraction method and has received more and more attention.

[0005] In the Chinese invention with publication number CN102869782A, it is disclosed that K4 capsular polysaccharide is prepared by fermentation of E. coli O5:K4:H4, and then the obtained K4 capsular polysaccharide is hydrolyzed to obtain 89% chondroitin with a purity of 90% and a molecular weight of about 45 kDa. However, the obtained chondroitin exists in a fructosylated form, and a large amount of chemical or enzymatic modification is required to finally obtain chondroitin sulfate. In the Chinese invention of CN106497845A, it is disclosed that the chondroitin synthesis pathway in Bacillus subtilis is optimized to increase the yield of chondroitin, but the structural composition of the obtained CS is relatively complex, and it is not possible to effectively synthesize the specific structure CS. Summary of the invention

[0006] The first object of the present invention is to provide a recombinant strain for producing chondroitin sulfate. The recombinant strain has excellent ability to efficiently synthesize CSA, and can achieve high yield and high purity preparation of CSA by fermentation without the need for additional chemical or enzyme modification, effectively solving the problems of complex preparation process, low yield and low purity of chondroitin sulfate in the existing microbial fermentation method, and has the advantages of simple operation, high biosafety of the obtained product, and easy large-scale production, and has good application prospects.

[0007] The second object of the present invention is to provide the use of the above-mentioned recombinant strain producing chondroitin sulfate in the preparation of chondroitin sulfate by microbial fermentation.

[0008] The third object of the present invention is to provide a method for preparing chondroitin sulfate.

[0009] Specifically, the recombinant strain for producing chondroitin sulfate provided by the present invention can synthesize and express UDP-glucosamine isomerase, UDP-glucose dehydrogenase, chondroitin polymerase, glucosamine synthetase, phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, glucose-6-phosphate isomerase, glucose phosphomutase, glucose-1-phosphate uridyltransferase, ATP sulfonase, sulfotransferase, pyruvate kinase and APS kinase.

[0010] Further, the recombinant strain has at least one of the following characteristics: the UDP-glucosamine isomerase includes an amino acid fragment as shown in SEQ ID NO: 10; the UDP-glucose dehydrogenase includes an amino acid fragment as shown in SEQ ID NO: 11; the chondroitin polymerase includes an amino acid fragment as shown in SEQ ID NO: 12; the glucosamine synthetase includes an amino acid fragment as shown in SEQ ID NO: 22; the phosphoglucosamine mutase includes an amino acid fragment as shown in SEQ ID NO: 23; the UDP-N-acetylglucosamine pyrophosphorylase includes an amino acid fragment as shown in SEQ ID NO: 24; the glucose-6-phosphate isomerase includes an amino acid fragment as shown in SEQ ID NO: 34; the glucose phosphomutase includes an amino acid fragment as shown in SEQ ID NO: 35; the glucose-1-phosphate uridyl transferase includes an amino acid fragment as shown in SEQ ID NO: 36; the ATP sulfonase includes an amino acid fragment as shown in SEQ ID NO: 49; the sulfotransferase includes an amino acid fragment as shown in SEQ ID NO: 50; the pyruvate kinase includes an amino acid fragment as shown in SEQ ID NO: The amino acid fragment shown in NO:51; the APS kinase includes the amino acid fragment whose sequence is shown in SEQ ID NO:52.

[0011] Furthermore, the recombinant strain is selected from one or more of Escherichia coli, Pichia pastoris and Saccharomyces cerevisiae.

[0012] Further, the recombinant strain has at least one of the following characteristics: the KfoA gene encoding the UDP-glucosamine isomerase includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 1; the KfoF gene encoding the UDP-glucose dehydrogenase includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 2; the KfoC gene encoding the chondroitin polymerase includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 3; the GlmS gene encoding the glucosamine synthetase includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 13; the GlmM gene encoding the phosphoglucosamine mutase includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 14; the GlmU gene encoding the UDP-N-acetylglucosamine pyrophosphorylase includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 15; the Pgi gene encoding the glucose-6-phosphate isomerase includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 25; the Pgm gene encoding the glucose phosphomutase includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 26; the GalU gene encoding the glucose-1-phosphate uridyltransferase includes a nucleotide fragment with a sequence as shown in SEQ ID NO: NO:27; the ATPS gene encoding the ATP sulfonase includes a nucleotide fragment with a sequence as shown in SEQ ID NO:37; the ST gene encoding the sulfotransferase includes a nucleotide fragment with a sequence as shown in SEQ ID NO:38; the pykA gene encoding the pyruvate kinase includes a nucleotide fragment with a sequence as shown in SEQ ID NO:39; the APSK gene encoding the APS kinase includes a nucleotide fragment with a sequence as shown in SEQ ID NO:40.

[0013] The present invention also provides the use of the above-mentioned recombinant strain producing chondroitin sulfate in preparing chondroitin sulfate by microbial fermentation.

[0014] The method for preparing chondroitin sulfate provided by the present invention comprises: taking the above-mentioned recombinant strain producing chondroitin sulfate and subjecting it to fermentation to obtain the chondroitin sulfate.

[0015] Furthermore, the fermentation treatment includes: S1, inoculating the recombinant strain into a fermentation medium for pre-culture to obtain a pre-treated culture solution; S2, adding an inducing expression agent to the pre-treated culture solution to induce expression to obtain a supernatant containing the chondroitin sulfate.

[0016] Furthermore, in step S1, the fermentation medium comprises glycerol, citric acid, potassium dihydrogen phosphate, magnesium sulfate, vitamin B1 and an organic nitrogen source in a mass ratio of (5-20):(1-4):(2-5):(1-3):(5-20).

[0017] Furthermore, in step S1, the organic nitrogen source is selected from one or more of soy peptone, yeast powder and urea.

[0018] Furthermore, in step S1, the pre-culture temperature is 35-40°C and the time is 1-10 hours.

[0019] Furthermore, in step S2, the temperature for inducing expression is 35-40° C. and the time is 48-72 hours.

[0020] Furthermore, in step S2, the induced expression also includes a feed treatment, and the feed treatment includes: monitoring the dissolved oxygen content of the pretreatment culture solution, and when the dissolved oxygen content is above 20%, taking a feed culture medium and adding it to the pretreatment culture solution to regulate the dissolved oxygen content of the pretreatment culture solution to 15-40%.

[0021] Furthermore, in step S2, the feed medium includes 250-1000 g / L of glycerol, 100-500 g / L of peptone and 5-20 g / L of magnesium sulfate.

[0022] Beneficial effects:

[0023] The recombinant strain provided by the present invention can simultaneously synthesize and express a large amount of UDP-glucosamine isomerase, UDP-glucose dehydrogenase, chondroitin polymerase, glucosamine synthetase, phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, glucose-6-phosphate isomerase, glucose phosphomutase, glucose-1-phosphate uridyl transferase, ATP sulfonase, sulfotransferase, pyruvate kinase and APS kinase. The above enzyme molecules cooperate with the original carbon metabolism pathway in the cells of the recombinant strain to jointly constitute an enzyme cascade pathway with the ability to efficiently synthesize CSA, thereby giving the strain the ability to efficiently synthesize and obtain high-purity CSA; the strain can be fermented to achieve high-yield and high-purity preparation of CSA, the operation is simple, the obtained product has high biological safety, and large-scale production is easy to achieve, and the strain has good application prospects. DETAILED DESCRIPTION

[0024] Based on a deep understanding of the metabolic pathway of chondroitin sulfate biosynthesis in cells, the inventors of the present invention have found through extensive and in-depth research that the ATPS-ST-pykA-APSK / GlmS-GlmM can be introduced into host cells commonly used in the field of genetic engineering technology such as Escherichia coli, Pichia pastoris and Saccharomyces cerevisiae. -GlmU-Pgi-Pgm-GalU / KfoA-KfoC-KfoF gene cluster, introduces or strengthens the expression of UDP-glucosamine isomerase, UDP-glucose dehydrogenase, chondroitin polymerase, glucosamine synthetase, phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, glucose-6-phosphate isomerase, glucose phosphomutase, glucose-1-phosphate uridyl transferase, ATP sulfonase, sulfotransferase, pyruvate kinase and APS kinase in the host cell, and together with the original carbon metabolism pathway in the host cell, constitutes a complete enzyme cascade pathway with the ability to efficiently synthesize CSA, so as to achieve high-quality supply of precursor chondroitin in the cell and high-efficiency sulfonation modification of specific sites of chondroitin, thereby achieving high-yield and high-purity preparation of CSA. Based on this, the technical solution of the present invention is obtained.

