Bacillus subtilis engineering bacteria with high chondroitin production and construction method and application thereof

Abstract

This invention relates to the field of bioengineering, and more particularly to a high-chondroitin-producing engineered Bacillus subtilis strain, its construction method, and its applications. The invention provides an engineered strain using Bacillus subtilis as a substrate strain, expressing an enzyme comprising UDP-N-acetylglucosamine C4 isomerase, chondroitin synthase, and one or more of the following enzyme combinations: UDP-glucose dehydrogenase; UTP-glucose-1-phosphate uridine transferase; glutamine-fructose-6-phosphate aminotransferase; UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase; and phosphogluconomutase. This invention, through metabolic engineering strategies, designs and constructs a stable, plasmid-free, high-chondroitin-producing engineered strain, overcoming existing yield bottlenecks and achieving efficient, safe, and sustainable production of chondroitin sulfate, providing technical support for its widespread application in the food, health product, and pharmaceutical fields.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and in particular to engineered Bacillus subtilis strains that produce high levels of chondroitin, their construction methods, and applications. Background Technology

[0002] Chondroitin sulfate (ChS / CS) is a naturally derived, bioactive macromolecule widely found in mammals and invertebrates, profoundly influencing various physiological processes, including anti-inflammatory effects, inhibition of tumor progression and metastasis, participation in angiogenesis, and antioxidant activity. Due to these significant bioactivities, chondroitin sulfate has gained recognition in the medical field and is now recommended by the European League Against Rheumatism (EULAR) as SYSADOA (Symptomatic Slow Acting Drug for OA) for the treatment of knee osteoarthritis. It is also recommended for the treatment of osteoarthritis in the knee, hip, and hand. As an important bioactive substance, the choice of commercial-scale industrial production methods for chondroitin sulfate is crucial for product quality, cost, and sustainability.

[0003] Currently, common production methods for chondroitin sulfate include animal tissue extraction, chemical synthesis, and enzymatic hydrolysis. However, these traditional methods all have limitations in practical applications. Animal tissue extraction is currently the main method for commercially and industrially obtaining chondroitin sulfate. It uses the cartilage of land animals such as cattle, pigs, and chickens, as well as marine animals such as sharks, rays, and bony fish, as raw materials. Although this method has abundant raw materials and mature extraction technology, it has many problems. On the one hand, there is a risk of viral transmission; animal diseases such as mad cow disease may be transmitted through the extraction process, raising concerns about the safety of use. On the other hand, different animal sources, parts, ages, and other factors can lead to significant differences in the type and content of chondroitin sulfate, affecting the uniformity and stability of product quality. Moreover, the extraction process requires complex purification steps to remove impurities such as proteins and fats, increasing production costs and difficulty. In addition, the extracted chondroitin sulfate may contain impurities such as animal-derived proteins, which may trigger immune responses after entering the human body. Chemical synthesis utilizes chemical reactions to progressively construct the molecular structure of chondroitin sulfate. Its advantages include high product purity and the ability to synthesize chondroitin sulfate derivatives with specific structures and properties as needed. However, this method is cumbersome, requiring multiple chemical reactions and strict condition control, making it complex and demanding in terms of equipment and technology. Furthermore, the chemical reagents used are expensive, and the production process generates significant waste, resulting in high treatment costs and making it difficult to reduce the final product cost. It also causes considerable environmental pollution, failing to meet the requirements of green development. Enzymatic hydrolysis utilizes specific enzymes, such as chondroitinase, to selectively degrade and extract chondroitin sulfate from animal tissues. Its reaction conditions are mild, typically carried out at room temperature and pressure, which helps maintain the biological activity and structural integrity of chondroitin sulfate. Moreover, the enzymes have high substrate specificity and catalytic activity, specifically acting on chondroitin sulfate, reducing damage and interference to other components and improving product purity, making it a relatively green production method. However, the high cost of enzymes limits their application in large-scale production. In addition, the enzymatic hydrolysis reaction is relatively slow, requiring a long reaction time to obtain sufficient product yield, affecting production efficiency. Furthermore, the stability of enzymatic hydrolysis products can be affected by factors such as enzyme activity and reaction conditions, requiring strict control of storage and transportation conditions to ensure product quality. Given the numerous challenges faced by animal tissue extraction, chemical synthesis, and enzymatic hydrolysis methods in chondroitin sulfate production (such as safety issues, high costs, environmental pollution, and low production efficiency), developing a more efficient, environmentally friendly, sustainable, and cost-effective production method has become an urgent priority. Driven by this demand, microbial fermentation has gradually become a research and application hotspot.

[0004] Microbial fermentation involves introducing genes related to the chondroitin sulfate synthesis pathway into microbial cells using genetic engineering, thereby synthesizing chondroitin sulfate through microbial fermentation. This method offers significant advantages: rapid microbial reproduction, high renewability, and sustainable production; furthermore, some commonly used industrial microbial strains are considered safe production hosts, resulting in safer chondroitin sulfate compared to animal tissue extraction. Moreover, once an efficient fermentation process is established, microbial fermentation holds the potential to significantly reduce production costs through large-scale fermentation, better meeting the demands of industrial production. However, this method requires complex genetic engineering operations and fermentation process optimization, placing high demands on technological research and development and production management. Most importantly, microbial fermentation is currently still in its developmental stage, with relatively low yields that are insufficient to meet the needs of large-scale industrial production.

[0005] In recent years, researchers at home and abroad have conducted in-depth and systematic studies on the production of chondroitin sulfate by microbial fermentation from multiple perspectives, including biochemical engineering and metabolic engineering. For example, some studies have successfully obtained a recombinant strain of Escherichia coli K4 that can directly produce chondroitin without a fructosylated backbone by inactivating the glycosyltransferase KfoE responsible for fructosylation modification of the chondroitin backbone in Escherichia coli K4. This achievement provides a new idea for improving the synthesis efficiency of chondroitin (CN103228781B). Chunlei Zhao et al. directly expressed key enzymes in the chondroitin synthesis pathway in Escherichia coli BL21 (DE3), which further promoted the technological development of chondroitin sulfate production by microbial fermentation (Reference 1: Zhao C, Li X, Guo L, Gao C, Song W, Wei W, Wu J, Liu L, Chen X. Reprogramming Metabolic Flux in Escherichia Coli to Enhance Chondroitin Production. Adv Sci (Weinh). 2024Mar;11(10):e2307351.). However, despite significant progress in these studies, *E. coli*, being an enteric pathogen, is susceptible to phage contamination during fermentation. Once infected, bacteriophages multiply rapidly within *E. coli*, eventually leading to cell lysis. This not only affects fermentation yield but may also introduce impurities such as phage proteins into the fermentation broth, increasing biosafety risks. Furthermore, contamination by other microorganisms can alter the physicochemical properties of the fermentation environment, interfering with *E. coli* growth and the synthesis of the target product, and even producing harmful metabolites such as toxins. These potential biosafety risks severely limit the application of *E. coli* in industries such as food and health supplements. The food and health supplement industries have extremely high requirements for the safety of raw materials; any risk of introducing pathogens or toxins is unacceptable. Therefore, ensuring the safety of the production process and products while increasing yield and reducing costs remains a key issue that needs to be addressed in the microbial fermentation production of chondroitin sulfate.

[0006] Given the biosafety risks posed by *E. coli*, the search for safer and more suitable alternative chassis cells for the food and health supplement industries is particularly urgent. Compared to *E. coli*, *Bacillus subtilis* does not secrete endotoxins or exotoxins, is recognized by the U.S. Food and Drug Administration (FDA) as a GRAS (Generally Recognized As Safe) strain, does not exhibit significant codon bias, and shows stronger tolerance to environmental stresses. It has been widely used in the production of food enzymes and chemicals. More importantly, *Bacillus subtilis* does not carry the enzyme genes for degrading chondroitin, making it an ideal candidate chassis for constructing chondroitin production strains. Studies have integrated the co-expression of key chondroitin synthesis genes kfoC and kfoA into the Bacillus subtilis 168 genome, and introduced the tuaD, gtaB, glmU, and glmS genes using the expression vector pP43NMK to enhance precursor supply. The engineered strain constructed using this method produced 6.06 g / L of chondroitin in a 3 L fed-batch fermentation process after 70 h (e.g., CN106497845B). However, while this result demonstrates the feasibility of the Bacillus subtilis platform and significantly improves yield, it still falls short of completely replacing traditional direct extraction methods and meeting the demands of large-scale industrial production. Therefore, how to further overcome the yield bottleneck in the Bacillus subtilis system has become a core challenge that urgently needs to be addressed for this technological approach. Summary of the Invention

[0007] In view of this, the present invention provides a high-chondroitin-producing Bacillus subtilis engineered strain, its construction method, and its applications. Through metabolic engineering strategies, the present invention designs and constructs a stable, plasmid-free, high-chondroitin-producing engineered strain, breaking through existing yield bottlenecks and achieving efficient, safe, and sustainable production of chondroitin sulfate, providing technical support for its widespread application in the food, health product, and pharmaceutical fields.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides an engineered strain using Bacillus subtilis as the substrate strain, expressing an enzyme comprising UDP-N-acetylglucosamine C4 isomerase, chondroitin synthase, and a combination of enzymes selected from one or more of the following:

[0010] (a) UDP-glucose dehydrogenase;

[0011] (b) UTP-glucose-1-phosphate uridine transferase;

[0012] (c) Glutamine-fructose-6-phosphate aminotransferase;

[0013] (d) UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase;

[0014] (e) Phosphoglucose mutase.

[0015] In some embodiments of the present invention, the chassis strain in the above-mentioned engineered strain is: Bacillus subtilis 168 (ATCC 23857).

[0016] In some embodiments of the present invention, the chassis strain in the above-described engineered strain expresses:

[0017] Enzyme combination 1 containing UDP-N-acetylglucosamine C4 isomerase, chondroitin synthase, and UDP-glucose dehydrogenase; or

[0018] Enzyme combination 2 comprising UDP-N-acetylglucosamine C4 isomerase, chondroitin synthase, UDP-glucose dehydrogenase, and UTP-glucose-1-phosphate uridine transferase; or

[0019] Enzyme combination 3 containing UDP-N-acetylglucosamine C4 isomerase, chondroitin synthase, UDP-glucose dehydrogenase, UTP-glucose-1-phosphate uridine transferase, and glutamine-fructose-6-phosphate aminotransferase; or

[0020] Enzyme combination 4 containing UDP-N-acetylglucosamine C4 isomerase, chondroitin synthase, UDP-glucose dehydrogenase, UTP-glucose-1-phosphate uridine transferase, glutamine-fructose-6-phosphate aminotransferase, and UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase; or

[0021] Enzyme combination 5 containing UDP-N-acetylglucosamine C4 isomerase, chondroitin synthase, UDP-glucose dehydrogenase, UTP-glucose-1-phosphate uridine transferase, glutamine-fructose-6-phosphate aminotransferase, UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase and phosphoglucose mutase.

[0022] In some embodiments of the present invention, the amino acid sequence of the UDP-N-acetylglucosamine C4 isomerase in the above-mentioned engineered strain is shown in SEQ ID NO:1;

[0023] The amino acid sequence of the chondroitin synthase is shown in SEQ ID NO:2;

[0024] The amino acid sequence of the UDP-glucose dehydrogenase is shown in SEQ ID NO:3;

[0025] The amino acid sequence of the UTP-glucose-1-phosphate uridine transferase is shown in SEQ ID NO:4;

[0026] The amino acid sequence of the glutamine-fructose-6-phosphate aminotransferase is shown in SEQ ID NO:5;

[0027] The amino acid sequence of the UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase is shown in SEQ ID NO:6;

[0028] The amino acid sequence of the glucose-phosphotransformase is shown in SEQ ID NO:7.

[0029] In some embodiments of the present invention, the sequence of SEQ ID NO:1 in the above-mentioned engineered strain is: MNILVTGGAGYIGSHTSLCLLNKGYNVVIIDNLINSSCESIRRIELIAKKKVTFYELNINNEKEVNQILKKHKFDCIMHFAGAKSVAESLIKPIFYYDNNVSGTLQLINCAIKNDVANFIFSSSATVYGESKIMPVTEDCHIGGTLNPYGTSKYISELMIRDIAKKYSDTNFLCLRYFNPTGAHESGMIGESPADIPSNLVPYILQVAMGKLEKLMVFGGDYPTKDGTGVRDYIHVMDLAEGHVAALSYLFRDNNTNYHVFNLGTGKGYSVLELVSTFEKISGVRIPYEIVSRRDGDIAESWSSPEKANKYLNWKAKRELETMLEDAWRWQMKNPNGYI.

