Corynebacterium glutamicum for producing medium molecular weight chondroitin as well as construction method and molecular marker of corynebacterium glutamicum

Abstract

The invention belongs to the technical field of biology, and particularly relates to corynebacterium glutamicum for producing medium-molecular-weight chondroitin as well as a construction method and a molecular marker of the corynebacterium glutamicum. The preservation number of the Corynebacterium glutamicum CgC26 strain provided by the invention is CGMCC (China General Microbiological Culture Collection Center) NO.31936, and the preservation number of the Corynebacterium glutamicum CgC26 strain is CGMCC NO.31936. And the medium molecular weight is 200 to 1000 kDa. The corynebacterium glutamicum CgC26 strain can synergistically utilize glucose and xylose to produce medium molecular weight chondroitin, the yield of chondroitin is as high as 9.59 + / -0.15 g / L, each g of glucose is converted into 0.09 g of chondroitin, the conversion rate is high, the carbon source utilization range of corynebacterium glutamicum can be widened, and the chondroitin production cost can be reduced.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to Corynebacterium glutamicum producing medium molecular weight chondroitin, a construction method thereof and a molecular marker. Background Art

[0002] Chondroitin sulfate is a glycosaminoglycan that is usually present as a proteoglycan on the cell surface and extracellular matrix. It has a variety of physiological functions, such as anti-inflammatory properties, anti-tumor effects, promotion of cell regeneration, antiviral activity, antioxidant properties, and interaction with cytokines. Chondroitin sulfate is synthesized from chondroitin by the action of sulfatase, and five different types, CS-O, CS-A, CS-C, CS-D, and CS-E, can be obtained depending on the position of the sulfate group. Chondroitin is a negatively charged linear acidic mucopolysaccharide composed of glucuronic acid and N-acetylgalactosamine, which are alternately connected by β-1,3 and β-1,4 glycosidic bonds catalyzed by chondroitin synthase.

[0003] Currently, chondroitin sulfate on the market is mainly extracted from animal tissues. This extraction process not only poses an environmental pollution risk, but also exposes the animal source to potential contamination with antibiotics, feces, inflammatory agents or pathogens. In addition, there is a risk of other glycosaminoglycans during the extraction process, resulting in poor product uniformity and difficulty in obtaining high-purity chondroitin sulfate. These challenges have seriously hindered the study of the structure-activity relationship of different chondroitin sulfate variants. Therefore, there is a growing interest in developing microbial cell factories to produce chondroitin sulfate. Recently, chondroitin sulfate has been successfully synthesized in microorganisms, but achieving the required balance between high-level sulfation and yield remains a challenge. A two-step synthesis method has emerged as a solution, which involves constructing a cell factory to synthesize chondroitin using a simple carbon source and then converting chondroitin to chondroitin sulfate by enzyme catalysis. Significant progress in sulfation engineering has made large-scale implementation of this strategy possible.

[0004] Corynebacterium glutamicum is a Gram-positive industrial microorganism with GRAS status, which is widely used in the production of amino acids, glycosaminoglycans, plant pigments, recombinant proteins, organic acids and other compounds. Current studies have shown that wild-type Corynebacterium glutamicum does not have a complete xylose metabolic pathway and cannot utilize xylose. The strains after metabolic modification have low efficiency in utilizing xylose. At present, the microbial synthesis of chondroitin has problems such as low yield, low conversion rate and high cost. The development of an engineered strain that synergistically utilizes glucose and xylose to produce chondroitin is crucial to broaden the scope of carbon source utilization of Corynebacterium glutamicum and reduce the production cost of chondroitin. Summary of the invention

[0005] The object of the present invention is to provide a Corynebacterium glutamicum for producing medium molecular weight chondroitin and a construction method and a molecular marker thereof. The Corynebacterium glutamicum can synergistically utilize glucose and xylose to produce chondroitin with high yield, high efficiency and low cost.

[0006] The invention provides a Corynebacterium glutamicum CgC26 strain for producing medium molecular weight chondroitin, with the preservation number being CGMCC NO.31936; the medium molecular weight is 200-1000 kDa.

[0007] The present invention provides the molecular marker sequence of the above-mentioned Corynebacterium glutamicum CgC26 strain, and the nucleotide sequence of the molecular marker sequence is shown in SEQ ID NO.4 and SEQ ID NO.5.

[0008] The present invention provides a primer set for detecting the above-mentioned molecular marker sequence, the primer set comprising: CgC26-1 having a nucleotide sequence as shown in SEQ ID NO.6;

[0009] CgC26-2 having a nucleotide sequence as shown in SEQ ID NO.7;

[0010] CgC26-3 having a nucleotide sequence as shown in SEQ ID NO.8;

[0011] The nucleotide sequence is CgC26-4 shown in SEQ ID NO.9.

[0012] The present invention provides a method for constructing the above-mentioned Corynebacterium glutamicum CgC26 strain, comprising the following steps: connecting an XI pathway module containing an RBS sequence and a Weimberg pathway module containing an RBS sequence to a backbone vector to construct a recombinant vector; transforming the recombinant vector into a chondroitin production strain to construct a Corynebacterium glutamicum CgC26 strain;

[0013] The XI pathway module includes the following genes: xylose isomerase gene, xylulokinase gene and xylose transporter gene;

[0014] The Weimberg pathway module includes the following genes: xylose dehydrogenase gene, xylose lactonase gene, 2-dehydro-3-deoxy-d-arabinonate dehydratase gene, xylonate dehydratase gene, 2,5-dioxopentanoate dehydratase gene and arabinose transporter gene.