[0025] In the present invention, the recombinant strain is capable of synthesizing and expressing UDP-glucosamine isomerase, UDP-glucose dehydrogenase, chondroitin polymerase, glucosamine synthetase, phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, glucose-6-phosphate isomerase, glucose phosphomutase, glucose-1-phosphate uridyltransferase, ATP sulfonase, sulfotransferase, pyruvate kinase and APS kinase.

[0026] In the present invention, the UDP-glucosamine isomerase refers to a class of enzyme molecules with the biological activity of catalyzing the conversion of UDP-N-acetylglucosamine into UDP-N-acetylglucosamine, limited to being able to achieve the corresponding catalytic activity, and the present invention does not impose any particular limitation on its specific amino acid sequence.

[0027] In some specific embodiments, the UDP-glucosamine isomerase preferably includes an amino acid fragment having a sequence as shown in SEQ ID NO: 10. At this time, the UDP-glucosamine isomerase has excellent enzymatic activity in the above host cell, and can efficiently and quickly catalyze the conversion of UDP-N-acetylglucosamine into UDP-N-acetylglucosamine in the cell, which is conducive to achieving a better CSA synthesis effect.

[0028] In the present invention, the coding gene of the UDP-glucosamine isomerase is the KfoA gene. Based on the amino acid sequence of the UDP-glucosamine isomerase, a person skilled in the art can easily obtain the nucleotide sequence of the KfoA gene. The present invention does not impose any particular limitation on the specific nucleotide sequence of the KfoA gene.

[0029] In some specific embodiments, the KfoA gene preferably includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 1. In this case, the KfoA gene can be efficiently expressed in Escherichia coli cells, thereby synthesizing a large amount of UDP-glucosamine isomerase with high enzyme activity in the cells, which is conducive to achieving a better CSA synthesis effect.

[0030] In the present invention, the UDP-glucose dehydrogenase refers to a class of enzyme molecules having the biological activity of catalyzing the conversion of UDP-glucose into UDP-glucuronic acid, and is limited to being able to achieve the corresponding catalytic activity. The present invention does not impose any particular limitation on its specific amino acid sequence.

[0031] In some specific embodiments, the UDP-glucose dehydrogenase preferably includes an amino acid fragment having a sequence as shown in SEQ ID NO: 11. In this case, the UDP-glucose dehydrogenase has excellent enzymatic activity in the above host cell, and can efficiently and quickly catalyze the conversion of UDP-glucose into UDP-glucuronic acid in the cell, which is conducive to achieving a better CSA synthesis effect.

[0032] In the present invention, the coding gene of the UDP-glucose dehydrogenase is the KfoF gene. Based on the amino acid sequence of the UDP-glucose dehydrogenase, those skilled in the art can easily obtain the nucleotide sequence of the KfoF gene. The present invention does not impose any particular limitation on the specific nucleotide sequence of the KfoF gene.

[0033] In some specific embodiments, the KfoF gene preferably includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 2. In this case, the KfoF gene can be efficiently expressed in Escherichia coli cells, thereby synthesizing a large amount of UDP-glucose dehydrogenase with high enzyme activity in the cells, which is conducive to achieving a better CSA synthesis effect.

[0034] In the present invention, the chondroitin polymerase refers to a class of enzyme molecules having the biological activity of catalyzing the synthesis of chondroitin from UDP-GlcA and UDP-GalNAc, and is limited to being able to achieve the corresponding catalytic activity. The present invention does not impose any particular limitation on its specific amino acid sequence.

[0035] In some specific embodiments, the chondroitin polymerase preferably includes an amino acid fragment as shown in SEQ ID NO: 12. In this case, the chondroitin polymerase has excellent enzymatic activity in the above host cells, and can efficiently and quickly catalyze UDP-GlcA and UDP-GalNAc to synthesize chondroitin in the cells, which is conducive to achieving a better CSA synthesis effect.

[0036] In the present invention, the gene encoding the chondroitin polymerase is the KfoC gene. Based on the amino acid sequence of the chondroitin polymerase, those skilled in the art can easily obtain the nucleotide sequence of the KfoC gene. The present invention does not impose any particular limitation on the specific nucleotide sequence of the KfoC gene.

[0037] In some specific embodiments, the KfoC gene preferably includes a nucleotide fragment as shown in SEQ ID NO: 3. In this case, the KfoC gene can be efficiently expressed in Escherichia coli cells, thereby synthesizing a large amount of chondroitin polymerase with high enzyme activity in the cells, which is conducive to achieving a better CSA synthesis effect.

[0038] In the present invention, the glucosamine synthase refers to a class of enzyme molecules with the biological activity of catalyzing the conversion of fructose-6-phosphate and glutamine into glucosamine-6-phosphate and glutamate, limited to the ability to achieve the corresponding catalytic activity, and the present invention does not impose any particular limitation on its specific amino acid sequence.

[0039] In some specific embodiments, the glucosamine synthetase preferably includes an amino acid fragment as shown in SEQ ID NO: 22. In this case, the glucosamine synthetase has excellent enzymatic activity in the above host cell, and can efficiently and quickly catalyze the conversion of fructose-6-phosphate and glutamine into glucosamine-6-phosphate and glutamate in the cell, which is conducive to achieving a better CSA synthesis effect.

[0040] In the present invention, the gene encoding the glucosamine synthase is the GlmS gene. Based on the amino acid sequence of the glucosamine synthase, those skilled in the art can easily obtain the nucleotide sequence of the GlmS gene. The present invention does not impose any particular limitation on the specific nucleotide sequence of the GlmS gene.

[0041] In some specific embodiments, the GlmS gene preferably includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 13. In this case, the GlmS gene can be efficiently expressed in Escherichia coli cells, thereby synthesizing a large amount of glucosamine synthase with high enzymatic activity in the cells, which is conducive to achieving a better CSA synthesis effect.

[0042] In the present invention, the phosphoglucosamine mutase refers to a class of enzyme molecules with biological activity of catalyzing the conversion of 6-P-glucosamine into 1-P-glucosamine, limited to the ability to achieve corresponding catalytic activity, and the present invention does not impose any particular limitation on its specific amino acid sequence.

[0043] In some specific embodiments, the phosphoglucosamine mutase preferably includes an amino acid fragment having a sequence as shown in SEQ ID NO: 23. In this case, the phosphoglucosamine mutase has excellent enzymatic activity in the above host cell, and can efficiently and quickly catalyze the conversion of 6-P-glucosamine into 1-P-glucosamine in the cell, which is conducive to achieving a better CSA synthesis effect.