[0030] In some embodiments of the present invention, in the above engineering strain, the sequence of SEQ ID NO: 2 is: MSLLAQAISKYNNKEYESALNLFEQAANIYGENLVRYHIIKCKHFLGLKNDLSDLNPSLKEYDLDLATQIMQLNQESSIDIATKNKLINQWQNITSKKSDSAAIKQVNSIPSDFPKDLILAPLPEGPNDFDWNKNVRKIRKSTHKVDNVGLSIVIPTFNRSHILEVTLSCLVNQESEYPFEVIVADDGSKEDICSVVRKYEKDLDIKYVRQKDYGYQLCAVRNLGLRTAKYDFVSILDCDMAPNPLWVQSYVKYLLEDDDVALIGPRKYVDTQNILPENFRSNKNLISNLPEVRTNNSVAGKEEGEISVDWRLEHFKRTDNLRLCDSPFRYFSGGNVAFSKKWLDKAGWFDEEFTHWGGEDNEFGYRLFKNGCFFRAVEGGMAYHQEPPGKENETDRAEGKKITINIVKEKVPYFYRKLQPIESALIHKVPLVSIYIPAYNCADTIKRCVDSALNQTITDLQVCICNDGSTDNTLDVINRLYGQNPRVKIISQENGGIASASNNAVKHASGFYIGQLDSDDYLEPDAVELCLAEFLKDRSLACVYTTNRNVNPDGSLIADGYNWPEFSREKLTTAMIVHHFRMFTARAWHLTSGFDENIENSVDLDIYLKLSEVGPFKHINKICYNRVLHGNNTSIKKLDMQKKNHFLVINRSLQRQGVDYYSYDAIDEDDASRRYVFKKNHEAQYQVSNLSSAAKLAFSLVYPDSLDRLLDKLHNILEYNPEASVIIAHVNPERLSQDVMQKVKTFEKENSIKVVINHELDYSNYKFRQVEYLVSNFNKLLRTGIEFDYIIFDNFDSLYVQHHSYDYIKKADIGMNFGKVNGYWQDKILSHQSLKQWTKQYLNKDIADIPIKGAAQGMFMSYEVAKPIFELLEQFLELCKANDDYPKYLAEEILFQLMTLILENRDGISLKRCNALTYMPWERKLQWTEQQIEEAKQGVGIAKNKFIIYQL。

[0031] In some embodiments of the present invention, among the above-described engineered strains, SEQ ID The sequence of NO:3 is: MKIAVAGVGYVGISIAILLSQKHDIIALDIDPKKVQLINKKISPICDPEIQKFLSNRKLNLYATTEKYEAYRDADYVIIATPTNYDPINNNFDTLSVESVACDVLSINPNATIIIKSTVPVGFTERLKRDLNTNNIIFSPEFLREGKALYDNLYPSRIVVGESSERARKFAELLSEGAIKKDIPILLTDS PEAEAIKLFANTYLAMRIAYFNELDTYASVHGLDTKQIIEGVSLDPRIGQHYNNPSFGYGGYCLPKDTKQSLANYRDVPQNLIQAIVDANTTRKDFVAED ILSRKPKVVGIYRLIMKAGSDNFRASSIQGVMKRLKAKGIEIVVYEPVLKEPYFFGSYVERDINSFKERVDVIVANRRTSELEDVSEKVYTRDLFGVDS.

[0032] In some embodiments of the present invention, the sequence of SEQ ID NO:4 in the above-mentioned engineered strain is: MAAINTKVKKAVIPVAGLGTRMLPATKAIPKEMLPLVDKPLIQYVVNECIAAGITEIVLVTHSSKNSIENHFDTSFELEAMLEKRVKRQLLDEVQSICPPHVTIMQVRQGLAKGLGHAVLCAHPVVGDEPVAVILPDVILDEYESDLSQDNLAEMIRRFDETGHSQIMVEPVADVTAYGVVDCKGVELAPGESVPMVGVVEKPKADVAPSNLAIVGRYVLSADIWPLLAKTPPGAGDEIQLTDAIDMLIEKETVEAYHMKGKSHDCGNKLGYMQAFVEYGIRHNTLGTEFKAWLEEEMGIKK.

[0033] In some embodiments of the present invention, in the above engineering strain, the sequence of SEQ ID NO:5 is: MCGIVGAIAQRDVAEILLEGLRRLEYRGYDSAGLAVVDAEGHMTRLRRLGKVQMLAQAAEEHPLHGGTGIAHTRWATHGEPSEVNAHPHVSEHIVVVHNGIIENHEPLREELKARGYTFVSETDTEVIAHLVNWELKQGGTLREAVLRAIPQLRGAYGTVIMDSRHPDTLLAARSGSPLVIGLGMGENFIASDQLALLPVTRRFIFLEEGDIAEITRRSVNIFDKTGAEVKRQDIESNLQYDAGDKGIYRHYMQKEIYEQPNAIKNTLTGRISHGQVDLSELGPNADELLSKVEHIQILACGTSYNSGMVSRYWFESLAGIPCDVEIASEFRYRKSAVRRNSLMITLSQSGETADTLAGLRLSKELGYLGSLAICNVPGSSLVRESDLALMTNAGTEIGVASTKAFTTQLTVLLMLVAKLSRLKGLDASIEHDIVHGLQALPSRIEQMLSQDKRIEALAEDFSDKHHALFLGRGDQYPIALEGALKLKEISYIHAEAYAAGELKHGPLALIDADMPVIVVAPNNELLEKLKSNIEEVRARGGQLYVFADQDAGFVSSDNMHIIEMPHVEEVIAPIFYTVPLQLLAYHVALIKGTDVDQPRNLAKSVTVE。

[0034] In some embodiments of the present invention, in the above-mentioned engineered strain, the sequence of SEQ ID NO:6 is: MSASDFSSAVVVLAAGAGTRMKSDLQKTLHSIGGRSLISHSLHAAAGLNPEIHVAVIGHGRDQVGPAVAQVAEELDREVLIAIQEEQNGTGHAVQCAMDQLEGFEGTIIVTNGDVPLLTDHTLSALLDAHVEVPTAVTLTMRLDDPTGYGRIVRNEEGEVTAIVEQKDASAEVQAIDEVNSGVFAFDAAILRSALAELKSDNAQGELYLTDVLGIARGEGHPVRAHTAADARELAGVNDRVQLAEAGAELNRRTVIAAMRGGATIVDPATTWIDVEVSIGRDVIIHPGTQLKGETVIGDRVEVGPDTTLTNMTIGDGASVIRTHGFDSTIGENATVGPFTYIRPGTTLGPEGKLGGFVETKKATIGRGSKVPHLTYVGDATIGEESNIGASSVFVNYDGENKHHTTIGSHVRTGSDTMFIAPVTVGDGAYSGAGTVIKDDVPPGALAVSGGRQRNIEGWVQKKRPGTAAQAAEAAQNVHNQEG.

[0035] In some embodiments of the present invention, the sequence of SEQ ID NO:7 in the above-mentioned engineered strain is: MTLLFGTDGVRGLANEVLTAPLALKLGAAAAHVLTAEKRVDGRRPVAIVGRDPRVSGEMLAAALSAGMASQGVDVIRVGVIPTPAVAFLTDDYGADMGVMISASHNPMPDNGIKFFSAGGHKLPDHVEDEIERVMDSLPAEGPTGHGVGRVIEEATDAQDRYLEHLKEAVPTSLEGIKIVVDAANGAASVVAPTAYEAAGATVIAIHNKPDSYNINMDC GSTHIDQVQAAVLKHGADLGLAHDGDADRCLAVDKDGNLVDGDQIMALLAIAMKENGELRKNTLVGTVMSNLGLKIAMDEAAGITLRTTKVGDRYVLEDLNAGGFSLGGEQSGHI VLPDHGTTGDGTLTGLSIMARMAETGKSLGELAQAMTVLPQVLINVPVSDKSTIVSHPSVVAAIAEAEELGATGRVLLRASGTEELFRVMVEAGDKEQARRIAGRLAAVVAEV.

[0036] In some embodiments of the present invention, the engineered strain described above expresses 1 to 4 (e.g., 1, 2, 3, or 4) copies of chondroitin synthase; preferably 1 to 3; more preferably 1, 2, or 3; even more preferably 2 or 3; and most preferably 3.

[0037] In some embodiments of the present invention, the preservation number of the above-mentioned engineered strain is: CGMCC No. 36960.

[0038] The present invention also provides an engineered strain, the preservation number of which is CGMCC No. 36960.

[0039] The present invention also provides a method for constructing the above-mentioned engineered strain, wherein the enzyme combination is expressed on a chassis strain to obtain the engineered strain.

[0040] The present invention also provides a nucleic acid molecule encoding the enzyme combination in the above-described engineered strain or the engineered strain obtained by the above-described preparation method.

[0041]

[0042]

[0043]

[0044] The sequence of the nucleic acid molecule encoding UTP-glucose-1-phosphate uridylyltransferase is shown in SEQ ID NO:11: ATGGCTGCCATTAATACGAAAGTCAAAAAAGCCGTTATCCCCGTTGCGGGATTAGGAACCAGGATGTTGCCGGCGACGAAAGCCATCCCGAAAGAGATGCTGCCACTTGTCGATAAGCCATTAATTCAATACGTCGTGAATGAATGTATTGCGGCTGGCATTACTGAAATTGTGCTGGTTACACACTCATCTAAAAACTCTATTGAAAACCACTTTGATACCAGTTTTGAACTGGAAGCAATGCTGGAAAAACGTGTAAAACGTCAACTGCTTGATGAAGTGCAGTCTATTTGTCCACCGCACGTGACTATTATGCAAGTTCGTCAGGGTCTGGCGAAAGGCCTGGGACACGCGGTATTGTGTGCTCACCCGGTAGTGGGTGATGAACCGGTAGCTGTTATTTTGCCTGATGTTATTCTGGATGAATATGAATCCGATTTGTCACAGGATAACCTGGCAGAGATGATCCGCCGCTTTGATGAAACGGGTCATAGCCAGATCATGGTTGAACCGGTTGCTGATGTGACCGCATATGGCGTTGTGGATTGCAAAGGCGTTGAATTAGCGCCGGGTGAAAGCGTACCGATGGTTGGTGTGGTAGAAAAACCGAAAGCGGATGTTGCGCCGTCTAATCTCGCTATTGTGGGTCGTTACGTACTTAGCGCGGATATTTGGCCGTTGCTGGCAAAAACCCCTCCGGGAGCTGGTGATGAAATTCAGCTCACCGACGCAATTGATATGCTGATCGAAAAAGAAACGGTGGAAGCCTATCATATGAAAGGGAAGAGCCATGACTGCGGTAATAAATTAGGTTACATGCAGGCCTTCGTTGAATACGGTATTCGTCATAACACCCTTGGCACGGAATTTAAAGCCTGGCTTGAAGAAGAGATGGGCATTAAGAAGTAA;

[0045]

[0046]

[0047]

[0048] This invention also provides the application of the above-mentioned engineered strains, the engineered strains obtained by the above-mentioned construction method, and / or the above-mentioned nucleic acid molecules in the preparation of chondroitin.

[0049] The present invention also provides a method for preparing chondroitin, comprising inoculating the above-mentioned engineered strain or the engineered strain obtained by the above-mentioned construction method, fermenting and culturing, collecting the fermentation broth, and obtaining the chondroitin.

[0050] In some embodiments of the present invention, in the above preparation method, the inoculation is carried out using a seed culture medium; the seed culture medium comprises: 40 g / L sucrose, 20 g / L yeast extract, 1.5 g / L anhydrous magnesium sulfate and 6.8 g / L potassium dihydrogen phosphate; the sucrose and the anhydrous magnesium sulfate can be prepared and used immediately.

[0051] In some embodiments of the present invention, the fermentation in the above preparation method uses a fermentation culture medium; the fermentation culture medium includes: 40 g / L sucrose, 20 g / L yeast extract, 1.5 g / L anhydrous magnesium sulfate, 6.8 g / L potassium dihydrogen phosphate, and trace elements.