[0015] As a preferred embodiment, the genome of the chondroitin producing strain includes: a chondroitin synthase gene, an isomerase gene, a 6-phosphoglucosamine synthetase gene and a UDP-glucose dehydrogenase gene.

[0016] As a preferred embodiment, the gene sequence of the chondroitin synthase gene is shown in SEQ ID NO.1;

[0017] The gene sequence of the isomerase gene is shown in SEQ ID NO.2;

[0018] The gene sequence of the 6-phosphoglucosamine synthetase gene is shown in SEQ ID NO.3;

[0019] The gene code of the UDP-glucose dehydrogenase gene is Gene ID: 1020791.

[0020] As a preferred embodiment, the primer pair of the XI pathway module containing the RBS sequence includes: PXI-F and PXI-R;

[0021] The gene sequence of PXI-F is as described in SEQ ID NO.32;

[0022] The gene sequence of PXI-R is as shown in SEQ ID NO.33.

[0023] As a preferred embodiment, the primer pair of the Weimberg pathway module containing the RBS sequence includes: trc XW-F and P trc XW-R;

[0024] The P trc The gene sequence of XW-F is as described in SEQ ID NO.34;

[0025] The P trc The gene sequence of XW-R is shown in SEQ ID NO.35.

[0026] The present invention provides the use of the Corynebacterium glutamicum CgC26 strain or the Corynebacterium glutamicum CgC26 strain constructed by the above-mentioned construction method in the production of medium molecular weight chondroitin.

[0027] The present invention also provides a method for producing medium molecular weight chondroitin, comprising the following steps:

[0028] Cultivating the Corynebacterium glutamicum CgC26 strain or the Corynebacterium glutamicum CgC26 strain constructed by the above construction method as a seed liquid;

[0029] The seed solution is used for fed-batch fermentation, and the fermentation solution contains the medium molecular weight chondroitin.

[0030] Beneficial effects: The present invention provides a Corynebacterium glutamicum CgC26 strain for producing medium molecular weight chondroitin, and the deposit number is CGMCC NO. 31936. The Corynebacterium glutamicum CgC26 strain of the present invention can synergistically utilize glucose and xylose to produce medium molecular weight chondroitin, and the yield of chondroitin is as high as 9.59±0.15g / L, and each g of glucose is converted into 0.09g of chondroitin, with a high conversion rate, which can broaden the scope of carbon source utilization of Corynebacterium glutamicum and reduce the production cost of chondroitin.

[0031] The present invention provides the molecular marker sequence of the above-mentioned Corynebacterium glutamicum CgC26 strain. The molecular marker sequences shown in SEQ ID NO.4 and SEQ ID NO.5 provided by the present invention are essential genes for the synergistic use of glucose and xylose to produce chondroitin synthesis, so the primer sets designed for these two nucleotide sequences can be used to identify the Corynebacterium glutamicum CgC26 strain.

[0032] The present invention provides a method for constructing the above-mentioned Corynebacterium glutamicum CgC26 strain, comprising the following steps: connecting an XI pathway module containing an RBS sequence and a Weimberg pathway module containing an RBS sequence to a backbone vector to construct a recombinant vector; transforming the recombinant vector into a chondroitin production strain to construct a Corynebacterium glutamicum CgC26 strain. The present invention relieves catabolism repression by point mutation of related genes in the xylose metabolic pathway, realizes the coordinated utilization of glucose and xylose, and optimizes the xylose metabolic pathway by constructing an RBS library to improve the metabolic rate of xylose.

[0033] The present invention also provides a method for producing medium molecular weight chondroitin, comprising the following steps: culturing the above-mentioned Corynebacterium glutamicum CgC26 strain or the Corynebacterium glutamicum CgC26 strain constructed by the above-mentioned construction method as a seed solution; fermenting the seed solution, wherein the fermentation solution contains the chondroitin. The method provided by the present invention can synergistically utilize glucose and xylose to produce medium molecular weight chondroitin of 200 to 1000 kDa, and the method has high yield, high efficiency and low cost.

[0034] Biological deposit information

[0035] The Corynebacterium glutamicum CgC26 strain was deposited in the General Microbiology Center of the China Culture Collection Administration, referred to as CGMCC, on September 10, 2023. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31936. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of pEC-XK99E-cgkfoCA, Ptac, saglmS and ugd plasmids;

[0037] Figure 2 This is the ESI-MS analysis result of the fermentation product after enzymatic hydrolysis;

[0038] Figure 3 The molecular weight of chondroitin inside and outside the cell is shown in the figure. The left one is inside the cell, and the right one is outside the cell.

[0039] Figure 4 The results of the generation and growth curve of Corynebacterium glutamicum containing XI pathway and Weimberg pathway are shown in Figure 1. A is the OD of each generation of 10 consecutive generations when the XI pathway is constructed. 600 , B is the growth of four different strains when constructing the XI pathway, and C is the OD of each generation of 10 consecutive passages when constructing the Weimberg pathway 600 , D is the growth of four different bacterial strains when the Weimberg pathway was constructed;

[0040] Figure 5 The fermentation kinetic curves of CgC26 in medium with different ratios of glucose and xylose; wherein A is the mass ratio of glucose to xylose of 10:0, B is the ratio of 7:3, C is the ratio of 5:5, D is the ratio of 3:7, E is the ratio of 0:10, and F is the legend of the detection items. DETAILED DESCRIPTION

[0041] The present invention provides a Corynebacterium glutamicum CgC26 strain for producing medium molecular weight chondroitin, with a deposit number of CGMCC NO.31936; the medium molecular weight is 200-1000 kDa. The Corynebacterium glutamicum CgC26 strain of the present invention is short rod to small rod shape under a microscope, sometimes slightly curved, blunt at both ends, unbranched, single or in figure eight arrangement, Gram-positive, spore-free, non-motile, and the colony is moist and round.