[0044] In the present invention, the gene encoding the phosphoglucosamine mutase is the GlmM gene. Based on the amino acid sequence of the phosphoglucosamine mutase, those skilled in the art can easily obtain the nucleotide sequence of the GlmM gene. The present invention does not impose any particular limitation on the specific nucleotide sequence of the GlmM gene.

[0045] In some specific embodiments, the GlmM gene preferably includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 14. In this case, the GlmM gene can be efficiently expressed in Escherichia coli cells, thereby synthesizing a large amount of phosphoglucosamine mutase with high enzyme activity in the cells, which is conducive to achieving a better CSA synthesis effect.

[0046] In the present invention, the UDP-N-acetylglucosamine pyrophosphorylase refers to a class of enzyme molecules having the biological activity of catalyzing the reaction of 1-phosphate glucose and UTP molecules to generate UDP-glucose, and is limited to being able to achieve the corresponding catalytic activity. The present invention does not impose any particular limitation on its specific amino acid sequence.

[0047] In some specific embodiments, the UDP-N-acetylglucosamine pyrophosphorylase preferably includes an amino acid fragment as shown in SEQ ID NO: 24. At this time, the UDP-N-acetylglucosamine pyrophosphorylase has excellent enzyme activity in the above host cells, and can efficiently and quickly catalyze the reaction of 1-phosphate glucose and UTP molecules to synthesize UDP-glucose in the cells, which is conducive to achieving a better CSA synthesis effect.

[0048] In the present invention, the coding gene of the UDP-N-acetylglucosamine pyrophosphorylase is the GlmU gene. Based on the amino acid sequence of the UDP-N-acetylglucosamine pyrophosphorylase, those skilled in the art can easily obtain the nucleotide sequence of the GlmU gene. The present invention does not impose any particular limitation on the specific nucleotide sequence of the GlmU gene.

[0049] In some specific embodiments, the GlmU gene preferably includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 15. In this case, the GlmU gene can be efficiently expressed in Escherichia coli cells, thereby synthesizing a large amount of UDP-N-acetylglucosamine pyrophosphorylase with high enzyme activity in the cells, which is conducive to achieving a better CSA synthesis effect.

[0050] In the present invention, the glucose-6-phosphate isomerase refers to a class of enzyme molecules with the biological activity of catalyzing the conversion of glucose-6-phosphate into fructose-6-phosphate, limited to being able to achieve the corresponding catalytic activity, and the present invention does not impose any particular limitation on its specific amino acid sequence.

[0051] In some specific embodiments, the glucose-6-phosphate isomerase preferably includes an amino acid fragment having a sequence as shown in SEQ ID NO: 34. In this case, the glucose-6-phosphate isomerase has excellent enzymatic activity in the above host cell, and can efficiently and quickly catalyze the conversion of glucose-6-phosphate into fructose-6-phosphate in the cell, which is conducive to achieving a better CSA synthesis effect.

[0052] In the present invention, the gene encoding glucose-6-phosphate isomerase is the Pgi gene. Based on the amino acid sequence of glucose-6-phosphate isomerase, those skilled in the art can easily obtain the nucleotide sequence of the Pgi gene. The present invention does not impose any particular limitation on the specific nucleotide sequence of the Pgi gene.

[0053] In some specific embodiments, the Pgi gene preferably includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 25. In this case, the Pgi gene can be efficiently expressed in Escherichia coli cells, thereby synthesizing a large amount of glucose-6-phosphate isomerase with high enzyme activity in the cells, which is conducive to achieving a better CSA synthesis effect.

[0054] In the present invention, the glucose phosphomutase refers to a class of enzyme molecules having the biological activity of catalyzing the conversion of 6-P-glucose into 1-P-glucose, and is limited to being able to achieve the corresponding catalytic activity. The present invention does not impose any particular limitation on its specific amino acid sequence.

[0055] In some specific embodiments, the phosphoglucomutase preferably includes an amino acid fragment having a sequence as shown in SEQ ID NO: 35. In this case, the phosphoglucomutase has excellent enzymatic activity in the above host cell, and can efficiently and quickly catalyze the conversion of 6-P-glucose into 1-P-glucose in the cell, which is conducive to achieving a better CSA synthesis effect.

[0056] In the present invention, the gene encoding the glucose phosphomutase is the Pgm gene. Based on the amino acid sequence of the glucose phosphomutase, those skilled in the art can easily obtain the nucleotide sequence of the Pgm gene. The present invention does not impose any particular limitation on the specific nucleotide sequence of the Pgm gene.

[0057] In some specific embodiments, the Pgm gene preferably includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 26. At this time, the Pgm gene can be efficiently expressed in Escherichia coli cells, thereby synthesizing a large amount of glucose phosphomutase with high enzyme activity in the cells, which is conducive to achieving a better CSA synthesis effect.

[0058] In the present invention, the glucose-1-phosphate uridyl transferase refers to a class of enzyme molecules with biological activity of catalyzing the conversion of 1-P-glucose into UDP-glucose, limited to being able to achieve the corresponding catalytic activity, and the present invention does not impose any particular limitation on its specific amino acid sequence.

[0059] In some specific embodiments, the glucose-1-phosphate uridyl transferase preferably includes an amino acid fragment as shown in SEQ ID NO: 36. In this case, the glucose-1-phosphate uridyl transferase has excellent enzyme activity in the above host cell, and can efficiently and quickly catalyze the conversion of 1-P-glucose into UDP-glucose in the cell, which is conducive to achieving a better CSA synthesis effect.

[0060] In the present invention, the gene encoding the glucose-1-phosphate uridyl transferase is the GalU gene. Based on the amino acid sequence of the glucose-1-phosphate uridyl transferase, those skilled in the art can easily obtain the nucleotide sequence of the GalU gene. The present invention does not impose any particular limitation on the specific nucleotide sequence of the GalU gene.

[0061] In some specific embodiments, the GalU gene preferably includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 27. In this case, the GalU gene can be efficiently expressed in Escherichia coli cells, thereby synthesizing a large amount of glucose-1-phosphate uridyl transferase with high enzyme activity in the cells, which is conducive to achieving a better CSA synthesis effect.

[0062] In the present invention, the ATP sulfonase refers to a class of enzyme molecules having the biological activity of catalyzing the reaction of sulfate and ATP to generate APS and PPi, and is limited to being able to achieve the corresponding catalytic activity. The present invention does not impose any particular limitation on its specific amino acid sequence.

[0063] In some specific embodiments, the ATP sulfonase preferably includes an amino acid fragment with a sequence as shown in SEQ ID NO: 49. In this case, the ATP sulfonase has excellent enzymatic activity in the above host cell, and can efficiently and quickly catalyze the reaction of sulfate and ATP to generate APS and PPi in the cell, which is conducive to achieving a better CSA synthesis effect.

[0064] In the present invention, the gene encoding the ATP sulfonase is the ATPS gene. Based on the amino acid sequence of the ATP sulfonase, those skilled in the art can easily obtain the nucleotide sequence of the ATPS gene. The present invention does not impose any particular limitation on the specific nucleotide sequence of the ATPS gene.

[0065] In some specific embodiments, the ATPS gene preferably includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 37. In this case, the ATPS gene can be efficiently expressed in Escherichia coli cells, thereby synthesizing a large amount of ATP sulfonase with high enzyme activity in the cells, which is conducive to achieving a better CSA synthesis effect.

[0066] In the present invention, the sulfotransferase refers to a class of enzyme molecules having the biological activity of catalyzing the reaction of PAPS and chondroitin sulfate to generate CSA, and is limited to being able to achieve the corresponding catalytic activity. The present invention does not impose any particular limitation on its specific amino acid sequence.