[0052] In some embodiments of the present invention, in the above preparation method, the content of each component in the trace elements (mother liquor) is as follows: ferrous sulfate heptahydrate 10 g / L, manganese sulfate tetrahydrate 0.5 g / L, calcium chloride 2 g / L, zinc sulfate heptahydrate 2.2 g / L, copper sulfate pentahydrate 1 g / L, ammonium molybdate tetrahydrate 0.1 g / L and sodium tetraborate decahydrate 0.02 g / L.

[0053] In some embodiments of the present invention, the dissolved oxygen content during fermentation in the above method is 15-30%;

[0054] When the dissolved oxygen level is below 20%, the dissolved oxygen level is increased by sequentially increasing the rotation speed and increasing the ventilation rate.

[0055] When the dissolved oxygen level is greater than 30%, the dissolved oxygen level is reduced by successively decreasing the ventilation rate and the rotation speed.

[0056] In some embodiments of the present invention, in the above method, when the dissolved oxygen level is below 20%, the rotation speed is increased by 100 rpm each time until the rotation speed is 1000 rpm; if the dissolved oxygen level is still below 20%, the ventilation rate is increased by 0.5 vvm each time until the ventilation rate is 2.5 vvm.

[0057] In some embodiments of the present invention, in the above method, when the dissolved oxygen content is below 30%, the dissolved oxygen content is increased to 30% by sequentially increasing the rotation speed, increasing the ventilation rate, and increasing the tank pressure, wherein:

[0058] The increase in rotational speed includes increasing the speed by 150 rpm each time, up to a speed of 900 rpm.

[0059] The increase in ventilation volume includes increasing it by 0.25 vvm each time until the ventilation volume reaches 2 vvm;

[0060] The increase in tank pressure includes: from 0.01 MPa to a tank pressure of 0.06 MPa.

[0061] In some embodiments of the present invention, the fermentation process further includes a fed-batch step; the fed-batch includes ammonia, sucrose, xylose, an antifoaming agent, and tryptone.

[0062] In some embodiments of the present invention, in the above method, the sucrose is added when the concentration of sucrose drops to 1 g / L during fermentation, and the residual sucrose concentration in the fermentation environment is controlled to be 0~2 g / L.

[0063] In some embodiments of the present invention, the xylose is added at least once, with a final concentration of 18 g / L.

[0064] In some embodiments of the present invention, the fermentation temperature is 37°C, the pH value is 7.0±0.05, and the time is 48~60h.

[0065] In some embodiments of the present invention, the feed in the above method comprises 25 wt% ammonia, 750 g / L sucrose, xylose with a final concentration of 18 g / L, 0.1 g / L defoamer, and 20% (w / v) tryptone.

[0066] The present invention also provides a product comprising: the above-described engineered strain, the engineered strain obtained by the above-described construction method, and / or the above-described nucleic acid molecule; said product is used to prepare chondroitin.

[0067] Biological deposit description: Biological material: DH003, classified and named: Bacillus subtilis, was deposited on December 09, 2025 at the China General Microbiological Culture Collection Center, Beijing, China, with accession number CGMCC No. 36960.

[0068] The beneficial effects of this invention include:

[0069] (1) Significantly high yield performance, breaking through the yield bottleneck of Bacillus subtilis platform.

[0070] This invention successfully constructed a high-chondroitin-producing Bacillus subtilis engineered strain by systematically designing and combining key enzymes (including KfoA, McCS, KfoF, GalU, GlmS, GlmU, GlmM, etc.) in the chondroitin synthesis pathway, and optimizing the McCS copy number (1 to 4). For example, strain DH003 achieved a chondroitin yield of 8.01 g / L in a 5 L fermenter; even when scaled up to a 50 L fermenter, it maintained a high yield of 7.5 g / L. Furthermore, using the same fermentation medium, fermentation conditions, and detection methods as the existing Bacillus subtilis platform (CN106497845B), the engineered strain DH003 constructed in this invention achieved a chondroitin yield of 9.86 g / L, significantly higher than the strain (which accumulated approximately 6.06 g / L of chondroitin), significantly breaking through the yield bottleneck of this system and laying a solid foundation for large-scale industrial production.

[0071] (2) It has good genetic stability and is suitable for long-term continuous fermentation.

[0072] This invention employs a genome integration expression strategy, constructing an engineered strain that does not rely on exogenous plasmids, thus avoiding the risks of plasmid loss or unstable expression. After eight consecutive passages, the chondroitin yield remained above 8 g / L in a 5 L fermenter, indicating that the strain possesses excellent genetic stability and production performance, fully meeting the process requirements for industrial continuous fermentation.

[0073] (3) The process has strong scalability and is promising for industrial application.

[0074] The fermentation process provided by this invention exhibits good yield consistency and process reproducibility at scales ranging from 5 L to 50 L, with no yield decline observed during scale-up, indicating that the metabolic engineering strategy and process parameters have high scalability. Furthermore, the fermentation cycle is reasonably controllable, the final product has a uniform structure and stable molecular weight distribution (weight-average molecular weight Mw approximately 53.2 kDa (62.82)%), and its quality meets relevant standards, demonstrating feasibility and economic viability for large-scale industrial production.

[0075] (4) High safety, suitable for food and health products.

[0076] This invention uses Bacillus subtilis as the substrate strain (such as Bacillus subtilis 168 (ATCC 23857)), which is recognized by the US FDA as a GRAS (Generally Recognized As Safe) strain, does not secrete endotoxins or exotoxins, and does not carry the chondroitin-degrading enzyme gene. Compared to potentially pathogenic bacteria such as Escherichia coli, the production strain of this invention fundamentally avoids biosafety risks and better meets the high standards of raw material safety requirements in the food, health product, and pharmaceutical industries.

[0077] (5) It is environmentally friendly and in line with the concept of green and sustainable development.

[0078] This invention utilizes microbial fermentation to produce chondroitin, avoiding the risks of viruses and immunogenic impurities associated with traditional animal extraction methods, as well as the environmental pollution problems caused by chemical synthesis. The fermentation process primarily consumes renewable carbon sources, resulting in a clean process with minimal waste, aligning with the development direction of green biomanufacturing.

[0079] In summary, this invention not only significantly improves the yield of chondroitin from the Bacillus subtilis platform, but also demonstrates significant advantages in terms of strain safety, genetic stability, process scalability, and product quality. It provides a reliable technical solution for the efficient, safe, and sustainable production of chondroitin and its sulfated derivatives, and has important industrial application value. Attached Figure Description

[0080] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0081] Figure 1 This diagram shows the chondroitin synthesis pathway.

[0082] Figure 2 Fermentation curve of engineered strain 3 in a 5 L fermenter is shown; where: A: BSG02; B: BSG031; C: BSG041; D: BSG051; E: BSG062; F: BSG072;

[0083] Figure 3 The fermentation curve of the engineered strain in Example 3 is shown in a 5 L fermenter; where: A: BSM012; B: DH003; C: BSM032;

[0084] Figure 4 Fermentation curve of engineered strain DH003 in a 50 L fermenter, as shown in Example 4;

[0085] Figure 5 HPLC chromatogram of chondroitin disaccharide after enzymatic hydrolysis of chondroitin sulfate sodium standard is shown.

[0086] Figure 6 The HPLC analysis chromatogram of chondroitin disaccharide after enzymatic hydrolysis of the fermentation product in Example 4 is shown.

[0087] Figure 7 This shows the mass spectrometry analysis of chondroitin disaccharide after enzymatic hydrolysis of the fermentation product in Example 4;

[0088] Figure 8 The molecular weight analysis spectrum of the fermentation products in Example 4 is shown.

[0089] Figure 9The fermentation curve of the engineered strain DH003 in a 3 L fermenter is shown in the control example 1. Detailed Implementation

[0090] This invention discloses an engineered Bacillus subtilis strain that produces high levels of chondroitin, its construction method, and its applications.

[0091] This invention provides a high-chondroitin-producing Bacillus subtilis engineered strain, its construction method, and its application. The aim is to design and construct a stable, plasmid-free, high-chondroitin-producing strain through metabolic engineering strategies, breaking through existing production bottlenecks and achieving efficient, safe, and sustainable production of chondroitin sulfate, thus providing technical support for its widespread application in the food, health product, and pharmaceutical fields.

[0092] Specifically, this invention provides an engineered Bacillus subtilis strain that uses Bacillus subtilis as a substrate strain and is capable of further expressing enzymes including UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), and enzymes selected from any one or more combinations of the following:

[0093] (1) UDP-glucose dehydrogenase (KfoF);

[0094] (2) UTP-glucose-1-phosphate uridine transferase (GalU);

[0095] (3) Glutamine-fructose-6-phosphate aminotransferase (GlmS);

[0096] (4) UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU).

[0097] (5) Glucose phosphate mutase (GlmM).

[0098] Furthermore, the engineered Bacillus subtilis strain uses Bacillus subtilis as the chassis strain and is capable of expressing enzymes comprising the following combination:

[0099]

[0100] It is understood that the expression of any of the above enzymes in the chassis strain is relatively independent, and the synergistic effect of each combination can positively promote the yield increase of the engineered Bacillus subtilis strain. Therefore, unless there is evidence in the prior art that when the enzymes in the above combinations coexist with other enzymes (whether known or unknown), the yield will significantly decrease due to specific interactions (such as competitive inhibition, metabolic pathway interference, or regulatory conflicts), the technical solution provided in this patent should be considered open-ended, i.e., "including but not limited to". In other words, unless there is clear and reproducible experimental data proving that the enzyme combination described in this patent, when used in conjunction with any other enzyme, will lead to a significant decrease in yield, the scope of protection of this patent covers free combinations including any of the above enzyme combinations with any other known or unknown enzymes.

[0101] In some preferred embodiments, the Bacillus subtilis engineered strain is a Bacillus subtilis chassis strain that further expresses an enzyme comprising a combination of UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), and UDP-glucose dehydrogenase (KfoF) (i.e., combination 1).

[0102] In some preferred embodiments, the Bacillus subtilis engineered strain further expresses UTP-glucose-1-phosphate uridine transferase (GalU), that is, the Bacillus subtilis engineered strain uses Bacillus subtilis as the chassis strain and further expresses an enzyme comprising a combination of UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), and UTP-glucose-1-phosphate uridine transferase (GalU) (i.e., combination 6).

[0103] In some other preferred embodiments, the Bacillus subtilis engineered strain further expresses glutamine-fructose-6-phosphate aminotransferase (GlmS), that is, the Bacillus subtilis engineered strain is a Bacillus subtilis chassis strain and further expresses an enzyme comprising a combination of UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), UTP-glucose-1-phosphate uridine transferase (GalU), and glutamine-fructose-6-phosphate aminotransferase (GlmS) (i.e., combination 16).

[0104] In some other preferred embodiments, the Bacillus subtilis engineered strain further expresses UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU), that is, the Bacillus subtilis engineered strain is a Bacillus subtilis chassis strain and further expresses an enzyme comprising a combination of UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), UTP-glucose-1-phosphate uridine transferase (GalU), glutamine-fructose-6-phosphate aminotransferase (GlmS), and UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU) (i.e., combination 26).

[0105] In some other preferred embodiments, the Bacillus subtilis engineered strain further expresses glucose-phosphotransferase (GlmM), that is, the Bacillus subtilis engineered strain is a Bacillus subtilis chassis strain and further expresses an enzyme comprising a combination of UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), UTP-glucose-1-phosphate uridine transferase (GalU), glutamine-fructose-6-phosphate aminotransferase (GlmS), UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU), and glucose-phosphotransferase (GlmM) (i.e., combination 31).

[0106] Furthermore, any of the above-mentioned Bacillus subtilis engineered strains can express 1 copy of McCS.

[0107] Furthermore, any of the above-mentioned Bacillus subtilis engineered strains can express 2 copies of McCS.

[0108] Furthermore, any of the above-mentioned Bacillus subtilis engineered strains can express 3 copies of McCS.

[0109] Furthermore, any of the above-mentioned Bacillus subtilis engineered strains can express 4 copies of McCS.

[0110] In some specific embodiments, the Bacillus subtilis engineered strain is based on Bacillus subtilis and further expresses an enzyme comprising a combination of UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), UTP-glucose-1-phosphate uridine transferase (GalU), glutamine-fructose-6-phosphate aminotransferase (GlmS), UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU), and phosphoglucose mutase (GlmM), wherein McCS is in 2 copies.

[0111] In other specific embodiments, the engineered Bacillus subtilis strain is based on Bacillus subtilis and further expresses an enzyme comprising a combination of UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), UTP-glucose-1-phosphate uridine transferase (GalU), glutamine-fructose-6-phosphate aminotransferase (GlmS), UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU), and phosphoglucose mutase (GlmM), wherein McCS is in 3 copies.