[0042] The present invention provides a method for constructing the above-mentioned Corynebacterium glutamicum CgC26 strain, comprising the following steps: connecting an XI pathway module containing an RBS sequence and a Weimberg pathway module containing an RBS sequence to a backbone vector to construct a recombinant vector; transforming the recombinant vector into a chondroitin production strain to construct a Corynebacterium glutamicum CgC26 strain;

[0043] The XI pathway module includes the following genes: xylose isomerase gene, xylulokinase gene and xylose transporter gene;

[0044] The Weimberg pathway module includes the following genes: xylose dehydrogenase gene, xylose lactonase gene, 2-dehydro-3-deoxy-d-arabinonate dehydratase gene, xylonate dehydratase gene, 2,5-dioxopentanoate dehydratase gene and arabinose transporter gene.

[0045] In the present invention, the genome of the chondroitin production strain may include: chondroitin synthase gene, isomerase gene, 6-phosphoglucosamine synthetase gene and UDP-glucose dehydrogenase gene. As a specific embodiment, the codon-optimized chondroitin synthase gene kfoC (as shown in SEQ ID NO.1) from E.coli K4, the acetyl glucosamine-4-epimerase gene kfoA (as shown in SEQ ID NO.2), the 6-phosphoglucosamine synthetase gene glmS (as shown in SEQ ID NO.3) from Staphylococcus aureus and the UDP-glucose dehydrogenase gene ugd (Gene ID: 1020791) of Corynebacterium glutamicum are sequentially connected to the pEC-XK99E vector by seamless cloning method to construct a chondroitin synthesis module; the chondroitin synthesis module is transformed into wild-type Corynebacterium glutamicum to construct a chondroitin production strain CgC25. The transformation of the present invention is common but not limited to the electroporation transformation under the conditions of 1800kV and 5ms in a 1mM electroporation cup.

[0046] In the present invention, the XI pathway module may include the following genes: a xylose isomerase gene, a xylulokinase gene, and a xylose transporter gene. As a specific embodiment, the xylose isomerase gene xylA (Gene ID: 948141), the xylulose kinase gene xylB (Gene ID: 948133), and the xylose transporter gene xylE (Gene ID: 948529) from E. coli K12 are cloned into DNA fragments, and each gene start codon carries 8 random base sequences (5'-AAAGGANNNNNNNN-3') (SEQ ID NO.36), the three gene fragments were sequentially connected to the pXMJ19 vector by seamless cloning, and a connection system containing a variety of different RBS sequence libraries was constructed; the connection system was transformed into Escherichia coli, the plasmid library was extracted, and the plasmid library was electroporated into wild-type Corynebacterium glutamicum, and after 10 passages every 12 hours, it was applied to a solid culture medium plate with xylose as a carbon source, and the genomic DNA of 10 strains with larger colonies was extracted after 48 hours of culture, and the RBS position was sequenced. The growth curve of different RBS sequence strains with xylose as a carbon source was measured, and the strain with the fastest growth rate and xylose consumption rate was selected, that is, the strain CgRXI-3 with high xylose utilization efficiency containing the xylose isomerization (XI) pathway. As a preferred embodiment, the genome of the strain CgRXI-3 constructed in Example 5 is used as a template, and primers PXI-F and PXI-R are used for PCR amplification to obtain the XI pathway module containing the RBS sequence. The gene sequence of PXI-F of the present invention is as described in SEQ ID NO.32; the gene sequence of PXI-R is as described in SEQ ID NO.33.

[0047] In the present invention, the Weimberg pathway module may include the following genes: xylose dehydrogenase gene, xylose lactonase gene, 2-dehydro-3-deoxy-d-arabinonate dehydratase gene, xylonate dehydratase gene, 2,5-dioxopentanoate dehydratase gene and arabinose transporter gene. As a specific embodiment, a gene cluster containing a xylose dehydrogenase gene xdh (Gene ID: 170658293), a xylose lactonase gene xylC (Gene ID: 292674602) and a 2-dehydro-3-deoxy-d-arabinonate dehydratase gene kdxD (Gene ID: 332011117) from Sphingomonas sanxanigenens, a xylose dehydratase gene xylD (Gene ID: 7329902) from Caulobacter crescentus, a 2,5-dioxopentanoate dehydratase gene aldH (Gene ID: 1021211) from Corynebacterium glutamicum and an arabinose transporter gene araE (Gene ID: 332011117) from E. coli K12 are used. ID: 947341), cloned into DNA fragments respectively, wherein the upstream of the start codon of xylD, aldH, and araE genes carries 8 random base sequences (5'-AAAGGANNNNNNNN-3') (SEQ ID NO.36), and the four gene fragments are sequentially connected to the pXMJ19 vector by seamless cloning to construct a connection system containing a variety of different RBS sequence libraries; the connection system is transformed into Escherichia coli, the plasmid library is extracted, and then the plasmid library is transformed into wild-type Corynebacterium glutamicum by electroporation, and after 10 passages every 12 hours, it is spread on a solid culture medium plate with xylose as a carbon source, and after culturing for 48 hours, the genomic DNA of 10 strains with larger colonies is extracted, the RBS position is sequenced, and the growth curve of strains with different RBS sequences with xylose as a carbon source is measured, and the strain with the fastest growth rate and xylose consumption rate is selected, that is, the strain CgRXW-1 with high xylose utilization efficiency containing the Weimberg pathway is selected. As a preferred embodiment, the genome of the constructed strain CgRXW-1 is used as a template, and primer pair P trc XW-F and P trc XW-R was PCR amplified to obtain the Weimberg pathway module containing the RBS sequence. trc The gene sequence of XW-F is as described in SEQ ID NO.34; trc The gene sequence of XW-R is shown in SEQ ID NO.35.