[0067] In some specific embodiments, the sulfotransferase preferably includes an amino acid fragment with a sequence as shown in SEQ ID NO: 50. In this case, the sulfotransferase has excellent enzymatic activity in the above host cell, and can efficiently catalyze the reaction of PAPS and chondroitin sulfate to generate CSA in the cell, which is conducive to achieving a better CSA synthesis effect.

[0068] In the present invention, the gene encoding the sulfotransferase is the ST gene. Based on the amino acid sequence of the sulfotransferase, those skilled in the art can easily obtain the nucleotide sequence of the ST gene. The present invention does not impose any particular limitation on the specific nucleotide sequence of the ST gene.

[0069] In some specific embodiments, the ST gene preferably includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 38. In this case, the ST gene can be efficiently expressed in Escherichia coli cells, thereby synthesizing a large amount of sulfotransferase with high enzyme activity in the cells, which is conducive to achieving a better CSA synthesis effect.

[0070] In the present invention, the pyruvate kinase refers to a class of enzyme molecules having the biological activity of catalyzing the conversion of phosphoenolpyruvate into pyruvate, and is limited to being able to achieve the corresponding catalytic activity. The present invention does not impose any particular limitation on its specific amino acid sequence.

[0071] In some specific embodiments, the pyruvate kinase preferably includes an amino acid fragment as shown in SEQ ID NO: 51. In this case, the pyruvate kinase has excellent enzymatic activity in the above host cell, and can efficiently and quickly catalyze the conversion of phosphoenolpyruvate into pyruvate in the cell, which is conducive to achieving a better CSA synthesis effect.

[0072] In the present invention, the coding gene of the pyruvate kinase is the pykA gene. Based on the amino acid sequence of the pyruvate kinase, those skilled in the art can easily obtain the nucleotide sequence of the pykA gene. The present invention does not impose any particular limitation on the specific nucleotide sequence of the pykA gene.

[0073] In some specific embodiments, the pykA gene preferably includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 39. In this case, the pykA gene can be efficiently expressed in Escherichia coli cells, thereby synthesizing a large amount of pyruvate kinase with high enzyme activity in the cells, which is conducive to achieving a better CSA synthesis effect.

[0074] In the present invention, the APS kinase refers to a class of enzyme molecules having the biological activity of catalyzing the conversion of APS into PAPS, and is limited to being able to achieve the corresponding catalytic activity. The present invention does not impose any particular limitation on its specific amino acid sequence.

[0075] In some specific embodiments, the APS kinase preferably includes an amino acid fragment as shown in SEQ ID NO: 52. In this case, the APS kinase has excellent enzymatic activity in the above host cells, and can efficiently and quickly catalyze the conversion of APS into PAPS in the cells, which is conducive to achieving a better CSA synthesis effect.

[0076] In the present invention, the gene encoding the APS kinase is the APSK gene. Based on the amino acid sequence of the APS kinase, those skilled in the art can easily obtain the nucleotide sequence of the APSK gene. The present invention does not impose any particular limitation on the specific nucleotide sequence of the APSK gene.

[0077] In some specific embodiments, the APSK gene preferably includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 40. In this case, the APSK gene can be efficiently expressed in Escherichia coli cells, thereby synthesizing a large amount of APS kinase with high enzyme activity in the cells, which is conducive to achieving a better CSA synthesis effect.

[0078] In the present invention, the host cell used to construct the recombinant strain is preferably Escherichia coli BL21 Star (DE3) and / or Escherichia coli BL21 (DE3), more preferably Escherichia coli BL21 Star (DE3). At this time, the glycerol metabolic pathway in the recombinant strain and the CSA synthetase cascade pathway composed of the above enzyme molecules have a more ideal CSA synthesis effect, which can use glycerol as the only carbon source, and while achieving high yield and high purity preparation of CSA, the obtained CSA also has an excellent sulfonation level.

[0079] Based on the technical effects of the above-mentioned recombinant strain producing chondroitin sulfate, the present invention also provides the use of the above-mentioned recombinant strain producing chondroitin sulfate in preparing chondroitin sulfate by microbial fermentation.

[0080] Based on the application of the above-mentioned recombinant strain producing chondroitin sulfate in preparing chondroitin sulfate by microbial fermentation, the present invention also provides a method for preparing chondroitin sulfate.

[0081] In the present invention, the preparation method specifically comprises: taking the above-mentioned recombinant strain producing chondroitin sulfate and subjecting it to fermentation to obtain the chondroitin sulfate.

[0082] In the present invention, the fermentation treatment specifically includes: S1, inoculating the recombinant strain into a fermentation medium for pre-culture to obtain a pre-treated culture solution; S2, adding an inducing expression agent to the pre-treated culture solution to induce expression to obtain a supernatant containing the chondroitin sulfate.

[0083] In the present invention, in step S1, the fermentation medium is used to provide the recombinant strain with the necessary nutrients, which can specifically be the above-mentioned fermentation medium for producing chondroitin sulfate, or other culture medium conventionally used in preparing chondroitin sulfate by microbial fermentation, and the present invention does not impose any particular limitation thereto.

[0084] In the present invention, the fermentation medium preferably includes glycerol, citric acid, potassium dihydrogen phosphate, magnesium sulfate, vitamin B1 and an organic nitrogen source; and the mass ratio of the glycerol, citric acid, potassium dihydrogen phosphate, magnesium sulfate, vitamin B1 and the organic nitrogen source is (5~20):(1~4):(2~5):(1~3):(5~20), such as 5:1:2:1:5, 8:1:3:1:17, 10:3:4:1:18, 20:4:5:3:20 or any value therebetween.

[0085] In the present invention, the organic nitrogen source is a type of reagent commonly used in the field of microbial technology, and the present invention does not impose any particular limitation on it. Specific examples include but are not limited to: one or more of soy peptone, yeast powder and urea.

[0086] In some specific embodiments, the fermentation medium preferably includes 5-15 g / L of glycerol, such as 5 g / L, 8 g / L, 9 g / L, 10 g / L, 15 g / L or any value therebetween; 0.1-5 g / L of citric acid, such as 0.1, 0.5, 0.8 g / L, 2 g / L, 3 g / L, 5 g / L or any value therebetween; 1-5 g / L of potassium dihydrogen phosphate, such as 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L or any value therebetween; any value therebetween; 0.1-5 g / L magnesium sulfate heptahydrate, such as 0.1 g / L, 0.5 g / L, 1 g / L, 3 g / L, 5 g / L or any value therebetween; 2-8 mg / L vitamin B1, such as 2 mg / L, 4 mg / L, 6 mg / L, 8 mg / L or any value therebetween; 5-20 g / L soy peptone, such as 5 g / L, 7 g / L, 9 g / L, 13 g / L, 18 g / L, 20 g / L or any value therebetween. At this time, the above recombinant strain is cultured using the fermentation medium, and the obtained CSA has better purity and sulfonation level.

[0087] In some specific embodiments, in step S1, the pre-culture conditions include a temperature of preferably 35-40°C, such as 35°C, 36°C, 37°C, 38°C, 39°C, 40°C or any value therebetween; and a time of preferably 1-10h, such as 1h, 3h, 5h, 7h, 9h, 10h or any value therebetween.

[0088] In the present invention, in step S2, the inducing expression agent refers to a type of substance that can induce the expression of the ATPS-ST-pykA-APSK / GlmS-GlmM-GlmU-Pgi-Pgm-GalU / KfoA-KfoC-KfoF gene cluster in recombinant strain cells, which is a type of substance commonly used in the field of genetic engineering technology. Specific examples include but are not limited to: isopropyl-β-D-thiogalactoside.