[0112] In other specific embodiments, the Bacillus subtilis engineered strain is based on Bacillus subtilis and further expresses an enzyme comprising a combination of UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), UTP-glucose-1-phosphate uridine transferase (GalU), glutamine-fructose-6-phosphate aminotransferase (GlmS), UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU), and phosphoglucose mutase (GlmM), wherein McCS is in 4 copies.

[0113] Furthermore, the engineered Bacillus subtilis strain is Bacillus subtilis DH003, which was deposited on December 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36960, located in Beijing, China, and classified as Bacillus subtilis.

[0114] This invention also provides a method for constructing a high-chondroitin-producing Bacillus subtilis engineered strain. The method uses Bacillus subtilis as the substrate strain and further expresses enzymes comprising UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), and any one or more of the following combinations:

[0115] (1) UDP-glucose dehydrogenase (KfoF);

[0116] (2) UTP-glucose-1-phosphate uridine transferase (GalU);

[0117] (3) Glutamine-fructose-6-phosphate aminotransferase (GlmS);

[0118] (4) UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU).

[0119] (5) Glucose phosphate mutase (GlmM).

[0120] Furthermore, the construction method uses Bacillus subtilis as the chassis strain and further expresses enzymes containing any of the aforementioned combinations (i.e., combinations 1-31).

[0121] In some preferred embodiments, the construction method uses Bacillus subtilis as the chassis strain and further expresses an enzyme (i.e., combination 1) containing a combination of UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), and UDP-glucose dehydrogenase (KfoF) in the chassis strain.

[0122] In some other preferred embodiments, the construction method uses Bacillus subtilis as the chassis strain and further expresses an enzyme (i.e., combination 6) comprising a combination of UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), and UTP-glucose-1-phosphate uridine transferase (GalU).

[0123] In some other preferred embodiments, the construction method uses Bacillus subtilis as the chassis strain and further expresses an enzyme comprising a combination of UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), UTP-glucose-1-phosphate uridine transferase (GalU), and glutamine-fructose-6-phosphate aminotransferase (GlmS) (i.e., combination 16).

[0124] In some other preferred embodiments, the construction method uses Bacillus subtilis as the chassis strain and further expresses an enzyme comprising a combination of UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), UTP-glucose-1-phosphate uridine transferase (GalU), glutamine-fructose-6-phosphate aminotransferase (GlmS), and UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU) (i.e., combination 26).

[0125] In some other preferred embodiments, the construction method uses Bacillus subtilis as the chassis strain and further expresses an enzyme comprising a combination of UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), UTP-glucose-1-phosphate uridine transferase (GalU), glutamine-fructose-6-phosphate aminotransferase (GlmS), UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU), and phosphoglucose mutase (GlmM) (i.e., combination 31).

[0126] Furthermore, the construction method described in any of the above embodiments expresses one copy of McCS in the chassis strain.

[0127] Furthermore, the construction method described in any of the above embodiments expresses two copies of McCS in the chassis strain.

[0128] Furthermore, the construction method described in any of the above-mentioned embodiments expresses 3 copies of McCS in the chassis strain.

[0129] Furthermore, the construction method described in any of the above-mentioned embodiments expresses 4 copies of McCS in the chassis strain.

[0130] In some specific embodiments, the construction method uses Bacillus subtilis as the chassis strain and further expresses an enzyme comprising a combination of UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), UTP-glucose-1-phosphate uridine transferase (GalU), glutamine-fructose-6-phosphate aminotransferase (GlmS), UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU), and phosphoglucose mutase (GlmM), wherein McCS is in 2 copies.

[0131] In other specific embodiments, the construction method uses Bacillus subtilis as the chassis strain and further expresses an enzyme comprising a combination of UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), UTP-glucose-1-phosphate uridine transferase (GalU), glutamine-fructose-6-phosphate aminotransferase (GlmS), UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU), and phosphoglucose mutase (GlmM), wherein McCS is in 3 copies.

[0132] In other specific embodiments, the construction method uses Bacillus subtilis as the chassis strain and further expresses an enzyme comprising a combination of UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), UTP-glucose-1-phosphate uridine transferase (GalU), glutamine-fructose-6-phosphate aminotransferase (GlmS), UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU), and phosphoglucose mutase (GlmM), wherein McCS is 4 copies.

[0133] Furthermore, the expression of UDP-N-acetylglucosamine C4 isomerase (KfoA) described in any of the above-mentioned methods involves in situ substitution of the ybcM gene with a gene fragment containing a nucleotide sequence capable of encoding UDP-N-acetylglucosamine C4 isomerase (KfoA).

[0134] Furthermore, the expression of chondroitin synthase (McCS) described in any of the above embodiments can be achieved by in situ substitution of nucleotides 1915 to 1980 of the amyE gene with a gene fragment containing a nucleotide sequence encoding chondroitin synthase (McCS) to obtain a McCS copy (defined as McCS copy A); or by in situ substitution of the promoter and the first 702 nucleotides of the nprE gene with a gene fragment containing a nucleotide sequence encoding chondroitin synthase (McCS) to obtain a McCS copy (defined as McCS copy B); or by inserting a gene fragment containing a nucleotide sequence encoding chondroitin synthase (McCS) between the terminators of the walL and desR genes to obtain a McCS copy (defined as McCS copy C).

[0135] Furthermore, the expression of UDP-glucose dehydrogenase (KfoF) described in any of the above-mentioned methods involves in situ substitution of the epr gene with a gene fragment containing a nucleotide sequence capable of encoding UDP-glucose dehydrogenase (KfoF).

[0136] Furthermore, the expression mode of UTP-glucose-1-phosphate uridine transferase (GalU) described in any of the above-mentioned embodiments is to replace the 37th to 270th nucleotides of the amyE gene in situ with a gene fragment containing a nucleotide sequence that can encode UTP-glucose-1-phosphate uridine transferase (GalU).

[0137] Furthermore, the expression mode of glutamine-fructose-6-phosphate aminotransferase (GlmS) described in any of the above-mentioned embodiments is to replace the 1212th to 1551st nucleotide sequences of the amyE gene with a gene fragment containing a nucleotide sequence that can encode glutamine-fructose-6-phosphate aminotransferase (GlmS) in situ.

[0138] Furthermore, the expression mode of UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU) described in any of the above-mentioned embodiments is to replace the promoter and the first 461 nucleotides of the rapE gene in situ with a gene fragment containing a nucleotide sequence capable of encoding UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU).

[0139] Furthermore, the expression of the phosphoglucopolysaccharide mutase (GlmM) described in any of the above embodiments involves in situ substitution of nucleotides 528 to 576 of the rapE gene with a gene fragment containing a nucleotide sequence encoding phosphoglucopolysaccharide mutase (GlmM).

[0140] Furthermore, the expression mode of UDP-N-acetylglucosamine C4 isomerase (KfoA) described in any of the above-mentioned embodiments is to replace the ybcM gene in situ with a gene fragment consisting of a P43 promoter and a nucleotide sequence capable of encoding UDP-N-acetylglucosamine C4 isomerase (KfoA).

[0141] Furthermore, the expression of chondroitin synthase (McCS) described in any of the above-mentioned methods can be as follows: a gene fragment consisting of a PxylR promoter, xylR gene, PxylA promoter, and a nucleotide sequence encoding chondroitin synthase (McCS) is used to replace nucleotides 1915 to 1980 of the amyE gene in situ to obtain a McCS copy (defined as McCS copy A); or a gene fragment consisting of a PxylR promoter, xylR gene, PxylA promoter, and a nucleotide sequence encoding chondroitin synthase (McCS) is used to replace the promoter and the first 702 nucleotides of the nprE gene in situ to obtain a McCS copy (defined as McCS copy B); or a gene fragment consisting of a PxylR promoter, xylR gene, PxylA promoter, and a nucleotide sequence encoding chondroitin synthase (McCS) is inserted between the walL and desR gene terminators to obtain a McCS copy (defined as McCS copy C).

[0142] Furthermore, the expression mode of UDP-glucose dehydrogenase (KfoF) described in any of the above-mentioned embodiments is to replace the epr gene in situ with a gene fragment consisting of a P43 promoter and a nucleotide sequence capable of encoding UDP-glucose dehydrogenase (KfoF).

[0143] Furthermore, the expression mode of UTP-glucose-1-phosphate uridine transferase (GalU) described in any of the above-mentioned embodiments is to replace the 37th to 270th nucleotides of the amyE gene in situ using a gene fragment consisting of a P43 promoter, a nucleotide sequence encoding UTP-glucose-1-phosphate uridine transferase (GalU) and a dps terminator.

[0144] Furthermore, the expression mode of glutamine-fructose-6-phosphate aminotransferase (GlmS) described in any of the above-mentioned embodiments involves in situ substitution of the 1212th to 1551st nucleotide sequences of the amyE gene using a gene fragment consisting of a P43 promoter, a nucleotide sequence encoding glutamine-fructose-6-phosphate aminotransferase (GlmS), and a clpP terminator.

[0145] Furthermore, the expression mode of UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU) described in any of the above-mentioned embodiments is to replace the promoter and the first 461 nucleotides of the rapE gene in situ with a gene fragment consisting of a P43 promoter, a nucleotide sequence encoding UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU) and a clpP terminator.

[0146] Furthermore, the expression of glucose-phosphodiesterase (GlmM) involves in situ substitution of nucleotides 528 to 576 of the rapE gene using a gene fragment consisting of a P43 promoter, a nucleotide sequence encoding glucose-phosphodiesterase (GlmM), and a dps terminator.

[0147] When expressing one McCS copy based on the substrate strain, any one of McCS copy A, McCS copy B, or McCS copy C can be selected for expression. When expressing two McCS copies based on the substrate strain, any two (repeated selections allowed) of McCS copy A, McCS copy B, or McCS copy C can be selected for expression. When expressing three McCS copies based on the substrate strain, any three (repeated selections allowed) of McCS copy A, McCS copy B, or McCS copy C can be selected for expression. When expressing four McCS copies based on the substrate strain, any four (repeated selections allowed) of McCS copy A, McCS copy B, or McCS copy C can be selected for expression.

[0148] Furthermore, the UDP-N-acetylglucosamine C4 isomerase (KfoA) involved in any of the above-mentioned items comprises an amino acid fragment with the sequence shown in SEQ ID NO.1.

[0149] Furthermore, the chondroitin synthase (McCS) involved in any of the above-mentioned items comprises an amino acid fragment with the sequence shown in SEQ ID NO.2.

[0150] Furthermore, the UDP-glucose dehydrogenase (KfoF) involved in any of the above-mentioned methods comprises an amino acid fragment with the sequence shown in SEQ ID NO.3.

[0151] Furthermore, the UTP-glucose-1-phosphate uridine transferase (GalU) involved in any of the above-mentioned items comprises an amino acid fragment with the sequence shown in SEQ ID NO.4.

[0152] Furthermore, any of the above-mentioned glutamine-fructose-6-phosphate aminotransferases (GlmS) comprises an amino acid fragment with the sequence shown in SEQ ID NO.5.

[0153] Furthermore, the UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU) involved in any of the above-mentioned items comprises an amino acid fragment with the sequence shown in SEQ ID NO.6.

[0154] Furthermore, any of the above-mentioned glucose-phosphotransmutases (GlmM) contains the amino acid fragment shown in SEQ ID NO.7.

[0155] Furthermore, the nucleotide sequence of any of the above that encodes UDP-N-acetylglucosamine C4 isomerase (KfoA) is shown in SEQ ID NO.8.

[0156] Furthermore, the nucleotide sequence of any of the above that encodes chondroitin synthase (McCS) is shown in SEQ ID NO.9.

[0157] Furthermore, the nucleotide sequence of any of the above that encodes UDP-glucose dehydrogenase (KfoF) is shown in SEQ ID NO.10.

[0158] Furthermore, the nucleotide sequence of any of the above that encodes UTP-glucose-1-phosphate uridine transferase (GalU) is shown in SEQ ID NO.11.

[0159] Furthermore, the nucleotide sequence of any of the above that encodes glutamine-fructose-6-phosphate aminotransferase (GlmS) is shown in SEQ ID NO.12.

[0160] Furthermore, the nucleotide sequence of any of the above that encodes UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU) is shown in SEQ ID NO.13.