[0048] In the present invention, the Weimberg pathway module containing the RBS sequence and the XI pathway module containing the RBS sequence are connected to a backbone vector to construct a recombinant vector; the recombinant vector is transformed into a chondroitin production strain to construct a Corynebacterium glutamicum CgC26 strain. As a specific embodiment, the Weimberg pathway module containing the RBS sequence and the XI pathway module containing the RBS sequence are connected to a pXMJ19 vector, and after sequencing verification, they are transformed into Corynebacterium glutamicum CgC25 by electroporation, and Corynebacterium glutamicum CgC26 that efficiently and synergistically utilizes glucose and xylose to synthesize chondroitin is successfully constructed.

[0049] The present invention provides the molecular marker sequence of the above-mentioned Corynebacterium glutamicum CgC26 strain, and the nucleotide sequence of the molecular marker sequence is shown in SEQ ID NO.4 and SEQ ID NO.5. In the present invention, the sequences of the two plasmids included in the construction process of strain CgC26 are part of their respective sequences, and the specific nucleotide sequences are shown in SEQ ID NO.4 and SEQ ID NO.5, with a total length of 1995bp and 1826bp respectively. The two sequences are respectively a sequence of a gene fragment inserted in the plasmid of the constructed chondroitin synthesis module and the plasmid of the xylose metabolism module except the plasmid backbone, and the sequences can respectively represent the connection order and partial gene sequence of the inserted genes in the two modules.

[0050] The present invention provides a primer set for detecting the above-mentioned molecular marker sequence, the primer set comprising: CgC26-1 having a nucleotide sequence as shown in SEQ ID NO.6; CgC26-2 having a nucleotide sequence as shown in SEQ ID NO.7; CgC26-3 having a nucleotide sequence as shown in SEQ ID NO.8; and CgC26-4 having a nucleotide sequence as shown in SEQ ID NO.9. In the present invention, the detection primer set can be designed according to the above-mentioned molecular marker sequence. As a specific implementation method, the specific method for detecting the above-mentioned Corynebacterium glutamicum CgC26 strain is as follows: (1) extracting the genome of the strain as a PCR template; (2) the PCR amplification system is calculated as 25 μL: 0.5 μL of 10-50 ng / μL DNA template, 0.4 μL of each 20 μM primer pair, 12.5 μL of premix Taq polymerase and the remainder of water; (3) the PCR reaction conditions are: 95°C pre-denaturation for 10 min; 94°C for 30-45 s, 53-63°C for 30-45 s, 72°C for 45-60 s, 35 cycles; 72°C extension for 10 min; (4) electrophoresis detection, gel recovery of the target fragment, and sending it to a sequencing company for sequencing verification, and the sequencing results are used to determine whether the Corynebacterium glutamicum CgC26 strain is contained.

[0051] The present invention also provides a method for producing medium molecular weight chondroitin, comprising the following steps: culturing the above-mentioned Corynebacterium glutamicum CgC26 strain or the Corynebacterium glutamicum CgC26 strain constructed by the above-mentioned construction method as a seed solution; using the seed solution for fed-batch fermentation, and the fermentation solution contains the medium molecular weight chondroitin. The method provided by the present invention can produce medium molecular weight chondroitin of 200 to 1000 kDa.

[0052] To further illustrate the present invention, the following detailed description of a Corynebacterium glutamicum producing medium molecular weight chondroitin and its construction method and molecular markers provided by the present invention are provided in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1 Construction of chondroitin synthesis module plasmid

[0054] (1) Using primers cgkfoC-F (SEQ ID NO.10) and cgkfoC-R (SEQ ID NO.11), the chondroitin synthase gene from E. coli K4 optimized according to the codon preference of Corynebacterium glutamicum was used as a template, and PCR was used to obtain the cgKfoC gene; the same method was used to obtain the acetyl glucosamine-4-epimerase gene cgkfoA by PCR using primers cgkfoA-F (SEQ ID NO.12) and cgkfoA-R (SEQ ID NO.13). The 6-phosphoglucosamine synthase gene glmS from Staphylococcus aureus was obtained by PCR using primers saglmS-F (SEQ ID NO.14) and saglmS-R (SEQ ID NO.15), and the UDP-glucose dehydrogenase gene ugd from Corynebacterium glutamicum was obtained by PCR using primers ugd-F (SEQ ID NO.16) and ugd-R (SEQ ID NO.17); the sequence information is shown in Table 4. (2) The PCR amplification system is: 0.5 μL of 10-50 ng / μL DNA template, 0.4 μL of each 20 μM primer pair, 12.5 μL of PrimeStar premix polymerase, and add water to 25 μL. (3) The PCR reaction conditions are: pre-denaturation at 98°C for 30 s; 98°C for 15 s, 54°C for 15 s, 72°C for 10-60 s, 35 cycles; extension at 72°C for 3 min. (4) PCR products are separated by agarose gel electrophoresis, and the target bands are recovered by gel to obtain the target fragments. (5) The above fragments are sequentially connected to the pEC-XK99E vector using the HiFi DNA Assembly Mastermix one-step method, heat-shock transformed into Escherichia coli competent cells, plated for screening, and verified. (6) Successful sequencing alignment indicates the successful construction of pEC-XK99E-cgkfoCA, Ptac, saglmS and ugd plasmids. The schematic diagram of the plasmid is shown in the figure. Figure 1 shown.