[0089] In some specific embodiments, in step S2, the amount of the inducing expression is preferably 0.01~1mmol / L, such as 0.01mmol / L, 0.05mmol / L, 0.1mmol / L, 0.15mmol / L, 0.2mmol / L, 0.5mmol / L, 0.8mmol / L, 1mmol / L or any value therebetween.

[0090] In some specific embodiments, in step S2, the conditions for inducing expression include a temperature of preferably 35-40°C, such as 35°C, 36°C, 37°C, 38°C, 39°C, 40°C or any value therebetween; and a time of preferably 48-72h, such as 48h, 54h, 60h, 66h, 72h or any value therebetween.

[0091] In the present invention, in step S2, the induced expression also includes the operation step of feeding treatment, and the feeding treatment specifically includes: monitoring the dissolved oxygen content of the pretreatment culture solution, and when the dissolved oxygen content is above 20%, taking the feed culture medium and adding it to the pretreatment culture solution to regulate the dissolved oxygen content of the pretreatment culture solution to 15-40%.

[0092] In some specific embodiments, the feed medium preferably includes 250-1000 g / L of glycerol, such as 250 g / L, 280 g / L, 300 g / L, 350 g / L, 500 g / L, 750 g / L, 1000 g / L or any value therebetween; 100-500 g / L of peptone, such as 100 g / L, 150 g / L, 200 g / L, 250 g / L, 300 g / L, 400 g / L, 500 g / L or any value therebetween; 5-20 g / L of magnesium sulfate, such as 5 g / L, 8 g / L, 10 g / L, 15 g / L, 20 g / L or any value therebetween.

[0093] Embodiments of the present invention are described in detail below, and the examples of the embodiments are intended to be used to explain the present invention, but should not be construed as limiting the present invention. In the embodiments, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be obtained commercially.

[0094] Preparation Example 1

[0095] This preparation example is used to illustrate the preparation of a recombinant strain, which includes a KfoA-KfoC-KfoF gene cluster. The preparation of the recombinant strain specifically includes:

[0096] 1. Construction of recombinant plasmid I containing the KfoA-KfoC-KfoF gene cluster: (1) Using a bacterial genomic DNA rapid extraction kit (purchased from Sangon Biotechnology (Shanghai)) and referring to the instructions, Escherichia coli K4 (ATCC23502) was treated to extract genomic DNA.

[0097] (2) The genomic DNA was amplified by PCR using the primer system shown in Table 1. The resulting PCR products were subjected to gel electrophoresis. The corresponding bands were recovered using the AxyPrep DNA Gel Recovery Kit (purchased from Aisgen) according to the instructions to obtain the KfoA gene (SEQ ID NO: 1), KfoC gene (SEQ ID NO: 2) and KfoF gene (SEQ ID NO: 3).

[0098] Table 1.

[0099]

[0100] The PCR amplification reaction system includes: 1 μL DNA template, 1 μL upstream and downstream primers, 25 μL 2× TransStar ® FastPfu Fly PCR SuperMix (Beijing Quanshijin Biotechnology, catalog number AS221-02) was added with ddH2O to make up the final volume to 50 μL.

[0101] The reaction program of PCR amplification included: 95°C for 3 min, 25 cycles (95°C for 20 s, 55°C for 20 s, 72°C for 2 min), and 72°C for 10 min.

[0102] (3) The expression vector pRSFduet-1 was digested with endonucleases Nde Ⅰ and Xho Ⅰ (purchased from New England Biolabs) according to the instructions, and then the homologous recombination seamless cloning kit (purchased from Beijing Quanshijin Biotechnology) was used to connect the digested expression vector pRSFduet-1 with the KfoA, KfoC and KfoF genes according to the instructions to obtain recombinant plasmid I;

[0103] Referring to the instruction manual, the recombinant plasmid I was transformed into Trans5ɑ competent cells (purchased from Beijing Quanshijin Biotechnology), then inoculated into LB medium, cultured at 37°C and 160 rpm with shaking overnight, and a large amount of recombinant plasmid I was extracted.

[0104] 2. Construction of recombinant strain: The recombinant plasmid I was transformed into chemically competent Escherichia coli BL21 Star (DE3), then inoculated into LB medium and cultured overnight at 37°C and 160 rpm to obtain a recombinant strain named C1.

[0105] The recombinant strain C1 obtained in this preparation example can simultaneously synthesize and express UDP-glucosamine isomerase (encoded by KfoA gene), UDP-glucose dehydrogenase (encoded by KfoF gene) and chondroitin polymerase (encoded by KfoC gene), and the specific amino acid sequences are shown in Table 2.

[0106] Table 2.

[0107]

[0108] Preparation Example 2

[0109] This preparation example is used to illustrate the preparation of a recombinant strain, which includes a GlmS-GlmM-GlmU / KfoA-KfoC-KfoF gene cluster. The preparation of the recombinant strain specifically includes:

[0110] 1. Construction of recombinant plasmid II containing the GlmS-GlmM-GlmU / KfoA-KfoC-KfoF gene cluster: (1) Use a bacterial genomic DNA rapid extraction kit and refer to the instructions to treat Escherichia coli K4 (ATCC23502) to extract genomic DNA.

[0111] (2) The genomic DNA was amplified by PCR using the primer system shown in Table 3. The resulting PCR products were subjected to gel electrophoresis. The corresponding bands were recovered using the AxyPrep DNA Gel Recovery Kit according to the instructions to obtain the GlmS gene (SEQ ID NO: 13), GlmM gene (SEQ ID NO: 14) and GlmU gene (SEQ ID NO: 15).

[0112] Table 3.

[0113]

[0114] The PCR amplification reaction system includes: 1 μL DNA template, 1 μL upstream and downstream primers, 25 μL 2× TransStar ® FastPfu Fly PCR SuperMix, make up to a final volume of 50 μL with ddH2O.

[0115] The reaction program of PCR amplification included: 95°C for 3 min, 25 cycles (95°C for 20 s, 55°C for 20 s, 72°C for 2 min), and 72°C for 10 min.

[0116] (3) using endonucleases Nco Ⅰ and Sac Ⅰ according to the instructions to digest the recombinant plasmid I obtained in Preparation Example 1, and then using a homologous recombination seamless cloning kit according to the instructions to connect the recombinant plasmid I with the GlmS, GlmM and GlmU genes to obtain recombinant plasmid II;

[0117] Referring to the instructions, the recombinant plasmid II was transformed into Trans5ɑ competent cells, and then inoculated into LB medium, cultured at 37°C and 160 rpm with shaking overnight, and a large amount of recombinant plasmid II was extracted.

[0118] 2. Construction of recombinant strain: The recombinant plasmid II was transformed into chemically competent Escherichia coli BL21 Star (DE3), then inoculated into LB medium and cultured overnight at 37°C and 160 rpm to obtain a recombinant strain named C2.

[0119] The recombinant strain C2 obtained in this preparation example can express UDP-glucosamine isomerase, UDP-glucose dehydrogenase and chondroitin polymerase, and can also express glucosamine synthetase (encoded by GlmS gene), phosphoglucosamine mutase (encoded by GlmM gene), and UDP-N-acetylglucosamine pyrophosphorylase (encoded by GlmU gene). The specific amino acid sequences are shown in Table 4.

[0120] Table 4.

[0121]

[0122] Preparation Example 3

[0123] This preparation example is used to illustrate the preparation of a recombinant strain, which includes a GlmS-GlmM-GlmU-Pgi-Pgm-GalU / KfoA-KfoC-KfoF gene cluster. The preparation of the recombinant strain specifically includes:

[0124] 1. Construction of recombinant plasmid III containing the GlmS-GlmM-GlmU-Pgi-Pgm-GalU / KfoA-KfoC-KfoF gene cluster: (1) Use a bacterial genomic DNA rapid extraction kit and refer to the instructions to treat Escherichia coli K4 (ATCC23502) to extract genomic DNA.