[0161] Furthermore, the nucleotide sequence of any of the above that encodes glucose-phosphodiesterase (GlmM) is shown in SEQ ID NO.14.

[0162] The present invention also provides an engineered Bacillus subtilis strain, which uses Bacillus subtilis as the chassis strain and can further express UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), and UDP-glucose dehydrogenase (KfoF), wherein McCS is 1 copy.

[0163] The present invention also provides another engineered Bacillus subtilis strain, which uses Bacillus subtilis as a chassis strain and can further express UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF) and UTP-glucose-1-phosphate uridine transferase (GalU), wherein McCS is 1 copy.

[0164] The present invention also provides another engineered Bacillus subtilis strain, which uses Bacillus subtilis as a chassis strain and is able to further express UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), UTP-glucose-1-phosphate uridine transferase (GalU), and glutamine-fructose-6-phosphate aminotransferase (GlmS), wherein McCS is 1 copy.

[0165] The present invention also provides another engineered Bacillus subtilis strain, which uses Bacillus subtilis as a chassis strain and is further able to express UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), UTP-glucose-1-phosphate uridine transferase (GalU), glutamine-fructose-6-phosphate aminotransferase (GlmS), and UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU), wherein McCS is 1 copy.

[0166] The present invention also provides another engineered Bacillus subtilis strain, which uses Bacillus subtilis as a chassis strain and is able to further express UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), UTP-glucose-1-phosphate uridine transferase (GalU), glutamine-fructose-6-phosphate aminotransferase (GlmS), UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU), and phosphoglucosuricase (GlmM), wherein McCS is 1 copy.

[0167] The present invention also provides another engineered Bacillus subtilis strain, which uses Bacillus subtilis as a chassis strain and is further able to express UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), UTP-glucose-1-phosphate uridine transferase (GalU), glutamine-fructose-6-phosphate aminotransferase (GlmS), UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU), and phosphoglucosuricase (GlmM), wherein McCS is in 2 copies.

[0168] The present invention also provides another engineered Bacillus subtilis strain, which uses Bacillus subtilis as a chassis strain and is further able to express UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), UTP-glucose-1-phosphate uridine transferase (GalU), glutamine-fructose-6-phosphate aminotransferase (GlmS), UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU), and phosphoglucopolysaccharide mutase (GlmM), wherein McCS is 3 copies.

[0169] The present invention also provides another engineered Bacillus subtilis strain, which uses Bacillus subtilis as a chassis strain and is further able to express UDP-N-acetylglucosamine C4 isomerase (KfoA), chondroitin synthase (McCS), UDP-glucose dehydrogenase (KfoF), UTP-glucose-1-phosphate uridine transferase (GalU), glutamine-fructose-6-phosphate aminotransferase (GlmS), UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU), and phosphoglucosuricase (GlmM), wherein McCS is 4 copies.

[0170] Furthermore, the Bacillus subtilis mentioned in any of the above items is Bacillus subtilis 168 (ATCC 23857).

[0171] The present invention also provides an engineered strain of Bacillus subtilis, DH003, which was deposited on December 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36960, located in Beijing, China, and classified as Bacillus subtilis.

[0172] The present invention also provides a composition comprising the engineered Bacillus subtilis strain described in any one of the preceding claims or the engineered Bacillus subtilis strain DH003 described in any one of the preceding claims.

[0173] Furthermore, the composition includes, but is not limited to, a direct-inoculation fermentation agent containing any of the above-mentioned engineered Bacillus subtilis strains or any of the above-mentioned engineered Bacillus subtilis strains DH003.

[0174] The present invention also provides the use of the engineered Bacillus subtilis strain described in any one of the preceding claims, or the engineered Bacillus subtilis strain DH003 described in any one of the preceding claims, or the composition described in any one of the preceding claims, in the production of chondroitin and chondroitin sulfate.

[0175] The present invention also provides a method for producing chondroitin, wherein the method comprises inoculating the engineered Bacillus subtilis strain described in any one of the above claims, or the engineered Bacillus subtilis strain DH003 described in any one of the above claims, or the combination described above, into a culture medium for fermentation to obtain the chondroitin.

[0176] The UDP-N-acetylglucosamine C4 isomerase (KfoA) described in this invention refers to an enzyme (EC 5.1.3.2) that can isomerize uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) at the 4 position to form uridine diphosphate N-acetylglucosamine (UDP-GalNAc).

[0177] The chondroitin synthase (McCS) described in this invention refers to a bifunctional glycosyltransferase (EC 2.4.1.175 / EC2.4.1.226) that can alternately transfer glucuronic acid and N-acetylgalactosamine from the UDP-activated monosaccharide precursor uridine diphosphate glucuronic acid (UDP-GlcA) and UDP-GalNAc to the non-reducing ends of the chondroitin glycan chain to polymerize and elongate the chondroitin glycan chain.

[0178] The UDP-glucose dehydrogenase (KfoF) described in this invention refers to an enzyme (EC 1.1.1.22) that can dehydrogenate UDP-glucose (UDP-Glc) at position 6 to form UDP-GlcA.

[0179] The UTP-glucose-1-phosphate uridine acyltransferase (GalU) described in this invention refers to an enzyme that can transfer uridine diphosphate (UDP) on uridine triphosphate (UTP) to glucose-1-phosphate to form UDP-Glc. The reaction produces pyrophosphate at the same time, so this enzyme is also called UDP-glucose pyrophosphoryltransferase (EC 2.7.7.9).

[0180] The glutamine-fructose-6-phosphate aminotransferase (GlmS) described in this invention refers to an aminotransferase (EC 2.6.1.16) that can transfer ammonia from glutamine to D-fructose-6-phosphate to form D-glucosamine-6-phosphate.

[0181] The UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU) described in this invention refers to a bifunctional enzyme (EC 2.3.1.157 / EC 2.7.7.23) that can transfer the acetyl group on acetyl-CoA to D-glucosamine 1-phosphate to form N-acetylglucosamine 1-phosphate, and further transfer the UDP on UTP to N-acetylglucosamine 1-phosphate to form UDP-GlcNAc.

[0182] The phosphoglucosuric enzyme (GlmM) described in this invention refers to an enzyme that can isomerize D-glucosamine 6-phosphate to D-glucosamine 1-phosphate through the classic ping-pong reaction mechanism. This enzyme can also catalyze the formation of glucose 6-phosphate to glucose 1-phosphate at a lower rate (EC 5.4.2.10).

[0183] It is understood that, based on the amino acid sequences of the various enzymes described above, those skilled in the art can easily obtain the nucleotide sequences of the enzymes. Therefore, the present invention does not impose any particular limitation on the nucleotide sequences that can encode various enzymes.

[0184] The P43 promoter described in this invention refers to the natural constitutive strong promoter of Bacillus subtilis, which continuously initiates gene transcription and expression during the logarithmic and stationary phases.

[0185] The PxylR promoter mentioned in this invention refers to the promoter of the Bacillus subtilis endogenous gene xylR (encoding a transcriptional repressor of xylose-utilizing enzymes).

[0186] The PxylA promoter described in this invention refers to a xylose-inducible promoter, which is insensitive to most metabolites and is inhibited by glucose.

[0187] The dps terminator mentioned in this invention refers to the special DNA sequence at the end of the transcription unit of the dps gene, which is responsible for terminating the RNA polymerase transcription process.

[0188] The clpP terminator mentioned in this invention refers to the special DNA sequence at the end of the transcription unit of the clpP gene, which is responsible for terminating the RNA polymerase transcription process.

[0189] The ybcM gene mentioned in this invention refers to an endogenous gene of Bacillus subtilis that encodes a nonfunctional putative protein. It has been identified as a neutral gene that can be edited without affecting the growth and production performance of the strain.

[0190] The xylR gene mentioned in this invention refers to an endogenous gene of Bacillus subtilis that encodes a transcriptional repressor of xylose-utilizing enzymes.

[0191] The amyE gene mentioned in this invention refers to an endogenous gene of Bacillus subtilis that encodes α-amylase, namely 1,4-α-D-glucanase, which randomly hydrolyzes α-1,4 glycosidic bonds, shortening long-chain molecules and producing a large number of small-molecule glucans with non-reducing ends.

[0192] The epr gene mentioned in this invention refers to an endogenous gene of Bacillus subtilis that encodes a serine protease, plays a role in swarming movement, and has been identified as a neutral gene that can be edited without affecting the growth and production performance of the strain.

[0193] The rapE gene described in this invention refers to an endogenous gene of Bacillus subtilis that encodes aspartate phosphatase E. As a component of a two-component signal transduction system, it regulates the transcriptional expression of the downstream gene Spo0F, thereby inhibiting its activity. It plays a role in the initiation stage of spore formation and has been identified as a neutral gene that can be edited without affecting the growth and production performance of the strain.

[0194] The nprE gene mentioned in this invention refers to an endogenous gene of Bacillus subtilis that encodes bacillus lysin, a metalloproteinase. It has been identified as a neutral gene, and its knockout does not affect the growth and production performance of the strain.

[0195] The wall gene mentioned in this invention refers to an endogenous gene of Bacillus subtilis that encodes a serine protease that binds to the cell wall. It has been identified as a neutral gene that can be edited without affecting the growth and production performance of the strain.

[0196] The desR gene mentioned in this invention refers to an endogenous gene of Bacillus subtilis that encodes a transcriptional regulatory protein that participates in the cold shock induction of Δ5-acetyl ester desaturase.

[0197] The method for detecting chondroitin yield involved in this invention can be found in the analytical methods on page 1595 of Part II of the Chinese Pharmacopoeia 2020. Specifically, the method for detecting chondroitin yield involved in this invention can be performed as follows: Take 10 mL of fermentation broth into a 50 mL centrifuge tube, add pure glacial acetic acid to adjust the pH to pH 3.8, place in an 85℃ water bath for acid hydrolysis for 4 h, after acid hydrolysis, cool to room temperature, adjust the pH to between 7.0 and 8.0 using 6 M sodium hydroxide (NaOH), and transfer to a 25 mL volumetric flask for final volume determination. Take 100 μL of the acid-hydrolyzed sample and mix it with 100 μL of chondroitin sulfate ABC enzyme (SIGMA-Aldrich, C2905-10UN). Add 800 μL of tris(hydroxymethyl)chloromethane buffer, mix thoroughly, and place in a 37℃ water bath for 1 h. After the enzymatic digestion is completed, boil in a water bath for 5 minutes, cool with cold water, centrifuge, and take 20 μL of the supernatant. Load the sample onto a Spherisorb SAX Column (Waters) for HPLC analysis at a detection wavelength of 232 nm. Quantification was performed using the external standard method. The sum of the peak areas of ΔDi-0S, ΔDi-4S, and ΔDi-6S after enzymatic hydrolysis of 10 g / L chondroitin sulfate sodium standard (China National Institutes for Food and Drug Control, 4386 / 10 / 12, batch number 140792-202003) was used as the peak area of ​​the reference standard. The peak area of ​​ΔDi-0S after enzymatic hydrolysis of the sample was used as the peak area of ​​the sample. The chondroitin content was calculated as (sample peak area / reference peak area × standard concentration 10 g / L × dilution factor 2.5).

[0198] The method for determining the molecular weight of chondroitin involved in this invention can be based on the analytical method of "Method for Determining the Molecular Weight of Low Molecular Weight Heparin", and uses a TSKgel G3000SWXL column to determine the molecular weight of chondroitin produced by fermentation.

[0199] The glucose determination method of the present invention is as follows: centrifuge the fermentation broth at 10,000 rpm for 5 min, take the supernatant, dilute it 20 times, and then measure it using a biosensor analyzer M-100 (Silman Technology).

[0200] The present invention will be further illustrated below with reference to the embodiments:

[0201] Example 1 Construction of engineered strains

[0202] In this embodiment, engineered strains BSG01, BSG02, BSG031, BSG041, BSG051, BSG062, BSG072, BSM012, DH003, and BSM032 were constructed.

[0203] 1. Construction of BSG01 strain:

[0204] a) Gene editing of Bacillus subtilis 168 (ATCC 23857) was performed using the CRISPR / Cas9 system;

[0205] b) The ybcM gene was replaced in situ with a gene fragment consisting of the P43 promoter and a nucleotide sequence that encodes UDP-N-acetylglucosamine C4 isomerase (KfoA) to express UDP-N-acetylglucosamine C4 isomerase (KfoA).

[0206] c) Obtain strain BSG01.