[0055] Example 2 Electroporation of the Chondroitin Synthesis Module Plasmid into Corynebacterium glutamicum

[0056] (1) Extract pEC-XK99E-cgkfoCA, Ptac, saglmS and ugd plasmids. (2) Take 1 μL of plasmid and add it to the competent cells of Corynebacterium glutamicum. After precooling for 10 minutes, electroporate at 1800V for 5ms to transform it into the cells. (3) After 3 hours of recovery, apply resistance plates for screening. (4) Pick a single colony and save it to successfully construct the chondroitin synthesis engineering strain CgC25. (5) Shake flask fermentation to extract chondroitin products.

[0057] Example 3 Extraction of chondroitin product from fermentation broth

[0058] 30 mL of fermentation broth was centrifuged at 12000 × g for 10 min to collect bacteria. The obtained supernatant was transferred to another tube for further extraction of extracellular chondroitin. At the same time, the precipitate was washed with PBS and then resuspended in distilled water. The high-pressure homogenization process was carried out for 8 cycles at 1100 bar to destroy the cells and release chondroitin. The generated cell debris was removed by centrifugation at 15000 × g for 20 min, and the intracellular chondroitin remained in the supernatant. Three volumes of anhydrous ethanol were added to the supernatant of the first and second centrifugations, and precipitated at 4 ° C for 6 hours. Subsequently, the precipitate was collected by centrifugation at 12000 × g for 10 min, and then an equal volume of distilled water was added to dissolve. After removing the insoluble matter, three volumes of anhydrous ethanol were added, and this step was repeated three times. After the obtained supernatant was freeze-dried, the chondroitin sample was obtained, which was then diluted in an appropriate ratio and determined using the sulfuric acid-carbazole method, and the strain containing the empty plasmid was used as a negative control.

[0059] Example 4 Identification of Products Extracted from Fermentation

[0060] The purified product was hydrolyzed by chondroitinase ABC at 37°C and then analyzed by liquid chromatography quadrupole time-of-flight mass spectrometry coupled to electrospray ionization mass spectrometry (ESI-MS) in negative scan mode.

[0061] The results are as follows Figure 2 As shown, the fermentation product after enzymatic hydrolysis is mainly chondroitin disaccharide with m / z of 378.10.

[0062] Example 5 Multi-angle laser scattering instrument-size exclusion chromatography combined determination of the molecular weight of the product

[0063] According to the method of patent CN202310886163.3, the molecular weight of the product was determined by multi-angle laser scattering instrument-size exclusion chromatography. Sample preparation: Weigh 10 mg of dry sample and fully dissolve it in 10 mL of ultrapure water, then filter it using a 0.22 μm filter membrane. Size exclusion chromatography conditions: Agilent 1260 liquid chromatography system equipped with Waters Ulturahudrogel TM Linear column; mobile phase is phosphate buffer (pH7.2); injection volume is 200μL; column temperature is room temperature; flow rate is set to 0.6mL / min; differential detector is used, and the detector temperature is 35℃. Multi-angle laser scattering instrument: MALLS, WyattTechnology DAWN HELEOS, Santa Barbara, CA, USA. Standard sample: bovine serum albumin BSA. Data acquisition and analysis software: ASTRA software.

[0064] The results are as follows Figure 3 As shown in Table 1, the weight average molecular weights of the intracellular and extracellular chondroitin products are 678.70±7.13 kDa and 587.20±4.13 kDa, respectively, which belong to the category of medium molecular weight chondroitin.

[0065] Table 1 Weight average molecular weight of intracellular and extracellular chondroitin

[0066] Chondroitin Weight average molecular weight (kDa) Intracellular chondroitin 678.70±7.13 Extracellular chondroitin 587.20±4.13

[0067] At the same time, this method can be used to distinguish high, medium and low molecular weight chondroitin products on the market.

[0068] Example 6 Construction of a strain with high xylose utilization efficiency containing a xylose isomerization (XI) pathway

[0069] (1) The xylose isomerase gene xylA from E. coli K12 was obtained by PCR using primers xylA-F (SEQ ID NO.18) and xylA-R (SEQ ID NO.19); the xylose isomerase gene xylB from E. coli K12 was obtained by PCR using primers xylB-F (SEQ ID NO.20) and xylB-R (SEQ ID NO.21); the xylose transporter gene xylE from E. coli K12 was obtained by PCR using primers xylE-F (SEQ ID NO.22) and xylE-R (SEQ ID NO.23); each gene start codon has 8 random base sequences (SEQ ID NO.36) upstream, and the sequence information is shown in Table 4. (2) The PCR system and procedure are consistent with those described in Example 1. (3) The above fragments were sequentially connected to the pXMJ19 vector using the HiFi DNA Assembly Mastermix one-step method, heat-shock transformed into E. coli competent cells, and plated. (4) Extract plasmids from all colonies on the plate, thereby successfully constructing a recombinant plasmid library containing random RBS. (5) Click-transform the above plasmid library into competent cells of Corynebacterium glutamicum. (6) Subculture every 12 hours after recovery, for 10 consecutive times. Figure 4 A is the OD of each generation 600 , Table 2 shows the specific values, OD 600 It represents the growth situation in each generation within 12 hours. The larger the value is, the greater the proportion of bacteria with fast growth rate in the bacterial community.