[0125] (2) The genomic DNA was amplified by PCR using the primer system shown in Table 5. The resulting PCR products were subjected to gel electrophoresis. The corresponding bands were recovered using the AxyPrep DNA Gel Recovery Kit according to the instructions to obtain the Pgi gene (SEQ ID NO: 25), the Pgm gene (SEQ ID NO: 26) and the GalU gene (SEQ ID NO: 27).

[0126] Table 5.

[0127]

[0128] The PCR amplification reaction system included: 1 μL of DNA template, 1 μL of upstream and downstream primers, 25 μL of 2×TransStar FastPfu® Fly PCR SuperMix, and ddH2O to a final volume of 50 μL.

[0129] The reaction program of PCR amplification included: 95°C for 3 min, 25 cycles (95°C for 20 s, 55°C for 20 s, 72°C for 2 min), and 72°C for 10 min.

[0130] (3) The recombinant plasmid II obtained in Preparation Example 2 was digested with endonuclease Sac Ⅰ, and then the recombinant plasmid II was connected with Pgi, Pgm and GalU genes using a homologous recombination seamless cloning kit to obtain recombinant plasmid III;

[0131] Referring to the instructions, the recombinant plasmid III was transformed into Trans5ɑ competent cells, and then inoculated into LB medium, cultured at 37°C and 160 rpm with shaking overnight, and a large amount of recombinant plasmid III was extracted.

[0132] 2. Construction of recombinant strain: The recombinant plasmid III was transformed into chemically competent Escherichia coli BL21 Star (DE3), then inoculated into LB medium and cultured overnight at 37°C and 160 rpm to obtain a recombinant strain named C3.

[0133] The recombinant strain C3 obtained in this preparation example can express glucose-6-phosphate isomerase (encoded by Pgi gene), glucose phosphomutase (encoded by Pgm gene) and glucose-1-phosphate uridyltransferase (encoded by GalU gene) on the basis of being able to express UDP-glucosamine isomerase, UDP-glucose dehydrogenase, chondroitin polymerase, glucosamine synthetase, phosphoglucosamine mutase and UDP-N-acetylglucosamine pyrophosphorylase. The specific amino acid sequences are shown in Table 6.

[0134] Table 6.

[0135]

[0136] Preparation Example 4

[0137] This preparation example is used to illustrate the preparation of a recombinant strain, which includes the ATPS-ST-pykA-APSK / GlmS-GlmM-GlmU-Pgi-Pgm-GalU / KfoA-KfoC-KfoF gene cluster. The preparation of the recombinant strain specifically includes:

[0138] 1. Construction of recombinant plasmid IV including ATPS-ST-pykA-APSK gene cluster: ATPS gene is derived from Saccharomyces cerevisiae, and the sequence obtained after codon optimization in Escherichia coli is shown in SEQ ID NO:37; ST gene is derived from human, and the sequence obtained after codon optimization in Escherichia coli is shown in SEQ ID NO:38; pykA gene is derived from Escherichia coli, and the sequence is shown in SEQ ID NO:39; APSK gene is derived from Escherichia coli, and the sequence is shown in SEQ ID NO:40, which were synthesized by Sangon Biotech (Shanghai) and loaded in pET24a vector.

[0139] (2) The pET24a vector was amplified by PCR using the primer system shown in Table 7. The resulting PCR products were subjected to gel electrophoresis. The corresponding bands were recovered using the AxyPrep DNA Gel Recovery Kit according to the instructions to obtain ATPS, ST, pykA and APSK genes.

[0140] Table 7.

[0141]

[0142] The PCR amplification reaction system included: 1 μL of DNA template, 1 μL of upstream and downstream primers, 25 μL of 2×TransStar FastPfu® Fly PCR SuperMix, and ddH2O to a final volume of 50 μL.

[0143] The reaction program of PCR amplification included: 95°C for 3 min, 25 cycles (95°C for 20 s, 55°C for 20 s, 72°C for 2 min), and 72°C for 10 min.

[0144] (3) Using the endonucleases Nde Ⅰ and Xho Ⅰ according to the instructions, the expression vector pCDFduet-1 was digested, and then the homologous recombination seamless cloning kit was used according to the instructions to connect the digested expression vector pCDFduet-1 with the ATPS, ST, pykA and APSK genes to obtain the recombinant plasmid IV;

[0145] Referring to the instructions, the recombinant plasmid IV was transformed into Trans5ɑ competent cells, and then inoculated into LB medium, cultured at 37°C and 160 rpm with shaking overnight, and a large amount of recombinant plasmid IV was extracted.

[0146] 2. Construction of recombinant strains: The recombinant plasmid IV and the recombinant plasmid III provided in Preparation Example 3 were transformed into chemically competent Escherichia coli BL21 Star (DE3), then inoculated into LB medium and cultured overnight at 37°C and 160 rpm to obtain a recombinant strain named C4.

[0147] The recombinant strain C4 obtained in this preparation example can express UDP-glucosamine isomerase, UDP-glucose dehydrogenase, chondroitin polymerase, glucosamine synthetase, phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, glucose-6-phosphate isomerase, glucose phosphomutase and glucose-1-phosphate uridyltransferase, and can also express ATP sulfonase (ATPS gene encoding), sulfotransferase (ST gene encoding), pyruvate kinase (pykA gene encoding) and APS kinase (APSK gene encoding), and the specific amino acid sequences are shown in Table 8.

[0148] Table 8.

[0149]

[0150] Preparation Example 5

[0151] This preparation example is used to illustrate the preparation of a recombinant strain, which includes the ATPS-ST-pykA-APSK / GlmS-GlmM-GlmU / KfoA-KfoC-KfoF gene cluster. The preparation specifically includes:

[0152] The recombinant plasmid II provided in Preparation Example 2 and the recombinant plasmid IV provided in Preparation Example 4 were transformed into chemically competent Escherichia coli BL21Star (DE3), then inoculated into LB medium, and cultured overnight at 37°C and 160 rpm to obtain a recombinant strain named C5.

[0153] The recombinant strain C5 obtained in this preparation example can simultaneously express UDP-glucosamine isomerase, UDP-glucose dehydrogenase, chondroitin polymerase, glucosamine synthetase, phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, ATP sulfonase, sulfotransferase, pyruvate kinase and APS kinase, and the specific amino acid sequences are shown in Tables 2, 4 and 8.

[0154] Preparation Example 6

[0155] This preparation example is used to illustrate the preparation of a recombinant strain, which includes an ATPS'-ST'-pykA'-APSK' / GlmS'-GlmM'-GlmU'-Pgi'-Pgm'-GalU' / KfoA'-KfoC'-KfoF' gene cluster, and its preparation specifically includes:

[0156] 1. Construction of recombinant plasmid V including GlmS'-GlmM'-GlmU'-Pgi'-Pgm'-GalU' / KfoA'-KfoC'-KfoF' gene cluster: Based on the amino acid sequences of the relevant enzymes shown in Tables 2, 4 and 6, Pichia codon optimization was performed to obtain the optimized genes, as shown in Table 9, and the genes were synthesized by Sangon Biotechnology (Shanghai) and loaded on the expression vector pPICZA (Ubao Bio, Catalog No. VT1338) to obtain recombinant plasmid V.

[0157] Table 9.