[0207] 2. Construction of BSG02 strain

[0208] a) Gene editing of Bacillus subtilis 168 (ATCC 23857) was performed using the CRISPR / Cas9 system;

[0209] b) The ybcM gene was replaced in situ with a gene fragment consisting of the P43 promoter and a nucleotide sequence that encodes UDP-N-acetylglucosamine C4 isomerase (KfoA) to express UDP-N-acetylglucosamine C4 isomerase (KfoA).

[0210] c) The 1915th to 1980th nucleotide sequences of the amyE gene are replaced in situ using a gene fragment consisting of the PxylR promoter, xylR gene, PxylA promoter and nucleotide sequence encoding chondroitin synthase (McCS) to express chondroitin synthase.

[0211] d) Obtain strain BSG02.

[0212] 3. Construction of BSG031 strain (combination 1)

[0213] a) Gene editing of Bacillus subtilis 168 (ATCC 23857) was performed using the CRISPR / Cas9 system;

[0214] b) The ybcM gene was replaced in situ with a gene fragment consisting of the P43 promoter and a nucleotide sequence that encodes UDP-N-acetylglucosamine C4 isomerase (KfoA) to express UDP-N-acetylglucosamine C4 isomerase (KfoA).

[0215] c) The 1915th to 1980th nucleotide sequences of the amyE gene are replaced in situ using a gene fragment consisting of the PxylR promoter, xylR gene, PxylA promoter and nucleotide sequence encoding chondroitin synthase (McCS) to express chondroitin synthase.

[0216] d) The epr gene was replaced in situ with a gene fragment consisting of the P43 promoter and a nucleotide sequence that encodes UDP-glucose dehydrogenase (KfoF) to express UDP-glucose dehydrogenase (KfoF).

[0217] e) Obtain strain BSG031.

[0218] 4. Construction of BSG041 strain (combination 6)

[0219] a) Gene editing of Bacillus subtilis 168 (ATCC 23857) was performed using the CRISPR / Cas9 system;

[0220] b) The ybcM gene was replaced in situ with a gene fragment consisting of the P43 promoter and a nucleotide sequence that encodes UDP-N-acetylglucosamine C4 isomerase (KfoA) to express UDP-N-acetylglucosamine C4 isomerase (KfoA).

[0221] c) The 1915th to 1980th nucleotide sequences of the amyE gene are replaced in situ using a gene fragment consisting of the PxylR promoter, xylR gene, PxylA promoter and nucleotide sequence encoding chondroitin synthase (McCS) to express chondroitin synthase.

[0222] d) The epr gene was replaced in situ with a gene fragment consisting of the P43 promoter and a nucleotide sequence that encodes UDP-glucose dehydrogenase (KfoF) to express UDP-glucose dehydrogenase (KfoF).

[0223] e) The 37th to 270th nucleotides of the amyE gene were replaced in situ using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding UTP-glucose-1-phosphate uridine transferase (GalU), and the dps terminator, to express UTP-glucose-1-phosphate uridine transferase (GalU).

[0224] f) The strain BSG041 was obtained.

[0225] 5. Construction of BSG051 strain (combination 16)

[0226] a) Gene editing of Bacillus subtilis 168 (ATCC 23857) was performed using the CRISPR / Cas9 system;

[0227] b) The ybcM gene was replaced in situ with a gene fragment consisting of the P43 promoter and a nucleotide sequence that encodes UDP-N-acetylglucosamine C4 isomerase (KfoA) to express UDP-N-acetylglucosamine C4 isomerase (KfoA).

[0228] c) The 1915th to 1980th nucleotide sequences of the amyE gene are replaced in situ using a gene fragment consisting of the PxylR promoter, xylR gene, PxylA promoter and nucleotide sequence encoding chondroitin synthase (McCS) to express chondroitin synthase.

[0229] d) The epr gene was replaced in situ with a gene fragment consisting of the P43 promoter and a nucleotide sequence that encodes UDP-glucose dehydrogenase (KfoF) to express UDP-glucose dehydrogenase (KfoF).

[0230] e) The 37th to 270th nucleotides of the amyE gene were replaced in situ using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding UTP-glucose-1-phosphate uridine transferase (GalU), and the dps terminator, to express UTP-glucose-1-phosphate uridine transferase (GalU).

[0231] f) The nucleotide sequence from 1212 to 1551 of the amyE gene was replaced in situ using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding glutamine-fructose-6-phosphate aminotransferase (GlmS), and the clpP terminator, to express glutamine-fructose-6-phosphate aminotransferase (GlmS).

[0232] g) The strain BSG051 was obtained.

[0233] 6. Construction of strain BSG062 (combination 26)

[0234] a) Gene editing of Bacillus subtilis 168 (ATCC 23857) was performed using the CRISPR / Cas9 system;

[0235] b) The ybcM gene was replaced in situ with a gene fragment consisting of the P43 promoter and a nucleotide sequence that encodes UDP-N-acetylglucosamine C4 isomerase (KfoA) to express UDP-N-acetylglucosamine C4 isomerase (KfoA).

[0236] c) The 1915th to 1980th nucleotide sequences of the amyE gene are replaced in situ using a gene fragment consisting of the PxylR promoter, xylR gene, PxylA promoter and nucleotide sequence encoding chondroitin synthase (McCS) to express chondroitin synthase.

[0237] d) The epr gene was replaced in situ with a gene fragment consisting of the P43 promoter and a nucleotide sequence that encodes UDP-glucose dehydrogenase (KfoF) to express UDP-glucose dehydrogenase (KfoF).

[0238] e) The 37th to 270th nucleotides of the amyE gene were replaced in situ using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding UTP-glucose-1-phosphate uridine transferase (GalU), and the dps terminator, to express UTP-glucose-1-phosphate uridine transferase (GalU).

[0239] f) The nucleotide sequence from 1212 to 1551 of the amyE gene was replaced in situ using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding glutamine-fructose-6-phosphate aminotransferase (GlmS), and the clpP terminator, to express glutamine-fructose-6-phosphate aminotransferase (GlmS).

[0240] g) The promoter and the first 461 nucleotides of the rapE gene were replaced in situ using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU), and the clpP terminator, to express UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU).

[0241] h) The strain BSG062 was obtained.

[0242] 7. Construction of BSG072 strain (combination 31, single copy of McCS)

[0243] a) Gene editing of Bacillus subtilis 168 (ATCC 23857) was performed using the CRISPR / Cas9 system;

[0244] b) The ybcM gene was replaced in situ with a gene fragment consisting of the P43 promoter and a nucleotide sequence that encodes UDP-N-acetylglucosamine C4 isomerase (KfoA) to express UDP-N-acetylglucosamine C4 isomerase (KfoA).

[0245] c) The 1915th to 1980th nucleotide sequences of the amyE gene are replaced in situ using a gene fragment consisting of the PxylR promoter, xylR gene, PxylA promoter and nucleotide sequence encoding chondroitin synthase (McCS) to express chondroitin synthase.

[0246] d) The epr gene was replaced in situ with a gene fragment consisting of the P43 promoter and a nucleotide sequence that encodes UDP-glucose dehydrogenase (KfoF) to express UDP-glucose dehydrogenase (KfoF).

[0247] e) The 37th to 270th nucleotides of the amyE gene were replaced in situ using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding UTP-glucose-1-phosphate uridine transferase (GalU), and the dps terminator, to express UTP-glucose-1-phosphate uridine transferase (GalU).

[0248] f) The nucleotide sequence from 1212 to 1551 of the amyE gene was replaced in situ using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding glutamine-fructose-6-phosphate aminotransferase (GlmS), and the clpP terminator, to express glutamine-fructose-6-phosphate aminotransferase (GlmS).

[0249] g) The promoter and the first 461 nucleotides of the rapE gene were replaced in situ using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU), and the clpP terminator, to express UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU).

[0250] h) The rapE gene was modified by in situ substitution of nucleotides 528 to 576 using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding glucose-1,4-dioxanone (GlmM), and the dps terminator, to express glucose-1,4-dioxanone (GlmM).

[0251] i) Obtain strain BSG072.

[0252] 8. Construction of strain BSM012 (combination 31, McCS double copy)

[0253] a) Gene editing of Bacillus subtilis 168 (ATCC 23857) was performed using the CRISPR / Cas9 system;

[0254] b) The ybcM gene was replaced in situ with a gene fragment consisting of the P43 promoter and a nucleotide sequence that encodes UDP-N-acetylglucosamine C4 isomerase (KfoA) to express UDP-N-acetylglucosamine C4 isomerase (KfoA).

[0255] c) The 1915th to 1980th nucleotide sequences of the amyE gene are replaced in situ using a gene fragment consisting of the PxylR promoter, xylR gene, PxylA promoter and nucleotide sequence encoding chondroitin synthase (McCS) to express chondroitin synthase.

[0256] d) The epr gene was replaced in situ with a gene fragment consisting of the P43 promoter and a nucleotide sequence that encodes UDP-glucose dehydrogenase (KfoF) to express UDP-glucose dehydrogenase (KfoF).

[0257] e) The 37th to 270th nucleotides of the amyE gene were replaced in situ using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding UTP-glucose-1-phosphate uridine transferase (GalU), and the dps terminator, to express UTP-glucose-1-phosphate uridine transferase (GalU).

[0258] f) The nucleotide sequence from 1212 to 1551 of the amyE gene was replaced in situ using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding glutamine-fructose-6-phosphate aminotransferase (GlmS), and the clpP terminator, to express glutamine-fructose-6-phosphate aminotransferase (GlmS).

[0259] g) The promoter and the first 461 nucleotides of the rapE gene were replaced in situ using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU), and the clpP terminator, to express UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU).

[0260] h) The rapE gene was modified by in situ substitution of nucleotides 528 to 576 using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding glucose-1,4-dioxanone (GlmM), and the dps terminator, to express glucose-1,4-dioxanone (GlmM).

[0261] i) The promoter and the first 702 nucleotides of the nprE gene were replaced in situ using a gene fragment consisting of the PxylA promoter and a nucleotide sequence that encodes chondroitin synthase (McCS) (a second McCS copy was obtained by expressing McCS copy B) to express double chondroitin synthase (McCS).

[0262] j) The strain BSM012 was obtained.

[0263] 9. Construction of strain DH003 (combination 31, three copies of McCS)

[0264] a) Gene editing of Bacillus subtilis 168 (ATCC 23857) was performed using the CRISPR / Cas9 system;

[0265] b) The ybcM gene was replaced in situ with a gene fragment consisting of the P43 promoter and a nucleotide sequence that encodes UDP-N-acetylglucosamine C4 isomerase (KfoA) to express UDP-N-acetylglucosamine C4 isomerase (KfoA).

[0266] c) The 1915th to 1980th nucleotide sequences of the amyE gene are replaced in situ using a gene fragment consisting of the PxylR promoter, xylR gene, PxylA promoter and nucleotide sequence encoding chondroitin synthase (McCS) to express chondroitin synthase.

[0267] d) The epr gene was replaced in situ with a gene fragment consisting of the P43 promoter and a nucleotide sequence that encodes UDP-glucose dehydrogenase (KfoF) to express UDP-glucose dehydrogenase (KfoF).

[0268] e) The 37th to 270th nucleotides of the amyE gene were replaced in situ using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding UTP-glucose-1-phosphate uridine transferase (GalU), and the dps terminator, to express UTP-glucose-1-phosphate uridine transferase (GalU).

[0269] f) The nucleotide sequence from 1212 to 1551 of the amyE gene was replaced in situ using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding glutamine-fructose-6-phosphate aminotransferase (GlmS), and the clpP terminator, to express glutamine-fructose-6-phosphate aminotransferase (GlmS).

[0270] g) The promoter and the first 461 nucleotides of the rapE gene were replaced in situ using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU), and the clpP terminator, to express UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU).

[0271] h) The rapE gene was modified by in situ substitution of nucleotides 528 to 576 using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding glucose-1,4-dioxanone (GlmM), and the dps terminator, to express glucose-1,4-dioxanone (GlmM).

[0272] i) The promoter and the first 702 nucleotides of the nprE gene were replaced in situ using a gene fragment consisting of the PxylA promoter and a nucleotide sequence that encodes chondroitin synthase (McCS) (a second McCS copy was obtained by expressing McCS copy B) to express double chondroitin synthase (McCS).

[0273] j) Insert a gene fragment consisting of the PxylA promoter and a nucleotide sequence encoding chondroitin synthase (McCS) between the walL and desR gene terminators (to obtain a third McCS copy using the McCS copy C expression method) to express triploid chondroitin synthase (McCS).