[0070] Table 2 Growth of each generation during the passage of the xylose isomerase (XI) pathway

[0071] Number of passages <![CDATA[OD 600 -1]]> <![CDATA[OD 600 -2]]> <![CDATA[OD 600 -3]]> First Generation 0.899 0.762 0.671 Second Generation 1.474 1.234 1.601 Third Generation 3.204 2.982 3.432 Fourth Generation 3.674 3.772 3.688 Fifth Generation 4.999 5.314 5.19 Sixth Generation 5.637 5.988 5.808 Seventh Generation 6.32 7.128 6.532 Eighth Generation 8.76 8.67 8.93 Ninth Generation 9.86 9.3 10.6 Tenth Generation 10.92 11.02 9.99

[0072] (7) Plate screening, select 10 larger colonies for sequencing, and determine the RBS sequence. Among them, there are three strains with different RBS. (8) Determine the growth curves of different RBS in the culture medium with xylose as the only carbon source. Figure 4Figure B shows the growth of three strains with different RBS and a control strain in 10 larger colonies, where the two lines represent OD600 and sugar content, respectively. The steeper the line, the better the growth and xylose utilization. It can be seen that the growth rate of the CgRXI-3 strain is the fastest. The RBS sequences upstream of the XylA, XylB and XylE genes of the CgRXI-3 strain are 5'-AAAGGACTCGGCGTATG-3' (SEQ ID NO.37), 5'-AAAGGACGGCGGTGATG-3' (SEQ ID NO.38) and 5'-AAAGGAGTCTGTGCATG-3' (SEQ ID NO.39), respectively; (9) The CgRXI-3 strain was preserved, and this strain is a strain with a high xylose utilization efficiency of the XI pathway module containing the RBS sequence.

[0073] Example 7 Construction of a strain with high xylose utilization efficiency containing the Weimberg pathway

[0074] (1) Using primers wei-F (SEQ ID NO.24) and wei-R (SEQ ID NO.25), PCR was used to obtain the gene cluster fragments of the xylose dehydrogenase gene xdh, xylose lactonase gene xylC and 2-dehydro-3-deoxy-d-arabinonate dehydratase gene kdxD from Sphingomonas sanxanigenens; using primers xylD-F (SEQ ID NO.26) and xylD-R (SEQ ID NO.27), PCR was used to obtain the xylonate dehydratase gene xylD from Caulobacter crescentus; using primers aldH-F (SEQ ID NO.28) and aldH-R (SEQ ID NO.29), PCR was used to obtain the 2,5-dioxopentanoate dehydratase gene aldH from Corynebacterium glutamicum; using primers araE-F (SEQ ID NO.30) and araE-R (SEQ ID NO.31), PCR was used to obtain the 2,5-dioxopentanoate dehydratase gene aldH from Corynebacterium glutamicum; using primers araE-F (SEQ ID NO.32) and araE-R (SEQ ID NO.33), PCR was used to obtain the 2,5-dioxopentanoate dehydratase gene aldH from Corynebacterium glutamicum; using primers araE-F (SEQ ID NO.34) and araE-R (SEQ ID NO.35), PCR was used to obtain the 2,5-dioxopentanoate dehydratase gene aldH from Corynebacterium glutamicum; using primers araE-F (SEQ ID NO.35) and araE-R (SEQ ID NO.36), PCR was used to obtain the 2,5-dioxopentanoate dehydratase gene aldH from Corynebacterium glutamicum; using primers araE- NO.31) PCR was used to obtain the xylose transporter gene araE from E.coli K12; wherein the upstream of the start codons of the xylD, aldH and araE genes carried 8 random base sequences (5'-AAAGGANNNNNNNN-3') (SEQ ID NO.36), and the sequence information is shown in Table 4. (2) The PCR system and procedure were consistent with those described in Example 1. (3) The above fragments were sequentially connected to the pXMJ19 vector using the HiFi DNA Assembly Mastermix one-step method, heat-shocked and transformed into competent Escherichia coli cells, and plated. (4) Plasmids were extracted from all colonies on the plate, thereby successfully constructing a recombinant plasmid library containing random RBS. (5) The above plasmid library was click-transformed into the competent Bacillus glutamicum. (6) After recovery, the cells were subcultured every 12 hours for 10 consecutive times. Figure 4 C in the middle is the OD of each generation 600 , Table 3 shows the specific values, OD 600 It represents the growth situation in each generation within 12 hours. The larger the value is, the greater the proportion of bacteria with fast growth rate in the bacterial community.