[0158]

[0159] 2. Construction of recombinant plasmid VI including ATPS'-ST'-pykA'-APSK' gene cluster: Based on the amino acid sequences of the relevant enzymes shown in Table 8, Pichia codon optimization was performed to obtain the optimized genes, as shown in Table 10. The genes were synthesized by Sangon Biotechnology (Shanghai) and loaded on the expression vector pPIC3.5K (Ubao Biotechnology, catalog number VT1342) to obtain recombinant plasmid VI.

[0160] Table 10.

[0161]

[0162] 2. Construction of recombinant strains: Recombinant plasmids V and VI were transformed into chemically competent Pichia pastoris GS115 by electroporation, and plated on MD plates (Thermo Fisher Scientific, catalog number R25001) containing 100 mg / L Zeocin to obtain a recombinant strain named C6.

[0163] The recombinant strain C6 obtained in this preparation example can simultaneously synthesize and express UDP-glucosamine isomerase, UDP-glucose dehydrogenase, chondroitin polymerase, glucosamine synthetase, phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, glucose-6-phosphate isomerase, glucose phosphomutase, glucose-1-phosphate uridyltransferase, ATP sulfonase, sulfotransferase, pyruvate kinase and APS kinase.

[0164] Example 1

[0165] This example is used to illustrate the ability of the recombinant strain provided in the above preparation example to synthesize chondroitin sulfate on a shake flask culture scale. The steps of synthesizing chondroitin sulfate by fermentation using the recombinant strain specifically include:

[0166] S1. Take 200 μL of the bacterial solution containing the above recombinant strain from the glycerol tube according to the inoculation amount of 1% and inoculate it into LB liquid culture medium, and culture it with shaking at 37°C and 200 rpm for 12 h to obtain seed solution.

[0167] S2, take 500 μL of seed solution and transfer it to a shake flask containing 50 mL of fermentation medium, and culture it at 37°C and 200 rpm for 4 h. At this time, the OD of the culture solution is 600 is 0.6-0.8; IPTG is added to the culture medium at a final concentration of 0.5 mmol / L, and the expression is induced at 37°C and 200 rpm for 48 hours, and the fermentation supernatant containing chondroitin sulfate is collected by centrifugation;

[0168] The fermentation medium includes 10 g / L glycerol, 2 g / L citric acid, 2.5 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate heptahydrate, 10 g / L soy peptone and 4.5 mg / L vitamin B1.

[0169] S3. Take 1 mL of the fermentation supernatant and mix it with 3 mL of anhydrous ethanol, perform alcohol precipitation overnight, then centrifuge at 12000 r / min for 10 min, collect the precipitate and add 1 mL of ultrapure water to reconstitute it to obtain the test solution; take 100 μL of the test solution and 800 μL of 50 mmol / L Tris-HCl buffer (pH 8.0), mix them thoroughly, add 100 μL of 1 U / L chondroitin ABC enzyme solution (Sigma-Aldrich, product number C3667), mix them, react in a 37°C water bath for 1 h, then heat at 100°C for 5 min, cool to room temperature with cold water, centrifuge at 10000 r / min for 20 min, collect the supernatant, filter it with a 0.45 μm filter membrane, perform HPLC detection, and calculate the CSA content by the external standard method as the sum of the areas of chondroitin disaccharide and 4-sulfated chondroitin disaccharide.

[0170] Among them, the conditions for HPLC detection include: liquid chromatograph with DAD detector; chromatographic column is ZORBAX SAX (4.6mm×250mm×5μm); mobile phase A is ultrapure water with pH=3.5 (pH adjusted with dilute hydrochloric acid), and mobile phase B is 2M sodium chloride solution with pH3.5 (pH adjusted with dilute hydrochloric acid); elution program is: 0~2min, 95% mobile phase A and 5% mobile phase B; 2~43min, 95% mobile phase A and 5% mobile phase B; 43~43.1min, 50% mobile phase A and 50% mobile phase B; 43.1~50min, 95% mobile phase A and 5% mobile phase B; injection volume is 20μL; flow rate is 1.0mL / min; column temperature is 40℃; detection wavelength is 232nm; running time is 50min.

[0171] S4. Take 100 μL of chondroitin ABC enzyme solution (1 U / L) and add it to 1 mL of fermentation supernatant. Incubate at 37°C for 12 h, inactivate chondroitin sulfate lyase ABC in a boiling water bath for 10 min, centrifuge at 12000×g for 20 min, and filter the supernatant. W A concentrator tube with a concentration of 3000 was used to obtain a disaccharide solution to be tested. The disaccharide solution to be tested was taken for UPLC-MS detection, and a standard curve was drawn with Δdi-4S and Δdi-0S standard solutions with concentrations of 0, 10 mg / L, 50 mg / L, 100 mg / L and 200 mg / L. The concentrations of Δdi-4S and Δdi-0S in the fermentation supernatant were obtained based on the standard curve, and the CSA sulfonation efficiency in the fermentation supernatant was calculated according to the following formula; the test results are shown in Table 11.

[0172] CSA sulfonation efficiency = Δdi-4S concentration / (Δdi-4S concentration + Δdi-0S concentration) × 100%

[0173] WATERS QUATTRO PREMIER XE was used for analysis in UPLC-MS detection, and the conditions included: the chromatographic column was CSHC18 (2.1×100mm, 1.7μm); the column temperature was 35°C; the mobile phase A was acetonitrile solution, and the mobile phase B was 1% formic acid solution, with the following gradient: 0~2min, 0%~5% mobile phase B; 2~4min, 5%~15% mobile phase B; 4~7min, 15%~40% mobile phase B; 7~8min, 40%~80% mobile phase B; 8~10min, 80%~0% mobile phase B; the flow rate was 0.3mL / min; the negative ion scanning mode was used, the scanning mass range was set to 100~1000m / z, the ESI ionization mode, the capillary voltage was 3.0kV, the ion source temperature was 100°C, the desolvation temperature was 400°C, and the collision energy was 6eV.

[0174] Table 11.

[0175]

[0176] It can be seen from the test results shown in Table 11 that compared with the recombinant strains C1~C3 and C5 provided in Preparation Examples 1~3 and 5, the recombinant strains C4 and C6 provided in Preparation Examples 4 and 6 are obtained by introducing the ATPS-ST-pykA-APSK / GlmS-GlmM-GlmU-Pgi-Pgm-GalU / KfoA-KfoC-KfoF gene cluster into Escherichia coli BL21 Star (DE3) or Pichia pastoris GS115 as host cells, and the enzyme molecules encoded by the above gene cluster constitute an enzyme cascade pathway with the ability to efficiently synthesize CSA, thereby giving the recombinant strains C4 and C6 the ability to efficiently synthesize CSA using glycerol as a carbon source. It is worth noting that in the recombinant strain C4 prepared with Escherichia coli BL21 Star (DE3) as the host cell, the glycerol metabolic pathway of Escherichia coli BL21 Star (DE3) itself and the enzyme molecules expressed by the introduced gene cluster interact with each other and cooperate to form a more ideal CSA synthase cascade pathway. While achieving high-yield and high-purity preparation of CSA, the sulfonation level of the obtained CSA is as high as 98%.

[0177] Example 2

[0178] This example is used to illustrate the ability of the recombinant strain provided in the above preparation example to synthesize chondroitin sulfate on a fermentation tank culture scale. The steps of synthesizing chondroitin sulfate by fermentation using the recombinant strain specifically include:

[0179] S1. Take 200 μL of the bacterial solution containing the above recombinant strain from the glycerol tube according to the inoculation amount of 10% and inoculate it into LB liquid culture medium, and culture it with shaking at 37°C and 200 rpm for 12 h to obtain seed solution.