[0274] (k) obtained strain DH003.

[0275] 10. Construction of strain BSM032 (combination 31, four copies of McCS)

[0276] a) Gene editing of Bacillus subtilis 168 (ATCC 23857) was performed using the CRISPR / Cas9 system;

[0277] b) The ybcM gene was replaced in situ with a gene fragment consisting of the P43 promoter and a nucleotide sequence that encodes UDP-N-acetylglucosamine C4 isomerase (KfoA) to express UDP-N-acetylglucosamine C4 isomerase (KfoA).

[0278] c) The 1915th to 1980th nucleotide sequences of the amyE gene are replaced in situ using a gene fragment consisting of the PxylR promoter, xylR gene, PxylA promoter and nucleotide sequence encoding chondroitin synthase (McCS) to express chondroitin synthase.

[0279] d) The epr gene was replaced in situ with a gene fragment consisting of the P43 promoter and a nucleotide sequence that encodes UDP-glucose dehydrogenase (KfoF) to express UDP-glucose dehydrogenase (KfoF).

[0280] e) The 37th to 270th nucleotides of the amyE gene were replaced in situ using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding UTP-glucose-1-phosphate uridine transferase (GalU), and the dps terminator, to express UTP-glucose-1-phosphate uridine transferase (GalU).

[0281] f) The nucleotide sequence from 1212 to 1551 of the amyE gene was replaced in situ using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding glutamine-fructose-6-phosphate aminotransferase (GlmS), and the clpP terminator, to express glutamine-fructose-6-phosphate aminotransferase (GlmS).

[0282] g) The promoter and the first 461 nucleotides of the rapE gene were replaced in situ using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU), and the clpP terminator, to express UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU).

[0283] h) The rapE gene was modified by in situ substitution of nucleotides 528 to 576 using a gene fragment consisting of the P43 promoter, a nucleotide sequence encoding glucose-1,4-dioxanone (GlmM), and the dps terminator, to express glucose-1,4-dioxanone (GlmM).

[0284] i) The promoter and the first 702 nucleotides of the nprE gene were replaced in situ using a gene fragment consisting of the PxylA promoter and a nucleotide sequence that encodes chondroitin synthase (McCS) (a second McCS copy was obtained by expressing McCS copy B) to express double chondroitin synthase (McCS).

[0285] j) Insert a gene fragment consisting of the PxylA promoter and a nucleotide sequence encoding chondroitin synthase (McCS) between the walL and desR gene terminators (to obtain a third McCS copy using the McCS copy C expression method) to express triploid chondroitin synthase (McCS).

[0286] k) Insert a gene fragment consisting of the PxylA promoter and a nucleotide sequence encoding chondroitin synthase (McCS) between the clpE and motA gene terminators (to obtain a fourth McCS copy using the McCS copy C expression method), so that it expresses four times the amount of chondroitin synthase (McCS).

[0287] l) The strain BSM032 was obtained.

[0288] The amino acid sequences and nucleotide coding sequences of each enzyme are shown in Table 1.

[0289] Table 1

[0290]

[0291] The following steps, taking the construction of the BSG01 strain as an example, are as follows:

[0292] (a) Preparation of Bacillus subtilis 168 / pCas9 competent cells

[0293] 1500 ng of pCas9 plasmid was transformed into Bacillus subtilis strain 168 using the Spizizen transformation method (procedure below). The transformed plasmid was spread onto solid LB medium (containing 10 mg / L chloramphenicol (CHL)) and incubated overnight at 37°C to obtain Bacillus subtilis strain 168 / pCas9. A single colony was picked and inoculated into 5 mL of LB liquid medium (10 mg / L CHL) to obtain Bacillus subtilis strain 168 / pCas9. The strain was then transformed into chemically competent cells using the Spizizen transformation method and used immediately.

[0294] (1) Solution preparation

[0295] 10×spizizen salts stock solution: 75 g dimethyl hydrogen phosphate trihydrate, 3 g potassium dihydrogen phosphate, 1 g ammonium sulfate, 0.1 g magnesium sulfate and 0.5 g sodium citrate, diluted with distilled water to 100 mL, sterilized at 115℃ for 20 min, and stored at room temperature.

[0296] Yeast powder solution: 0.1 g yeast powder, dilute with distilled water to 1 mL. Sterilize at 115℃ for 20 min, store at 4℃.

[0297] Hydrolyzed casein solution: 0.1 g hydrolyzed casein, diluted to 10 mL with distilled water. Sterilize at 115℃ for 20 min, store at room temperature.

[0298] Magnesium sulfate solution: 0.6 g MgSO4, diluted to 50 mL with distilled water. Sterilize at 115℃ for 20 min, store at room temperature.

[0299] Glucose solution: 10g glucose, diluted with distilled water to 50 mL. Sterilize at 115℃ for 20 min, store at room temperature.

[0300] Calcium chloride solution: 0.056 g calcium chloride, diluted to 5 mL with distilled water. Sterilize at 115℃ for 20 min, store at room temperature.

[0301] L-Trp solution: 0.025 g L-Trp, diluted to 10 mL with distilled water. Filter sterilize and store at -20°C.

[0302] (2) Working culture medium

[0303] GM 1 solution (5 mL): 0.5 mL 10×spizizen salts stock solution, 0.05 mL yeast extract solution, 0.125 mL glucose solution, 0.1 mL hydrolyzed casein solution, 0.1 mL L-Trp solution, and sterile distilled water to a final volume of 5 mL.

[0304] GM 2 solution (20 mL): 2 mL 10×spizizen salts, 0.1 mL yeast powder solution, 0.5 mL glucose solution, 0.08 mL hydrolyzed casein solution, 0.4 mL L-Trp solution, 0.1 mL calcium chloride solution, 0.25 mL magnesium sulfate solution, and sterile distilled water to a final volume of 20 mL.

[0305] (3) Preparation of competent cells

[0306] Take 1 μL of freshly cultured Bacillus subtilis engineered bacterial culture and inoculate it into 2.5 mL of GM 1 solution. Incubate overnight at 37°C with shaking at 220 rpm. Inoculate the overnight culture into 2.5 mL of fresh GM 1 solution at a 10% inoculation rate and incubate at 37°C with shaking at 220 rpm for 5 h. Inoculate the second culture into 5 mL of GM 2 solution at a 10% inoculation rate and incubate at 37°C with shaking at 220 rpm for 90 min. Take 1 mL of the culture, centrifuge at 5000 rpm at room temperature for 5 min, and resuspend the bacterial pellet with 1 / 10 volume of the supernatant. This is the competent Bacillus subtilis engineered bacterial cell, which should be prepared and used immediately.

[0307] (4) Plasmid transformation

[0308] Add the plasmid to the Bacillus subtilis engineered competent cell suspension to a final concentration of 150 ng / mL, mix well, and incubate at 37℃ with shaking at 220 r / min for 2-4 h. Spread the mixture onto LB solid medium with the corresponding resistance.

[0309] (ii) Constructing the kfoA expression frame

[0310] (1) Amplify upstream homologous arm fragment

[0311] PCR amplification system: 1 μL of B. subtilis 168 genomic DNA template (50 ng / μL), 1 μL each of kfoA-F1 and kfoA-R1 (10 μM) primers (Table 2), 25 μL of 2×PrimeSTAR MAX Premix, and 2 μL of ddH2O2.

[0312] PCR reaction conditions: pre-denaturation 98℃ for 5 min; 30 cycles: 98℃ for 10 sec, 57℃ for 15 sec, 72℃ for 1 min; termination at 4℃. PCR products were verified by 1% agarose gel electrophoresis and then purified using the FastPure Gel DNA Extraction Mini Kit to obtain the upstream homologous arm fragment.

[0313] (2) Amplification of downstream homologous arm fragments

[0314] Referring to the upstream homologous arm fragment, the primers were kfoA-F2 and kfoA-R2 (Table 2).

[0315] (3) Amplify the kfoA fragment

[0316] Referencing upstream homologous arm fragments, the E. coli K4 genome was used as a template, and the primers were kfoA-F3 and kfoA-R3 (Table 2).

[0317] (4) Amplify the P43 promoter fragment

[0318] Referencing the upstream homologous arm fragment, the template was the B. subtilis168 genome, and the primers were P43-F1 and P43-R1 (Table 2).

[0319] Donor DNA was obtained using overlap PCR. The PCR amplification system consisted of 1 μL each of the upstream and downstream homologous arms, the kfoA gene fragment, and the P43 promoter fragment (50 ng / μL), 1 μL each of primers kfoA-F1 and kfoA-R2 (10 μM), 25 μL of 2×PrimeSTAR MAX Premix, and 19 μL of ddH2O2. PCR reaction conditions were as follows: pre-denaturation at 98℃ for 5 min, 30 cycles: 98℃ for 10 sec, 57℃ for 15 sec, 72℃ for 1 min; termination at 4℃.

[0320] (III) Construction of pTarget-kfoA plasmid

[0321] Based on the CRISPR / Cas9 gene editing principle, a specific guide sgRNA sequence was used, which was synthesized by Suzhou Genewise Biotechnology Co., Ltd. and ligated into the pTarget plasmid (preserved in the laboratory) to obtain the pTarget-kfoA plasmid.

[0322] (iv) Integration of the kfoA gene

[0323] Bacillus subtilis 168 / pCas9 strain was transformed into competent cells using the Spizizen transformation method. 2000 ng Donor DNA and 1500 ng pTarget-kfoA were added to the strain, and the cells were then transformed into Bacillus subtilis 168 / pCas9 strain using the Spizizen transformation method. The cells were plated on solid LB medium (containing 10 mg / L chloroquine, 50 mg / L kanamycin, and 50 mg / L isopropyl-β-D-thiogalactopyranoside (IPTG)) and incubated overnight at 30°C. Several single clones were picked from the transformation plates as templates, and positive clones were verified by colony PCR using kfoA-TF / kfoA-TR primers. The amplified fragment size was 3533 bp, and the amplified product was sequenced for verification.

[0324] (v) pTarget-kfoA plasmid elimination

[0325] Positive clones were inoculated into 5 mL of LB liquid medium (100 mg / L chl), followed by inoculation into LB (100 mg / L chl) liquid medium, and cultured at 42°C with shaking at 220 rpm for 12 h. The 12-h culture was then streaked onto LB (100 mg / L chl) solid medium and incubated at 42°C for 12 h. After incubation, the streaked colonies were numbered, and each numbered colony (only half its size was selected) was inoculated into the corresponding area on LB (50 mg / L kana) solid medium and incubated overnight at 30°C. Colonies that could not grow in the corresponding area on LB (50 mg / L kana) solid medium were considered successfully eliminated pTarget-kfoA strains. Strains eliminated by pTarget-kfoA were considered successfully edited strains. Strains that successfully eliminated the pTarget-X plasmid can be used as starting strains for the next round of gene editing or can continue to eliminate the pCas9 plasmid for fermentation.

[0326] (vi) pCas9 plasmid elimination

[0327] Colonies containing the repA-cas9 plasmid were inoculated into 5 mL of LB liquid medium containing 0.005% sodium dodecyl sulfate (SDS) and cultured at 37°C with shaking at 220 rpm for 12 h. After the culture was complete, 1 μL of the bacterial culture was inoculated into 5 mL of LB liquid medium and cultured at 37°C with shaking at 220 rpm for 12 h, repeated 7 times. The cultured bacterial culture was then streaked onto LB solid medium. After the culture was complete, the streaked colonies were numbered, and each numbered colony (only half the size of the colony was picked) was inoculated into the corresponding area on LB (10 mg / L chl) solid medium and cultured overnight at 37°C. The colonies that could not grow in the corresponding area on the LB (10 mg / L chl) solid medium were the successfully eliminated pCas9 strain.

[0328] The subsequent construction of engineered strains followed the above procedure, iteratively integrating the McCS (1-4 copies), KfoF, GalU, GlmS, GlmU, and GlmM genes into the genome of the engineered strains in sequence. The relevant primers are shown in Table 2. The sizes of the amplified fragments identified by PCR of positive clones were 6124 bp, 3364 bp, 3769 bp, 3843 bp, 3625 bp, 3465 bp, 5016 bp, and 4972 bp, respectively. Among them, the PxylR promoter, xylR gene, and PxylA promoter were amplified using xylR-F1 and xylA-R1.