[0075] Table 3 Growth of each generation during the passage of constructs containing the Weimberg pathway

[0076] Number of passages <![CDATA[OD 600 -1]]> <![CDATA[OD 600 -2]]> <![CDATA[OD 600 -3]]> First Generation 0.641 0.592 0.634 Second Generation 1.212 1.309 1.234 Third Generation 2.451 2.045 2.121 Fourth Generation 3.009 3.322 2.867 Fifth Generation 4.109 3.789 4.086 Sixth Generation 4.537 4.423 4.668 Seventh Generation 5.031 5.321 4.903 Eighth Generation 5.903 6.421 5.832 Ninth Generation 7.076 7.883 6.782 Tenth Generation 7.461 7.892 7.242

[0077] (7) Plate screening, select 10 larger colonies for sequencing, and determine the RBS sequence. There are two strains with different RBS. (8) Determine the growth curves of different RBS in the culture medium with xylose as the only carbon source. Figure 4Figure D shows the growth of two different RBS strains and a control strain in 10 larger colonies. The CgRXW-1 strain had the fastest growth rate. The RBS sequences upstream of the XylD, AraE and AldH genes of the CgRXW-1 strain were 5'-AAAGGATCGAGGAGATG-3' (SEQ ID NO. 40), 5'-AAAGGACTACCTGAATG-3' (SEQ ID NO. 41) and 5'-AAAGGACGTGCGGTATG-3' (SEQ ID NO. 42), respectively. (9) The CgRXW-1 strain was selected for preservation. This strain is a strain with a high xylose utilization efficiency containing the Weimberg pathway.

[0078] Example 8 Construction of a strain with high xylose utilization efficiency containing XI pathway and Weimberg pathway

[0079] (1) Using the genome of strain CgRXI-3 constructed in Example 5 as a template, primers PXI-F (SEQ ID NO.32) and PXI-R (SEQ ID NO.33) were used for PCR amplification to obtain the XI pathway module containing the RBS sequence, and the sequence information is shown in Table 4. (2) Using the genome of strain CgRXW-1 constructed in Example 6 as a template, primers PtrcXW-F (SEQ ID NO.34) and PtrcXW-R (SEQ ID NO.35) were used for PCR amplification to obtain the Weimberg pathway module containing the RBS sequence, and the sequence information is shown in Table 4. (3) The above fragments were sequentially connected to the pXMJ19 vector using the HiFi DNA Assembly Mastermix one-step method, heat-shocked and transformed into Escherichia coli competent cells, and plated. (4) Successful sequencing alignment indicates the successful construction of the pEC-XK99E-XI-XW plasmid. (5) The pEC-XK99E-XI-XW plasmid was transformed into the chondroitin-producing Corynebacterium glutamicum CgC25 by electroporation according to the method in Example 2, and the Corynebacterium glutamicum CgC26 strain containing the XI pathway and the Weimberg pathway that can synergistically utilize glucose and xylose to produce medium molecular weight chondroitin was successfully constructed. The deposit number is CGMCC NO.31936.

[0080] Example 9 Identification of strains that synergistically utilize glucose and xylose to produce medium molecular weight chondroitin

[0081] According to the sequences of the two plasmids included in the construction process of strain CgC26, and using a part of each sequence as a molecular marker, the specific nucleotide sequences are shown in SEQ ID NO.4 and SEQ ID NO.5, with a total length of 1995 bp and 1826 bp, respectively. The identification primer pairs are CgC26-1 (SEQ ID NO.6), CgC26-2 (SEQ ID NO.7), CgC26-3 (SEQID NO.8), and CgC26-4 (SEQ ID NO.9), and the sequence information is shown in Table 4.

[0082] The specific identification methods are as follows:

[0083] (1) Extract the genome of strain CgC26 or the genome of the bacterial solution of strain CgC26 as a PCR template. (2) The PCR amplification system is: 0.5 μL of 10-50 ng / μL DNA template, 0.4 μL of each 20 μM primer pair, 12.5 μL of premix Taq polymerase, and add water to 25 μL. (3) The PCR reaction conditions are: 95°C pre-denaturation for 10 min; 94°C for 30-45 s, 53-63°C for 30-45 s, 72°C for 45-60 s, 35 cycles; 72°C extension for 10 min. (4) Electrophoresis detection, gel recovery of target fragments. The target fragments were sent to a sequencing company for sequencing, and the full lengths were 1995 bp and 1826 bp, respectively. The sequencing results were consistent with the sequences, that is, the specific molecular markers of the strains that synergistically utilize glucose and xylose to produce medium molecular weight chondroitin were obtained.

[0084] Table 4 Sequence information corresponding to each primer

[0085]

[0086]

[0087] Example 10 Production of medium molecular weight chondroitin using a 5L fermenter

[0088] (1) Streak the CgC26 strain of Corynebacterium glutamicum frozen at -80℃ onto a nutrient-rich agar plate and culture at 30℃ for 48h. (2) Pick a single colony into a conical flask containing 50mL of seed culture medium and culture at 30℃ and 250rpm until the end of the logarithmic phase as seed solution. (3) Transfer the seed solution to a 5L fermentation tank containing 2.5L of fermentation medium. Use ammonia water to maintain the pH range of 6.8-7.0 during the fermentation process. Define the DO at the initial fermentation as 100%, and maintain the DO not less than 10% by adjusting the rotation speed and ventilation volume. (4) Take samples every 6h during the fermentation process to monitor the cell density, residual sugar and chondroitin production of the fermentation broth. The other components of the fermentation medium remain unchanged, the initial total sugar concentration is 40g / L, and glucose and xylose with a concentration of 60% are continuously added during the fermentation process. The feed amount is controlled by controlling the flow rate to maintain the total sugar concentration of about 10g / L. The growth of chondroitin synthesis was statistically analyzed using different ratios of glucose and xylose as carbon sources. The results are as follows Figure 5 As shown in Table 5. The mass ratios of glucose and xylose are 10:0, 7:3, 5:5, 3:7, and 0:10, respectively. (5) The method for determining the chondroitin production is as shown in Example 3; the fermentation broth is diluted to an appropriate multiple with distilled water, and the OD600 is measured using a spectrophotometer with water as a blank control, and then converted into cell density according to the dilution multiple; the concentrations of glucose and xylose are determined by the method described in patent CN201810737183.3.