[0180] S2. Take 5 mL of seed liquid and transfer it to a fermenter containing 6 L of fermentation medium. Stir and cultivate at a temperature of 37 ° C, a pH of 7.0, a dissolved oxygen content of 25%, and a ventilation rate of 1:1 (VVM). When the dissolved oxygen increases, feed medium is added at a constant dissolved oxygen content of 30% to keep the dissolved oxygen of the culture medium above 25%. When the OD of the culture medium reaches 0.5, the culture medium is stirred and cultured at a temperature of 37 ° C, a pH of 7.0, a dissolved oxygen content of 25%, and a ventilation rate of 1:1 (VVM). When the dissolved oxygen increases, feed medium is added at a constant dissolved oxygen content of 30% to keep the dissolved oxygen of the culture medium above 25%. 600 =30, add IPTG to the culture medium at a final concentration of 0.1 mmol / L, and induce expression at 35°C for 24 h. When the dissolved oxygen increases, start feeding, and feed the culture medium at a constant dissolved oxygen of 30% to maintain the dissolved oxygen above 25% until the end of fermentation. Centrifuge and collect the fermentation supernatant containing chondroitin sulfate.

[0181] The fermentation medium includes 10 g / L glycerol, 2 g / L citric acid, 2.5 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate heptahydrate, 10 g / L soy peptone and 4.5 mg / L vitamin B1, and the feed medium includes 500 g / L glycerol, 200 g / L peptone and 10 g / L magnesium sulfate heptahydrate. The feed medium includes 500 g / L glycerol, 200 g / L peptone and 10 g / L magnesium sulfate heptahydrate.

[0182] S3. The CSA concentration and CSA sulfonation level in the fermentation supernatant were detected according to the method provided in Example 1. The test results are shown in Table 12.

[0183] Table 12.

[0184]

[0185] From the test results shown in Table 12, it can be seen that compared with the recombinant strains C1-C3 and C5 provided in Preparation Examples 1-3 and 5, the recombinant strains C4 and C6 provided in Preparation Examples 4 and 6 have an enzyme cascade pathway capable of efficiently synthesizing CSA in the cells, so that when the fermentation scale is expanded, glycerol can still be well utilized as a carbon source to efficiently synthesize high-purity CSA.

[0186] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

[0187] The amino acid and nucleotide sequences involved in the present invention are specifically shown in Table 13.

[0188] Table 13.

[0189]

Claims

1. A recombinant strain producing chondroitin sulfate, characterized in that: The recombinant strain can express UDP-glucosamine isomerase, UDP-glucose dehydrogenase, chondroitin polymerase, glucosamine synthetase, phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, glucose-6-phosphate isomerase, glucose phosphomutase, glucose-1-phosphate uridyltransferase, ATP sulfonase, sulfotransferase, pyruvate kinase and APS kinase, and the recombinant strain is Escherichia coli or Pichia pastoris.

2. The recombinant strain producing chondroitin sulfate according to claim 1, characterized in that: The recombinant strain has at least one of the following characteristics: The UDP-glucosamine isomerase comprises an amino acid fragment as shown in SEQ ID NO: 10; The UDP-glucose dehydrogenase comprises an amino acid fragment as shown in SEQ ID NO: 11; the chondroitin polymerase comprises an amino acid fragment as shown in SEQ ID NO: 12; The glucosamine synthetase comprises an amino acid fragment as shown in SEQ ID NO: 22; The phosphoglucosamine mutase comprises an amino acid fragment as shown in SEQ ID NO: 23; The UDP-N-acetylglucosamine pyrophosphorylase comprises an amino acid fragment as shown in SEQ ID NO: 24; The glucose-6-phosphate isomerase comprises an amino acid fragment as shown in SEQ ID NO:34; The phosphoglucomutase comprises an amino acid fragment as shown in SEQ ID NO:35; The glucose-1-phosphate uridyl transferase comprises an amino acid fragment as shown in SEQ ID NO:36; The ATP sulfonase comprises an amino acid fragment as shown in SEQ ID NO:49; The sulfotransferase comprises an amino acid fragment as shown in SEQ ID NO:50; The pyruvate kinase comprises an amino acid fragment as shown in SEQ ID NO:51; The APS kinase comprises an amino acid fragment whose sequence is shown in SEQ ID NO:

52.

3. The recombinant strain producing chondroitin sulfate according to claim 1, characterized in that: The recombinant strain has at least one of the following characteristics: The KfoA gene encoding the UDP-glucose amino isomerase includes a nucleotide fragment whose sequence is shown in SEQ ID NO: 1; The KfoF gene encoding the UDP-glucose dehydrogenase includes a nucleotide fragment whose sequence is shown in SEQ ID NO: 2; The KfoC gene encoding the chondroitin polymerase includes a nucleotide fragment whose sequence is shown in SEQ ID NO: 3; The GlmS gene encoding the glucosamine synthase includes a nucleotide fragment whose sequence is shown in SEQ ID NO: 13; The GlmM gene encoding the phosphoglucosamine mutase includes a nucleotide fragment whose sequence is shown in SEQ ID NO: 14; The GlmU gene encoding the UDP-N-acetylglucosamine pyrophosphorylase includes a nucleotide fragment whose sequence is shown in SEQ ID NO: 15; The Pgi gene encoding the glucose-6-phosphate isomerase includes a nucleotide fragment whose sequence is shown in SEQ ID NO: 25; The Pgm gene encoding the phosphoglucomutase includes a nucleotide fragment as shown in SEQ ID NO: 26; The GalU gene encoding the glucose-1-phosphate uridyl transferase includes a nucleotide fragment whose sequence is shown in SEQ ID NO: 27; The ATPS gene encoding the ATP sulfonase includes a nucleotide fragment whose sequence is shown in SEQ ID NO: 37; The ST gene encoding the sulfotransferase includes a nucleotide fragment whose sequence is shown in SEQ ID NO: 38; The pykA gene encoding the pyruvate kinase includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 39; The APSK gene encoding the APS kinase includes a nucleotide fragment whose sequence is shown in SEQ ID NO:

40.

4. Use of the recombinant strain producing chondroitin sulfate according to any one of claims 1 to 3 in preparing chondroitin sulfate by microbial fermentation.

5. A method for preparing chondroitin sulfate, characterized in that: The preparation method comprises: taking the recombinant strain producing chondroitin sulfate according to any one of claims 1 to 3 and subjecting it to fermentation to obtain the chondroitin sulfate.

6. The method for preparing chondroitin sulfate according to claim 5, characterized in that: The fermentation treatment comprises: S1, inoculating the recombinant strain into a fermentation medium for pre-culture to obtain a pre-treated culture solution; S2, adding an inducing expression agent to the pre-treated culture solution for inducing expression to obtain a supernatant containing the chondroitin sulfate.

7. The method for preparing chondroitin sulfate according to claim 6, characterized in that: In step S1, the pre-culture temperature is 35-40° C. and the time is 1-10 hours.

8. The method for preparing chondroitin sulfate according to claim 6, characterized in that: In step S2, the induction expression temperature is 35-40°C and the time is 48-72 hours.

9. The method for preparing chondroitin sulfate according to claim 6, characterized in that: In step S2, the induced expression also includes a feed treatment, which includes: monitoring the dissolved oxygen content of the pretreatment culture solution, and when the dissolved oxygen content is above 20%, taking a feed culture medium and adding it to the pretreatment culture solution to regulate the dissolved oxygen content of the pretreatment culture solution to 15-40%.

10. The method for preparing chondroitin sulfate according to claim 9, characterized in that: The feed medium includes 250-1000 g / L of glycerol, 100-500 g / L of peptone and 5-20 g / L of magnesium sulfate.

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