[0089] Table 5 Fermentation of CgC26 in medium with different glucose and xylose ratios in a 5L fermenter

[0090]

[0091]

[0092] from Figure 5 It can be seen that when glucose is the only carbon source, the yield of chondroitin is higher than that of mixed carbon sources, with a yield of 9.59±0.15 g / L, which is 29.6% higher than the 7.4 g / L yield reported in the literature Biosynthesis of non-sulfated high-molecular-weight glycosaminoglycans and specific-sized oligosaccharides published by Hu et al. in 2022. In this study, each gram of glucose was converted into 0.09 grams of chondroitin, which is much higher than the approximately 0.04 grams reported in the literature. Figure 5 The results shown in B, C, and D show that glucose and xylose are consumed simultaneously. Specifically, when the ratio of glucose to xylose is 7:3 ( Figure 5 In Figure B), the two sugars are utilized almost simultaneously, indicating that glucose is consumed faster than xylose. This observation is consistent with the 5:5 ratio of glucose to xylose ( Figure 5 Further support was also received for C).

[0093] Table 5 details the specific parameters using different ratios of mixed sugars as carbon sources, including maximum specific growth rate, final cell density, and cell density at 3 hours. When glucose was used as the sole carbon source, the maximum specific growth rate was 0.58 h-1, which was higher than that of mixed sugars and xylose. The maximum growth rate of mixed sugars exceeded that of xylose. In mixed sugar medium, the cell density after 3 hours of culture exceeded that of single glucose and xylose, indicating that the addition of xylose helps shorten the lag phase.

[0094] It can be seen that the Corynebacterium glutamicum CgC26 strain provided by the present invention can synergistically utilize glucose and xylose to produce medium molecular weight chondroitin, which is crucial for broadening the carbon source utilization range of Corynebacterium glutamicum and reducing the production cost of chondroitin.

[0095] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A Corynebacterium glutamicum CgC26 strain producing medium molecular weight chondroitin, characterized in that: The deposit number is CGMCCNO.31936; the medium molecular weight is 200-1000 kDa.

2. The molecular marker sequence of the Corynebacterium glutamicum CgC26 strain according to claim 1, characterized in that The nucleotide sequences of the molecular marker sequences are shown in SEQ ID NO.4 and SEQ ID NO.

5.

3. A primer set for detecting the molecular marker sequence according to claim 2, characterized in that: The primer set includes: CgC26-1 having a nucleotide sequence as shown in SEQ ID NO.6; CgC26-2 having a nucleotide sequence as shown in SEQ ID NO.7; CgC26-3 having a nucleotide sequence as shown in SEQ ID NO.8; The nucleotide sequence is CgC26-4 shown in SEQ ID NO.

9.

4. The method for constructing the Corynebacterium glutamicum CgC26 strain according to claim 1, characterized in that: The method comprises the following steps: connecting an XI pathway module containing an RBS sequence and a Weimberg pathway module containing an RBS sequence to a backbone vector to construct a recombinant vector; transforming the recombinant vector into a chondroitin production strain to construct a Corynebacterium glutamicum CgC26 strain; The XI pathway module includes the following genes: xylose isomerase gene, xylulokinase gene and xylose transporter gene; The Weimberg pathway module includes the following genes: xylose dehydrogenase gene, xylose lactonase gene, 2-dehydro-3-deoxy-d-arabinonate dehydratase gene, xylonate dehydratase gene, 2,5-dioxopentanoate dehydratase gene and arabinose transporter gene.

5. The construction method according to claim 4, characterized in that: The genome of the chondroitin production strain comprises: a chondroitin synthase gene, an isomerase gene, a 6-phosphoglucosamine synthetase gene and a UDP-glucose dehydrogenase gene.

6. The construction method according to claim 5, characterized in that: The gene sequence of the chondroitin synthase gene is shown in SEQ ID NO.1; The gene sequence of the isomerase gene is shown in SEQ ID NO.2; The gene sequence of the 6-phosphoglucosamine synthetase gene is shown in SEQ ID NO.3; The gene code of the UDP-glucose dehydrogenase gene is Gene ID: 1020791.

7. The construction method according to claim 4, characterized in that: The primer pair for the XI pathway module containing the RBS sequence includes: PXI-F and PXI-R; The gene sequence of PXI-F is as described in SEQ ID NO.32; The gene sequence of PXI-R is as shown in SEQ ID NO.

33.

8. The construction method according to claim 4, characterized in that: The primer pair of the Weimberg pathway module containing the RBS sequence includes: trc XW-F and P trc XW-R; The P trc The gene sequence of XW-F is as described in SEQ ID NO.34; The P trc The gene sequence of XW-R is shown in SEQ ID NO.

35.

9. Use of the Corynebacterium glutamicum CgC26 strain according to claim 1 or the Corynebacterium glutamicum CgC26 strain constructed by the construction method according to any one of claims 4 to 8 in the production of medium molecular weight chondroitin.

10. A method for producing medium molecular weight chondroitin, characterized in that: The following steps are involved: Cultivating the Corynebacterium glutamicum CgC26 strain according to claim 1 or the Corynebacterium glutamicum CgC26 strain constructed by the construction method according to any one of claims 4 to 8 as a seed liquid; The seed solution is used for fed-batch fermentation, and the fermentation solution contains the medium molecular weight chondroitin.

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