[0329] Following the construction method of BSG01 strain, engineered Bacillus subtilis strains BSG02, BSG031, BSG041, BSG051, BSG062, BSG072, BSM012, DH003, and BSM032 were constructed. The sgRNAs, primers, and corresponding sequences involved are shown in Table 2.

[0330] Table 2 sgRNA, primers and sequences

[0331]

[0332]

[0333]

[0334]

[0335]

[0336] Example 2: Shake-flask fermentation of engineered strains

[0337] The engineered Bacillus subtilis strains BSG01, BSG02, BSG031, BSG041, BSG051, BSG062, BSG072, BSM012, DH003, and BSM032 were subjected to shake-flask fermentation. Single clones of each engineered strain were inoculated into 250 mL shake flasks containing 50 mL of LB medium (formulation shown in Table 3) and cultured at 37℃ and 220 rpm for 10 h. A 5% inoculum (v / v) of the seed culture was transferred to 500 mL shake flasks containing 50 mL of fermentation medium (formulation shown in Table 4). Fermentation conditions were: 220 rpm, 37℃, initial pH controlled at 7.0, xylose added after 4 h of fermentation to a final concentration of 18 g / L, and fermentation continued for 48 h. Each strain was replicated three times. After fermentation, the fermentation broth was collected, and the chondroitin yield was determined by HPLC. Bacillus subtilis 168 (ATCC 23857) was used as a control.

[0338] Table 3 LB medium formulation

[0339]

[0340] Table 4 Fermentation medium formulation

[0341]

[0342] The chondroitin yields of each strain during shake-flask fermentation are shown in Table 5. After 48 hours of fermentation, the chondroitin yields of the chassis strain Bacillus subtilis 168, engineered strains BSG01, BSG02, BSG031, BSG041, BSG051, BSG062, BSG072, BSM012, DH003, and BSM032 were 0 g / L, 0 g / L, 0.054 g / L, 0.073 g / L, 0.136 g / L, 0.196 g / L, 0.226 g / L, 0.630 g / L, 0.737 g / L, 0.850 g / L, and 0.816 g / L, respectively.

[0343] Table 5 Results of chondroitin shake-flask fermentation

[0344]

[0345] As can be clearly seen from the results in Table 5, the wild-type Bacillus subtilis 168, serving as the chassis strain, produced 0 g / L of chondroitin, verifying that wild-type Bacillus subtilis lacks the ability to synthesize chondroitin. This constitutes the necessary starting point for the metabolic engineering modification in this invention. Guided by the strategy designed in this invention, the most basic synthetic pathway was successfully constructed by first integrating and expressing UDP-N-acetylglucosamine C4 isomerase (KfoA) and one copy of chondroitin synthase (McCS) into the genome, achieving a breakthrough in the production of chondroitin from scratch (BSG02: 0.054 g / L).

[0346] Subsequently, through the systematic introduction and overexpression of a series of key enzymes designed in this invention, chondroitin production achieved a significant stepwise increase: the sequential introduction of UDP-glucose dehydrogenase (KfoF) increased production by 35% (BSG031, 0.073 g / L); the introduction of UTP-glucose-1-phosphate uridine transferase (GalU) brought a significant increase of 86% (BSG041, 0.136 g / L); the addition of glutamine-fructose-6-phosphate aminotransferase (GlmS) further increased production by 44% (BSG051, 0.196 g / L); the continued introduction of UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase (GlmU) increased production by another 15% (BSG062, 0.226 g / L); and the introduction of phosphogluconomutase (GlmM) drove a dramatic 178% increase in production (BSG072, 0.630 g / L).

[0347] Building upon the enhanced precursor supply described above, this invention further increases the copy number of chondroitin synthase (McCS). For example, increasing the McCS copy number to 2 (BSM012) and 3 (DH003) resulted in yields of 0.737 g / L and 0.850 g / L, respectively, representing additional increases of 17% and 35% compared to the BSG072 strain containing only 1 copy. Although increasing the McCS copy number to 4 (BSM032) resulted in a yield of 0.816 g / L, a 4% decrease compared to the DH003 strain with 3 copies, a high expression level was still maintained.

[0348] Example 3: Fermentation of engineered strains in a 5 L fermenter

[0349] This embodiment takes BSG02, BSG031, BSG041, BST051, BSG062, BSG072, BSM012, DH003, and BSM032 as examples to study the chondroitin yield after scale-up fermentation.

[0350] Bacillus subtilis 168 (ATCC 23857) and engineered strains BSG031, BSG041, BST051, BSG062, BSG072, BSM012, DH003, and BSM032 were inoculated into LB liquid medium and activated overnight at 37°C. A 5% inoculum (v / v) was then transferred to a 500 mL shake flask containing 100 mL of the seed culture medium shown in Table 6 and incubated at 37°C and 220 rpm for 4.5 h, with an OD600 > 12. The seed culture was then transferred at a 5% inoculum (v / v) to a 5 L T&JIntelli-FermA benchtop fermenter (purchased from Shanghai Dibier Company) containing 1.8 L of the fermentation medium shown in Table 7, with 0.1 g / L of antifoaming agent added. Fermentation was carried out according to the control process shown in Table 8 for 48 h. Each strain was replicated three times. After fermentation, the fermentation broth was collected, and the chondroitin yield was determined by HPLC. The results are shown in Table 9, and the fermentation curves are shown in Table 8. Figure 2 and Figure 3 As shown.

[0351] Table 6 Seed Culture Medium Formulation

[0352]

[0353] Table 7 Fermentation medium formulation

[0354]

[0355] Table 8.5 L-tank fermentation control process

[0356]

[0357] Table 9. Results of Chondroitin Fermentation in 5 L Tanks

[0358]

[0359] The results of Examples 2 and 3 show that with the introduction of the enzyme / combination provided by this invention and the increase in the copy number of chondroitin synthase (McCS) into the metabolic pathway, the chondroitin production of the engineered strain can show a significant increasing trend. However, when the copy number of chondroitin synthase (McCS) increases to 4, the chondroitin production decreases, but still maintains a high expression level. In summary, the enzyme / combination provided by this invention can significantly enhance the chondroitin expression of Bacillus subtilis engineered strains, which lays an industrial foundation for obtaining chondroitin through Bacillus subtilis fermentation.

[0360] Example 4: Fermentation of chondroitin by engineered Bacillus subtilis strain DH003 in a 50 L fermenter

[0361] The engineered strain DH003 was selected and activated in LB medium according to the method in Example 3. First, a 5% inoculum was added to a seed shaker flask. The seed culture medium formulation is shown in Table 6. The flask was incubated at 37℃ and 220 rpm for 4.5 h, with an OD600 > 12. Then, a 5% inoculum was added to a 15 L seed tank. The seed culture medium formulation is shown in Table 6. The flask was incubated at 37℃ for 2.5 h, with dissolved oxygen controlled at 30-40%. When dissolved oxygen was below 20%, the rotation speed, air volume, and tank pressure were increased sequentially, with an OD600 > 12. The prepared seed culture was transferred at a 5% inoculum to a 50 L fermenter (purchased from Bailun Biotechnology) containing 25 L of fermentation medium as shown in Table 7. 0.1 g / L antifoaming agent was added, and fermentation was carried out according to the control process shown in Table 10 for 48 h. This process was repeated three times. After fermentation, the fermentation broth was collected, and the chondroitin yield was determined by HPLC. The results are shown in Table 11, and the fermentation curve is shown in Table 11. Figure 4 As shown.

[0362] Table 10 50 L Tank Fermentation Control Process

[0363]

[0364] The chondroitin content in the fermentation broth was detected by HPLC. The chromatogram of chondroitin disaccharide after enzymatic hydrolysis of chondroitin sulfate sodium standard is shown below. Figure 5 As shown, the chondroitin disaccharide spectrum after enzymatic hydrolysis of the fermentation product chondroitin is as follows. Figure 6 As shown, the chondroitin content in the fermentation broth was calculated to be 7.50 ± 0.29 g / L.

[0365] Scale-up culture results showed that the chondroitin production of the Bacillus subtilis engineered strain DH003 provided by this invention was 8.01±0.16 g / L in a 5L fermenter and 7.50±0.29 g / L in a 50L fermenter. This indicates that the Bacillus subtilis engineered strain provided by this invention can achieve high-yield expression of chondroitin. Furthermore, after a 10-fold volume increase (5 L→50 L), the chondroitin production remained stable without a significant decline trend. This demonstrates that the metabolic pathway design strategy of this invention is reasonable and possesses excellent stability and scalability, making it feasible for industrial-scale chondroitin production.

[0366] In this embodiment, the chondroitin obtained from 50L fermentation was further identified by mass spectrometry (LC-MS). The mass spectrum of chondroitin disaccharide after enzymatic hydrolysis is shown below. Figure 7 As shown, its mass-to-charge ratio (m / z) is 378, which is similar to the theoretical molecular weight (C) of chondroitin disaccharide (ΔDi-OS). 14 H 21 NO 11 [MH] -The m / z = 378.103 is completely consistent, which indicates that the chondroitin structure obtained by fermentation using the Bacillus subtilis engineered strain provided by this invention is correct.

[0367] (3) Detection of chondroitin molecular weight

[0368] The results are as follows Figure 8 As shown in Table 11, the chondroitin produced by fermentation has a 64.82% weight-average molecular weight Mw = 53.215 kDa and a retention time of 12.444 min; and a 35% weight-average molecular weight Mw = 12.166 kDa and a retention time of 17.127 min.

[0369] Table 11 Results of chondroitin molecular weight analysis

[0370]

[0371] Based on the shake-flask level validation, this embodiment further scaled up the selected strain DH003 in 5 L and 50 L fermenters for fermentation validation. The results showed that this engineered strain exhibited excellent production performance and process scalability under controlled fermentation conditions. In a 5 L tank, after 48 hours of fermentation, the chondroitin yield reached 8.01 ± 0.16 g / L; after scaling up to a 50 L tank, the yield stabilized at 7.50 ± 0.29 g / L. This result not only significantly exceeded the highest reported yield (approximately 6.06 g / L) of the existing Bacillus subtilis platform, but more importantly, the yield did not significantly decrease and remained stable during a 10-fold increase in fermentation volume. This fully demonstrates that the metabolic pathway and supporting fermentation process constructed in this invention possess high stability and scalability, perfectly meeting the core requirements of industrial production for yield and process stability.

[0372] Further product analysis showed that the mass spectrometry characteristics (m / z 378) of the disaccharide fraction obtained from fermentation after enzymatic hydrolysis were completely consistent with the standard, confirming the correctness of the product structure. Molecular weight distribution analysis showed that the weight-average molecular weight (Mw) of the product was approximately 53.215 kDa (94.28%), and the distribution was concentrated, indicating that the chondroitin produced by this invention has high structural uniformity and its quality meets the requirements of relevant application fields for product molecular weight.

[0373] In summary, the Bacillus subtilis engineered strain (especially DH003) and its fermentation process provided by this invention can achieve efficient, stable, and large-scale production of chondroitin. This marks a key breakthrough in the microbial fermentation technology for producing chondroitin based on the safe GRAS host Bacillus subtilis, laying a solid technical foundation for its replacement of traditional animal extraction methods and achieving green and safe industrial production.

[0374] Example 5: Stability Verification of Engineered Strains

[0375] The engineered strain DH003 was used for chondroitin fermentation in a 5 L fermenter as described in Example 3. After fermentation, an appropriate amount of fermentation broth was taken, diluted with sterile water, and spread onto LB agar plates. The plates were incubated at 37°C until single colonies appeared. Single colonies were picked and identified by PCR before being used for the next generation of chondroitin fermentation. The strain was continuously passaged for eight generations, and the chondroitin yield in each generation was measured. The results showed that the chondroitin yield remained above 8 g / L after eight passages. This indicates that the engineered strain DH003 provided by this invention exhibits good genetic stability during continuous passage, with no loss or attenuation of the exogenous gene, and the chondroitin synthesis capacity consistently maintained above 8 g / L, fully meeting the requirements for long-term continuous fermentation of industrial strains.

[0376] In summary, this invention addresses the limitations of existing chondroitin production methods (animal extraction, chemical synthesis, and enzymatic hydrolysis) in terms of safety, cost, environmental impact, and efficiency, as well as the core challenges of insufficient yield and the need to improve chassis safety in microbial fermentation methods. It proposes a safe and efficient metabolic engineering solution based on Bacillus subtilis 168. Through systematic metabolic pathway design, key enzymes essential for chondroitin synthesis (KfoA and McCS) and a series of precursor supply enhancement enzymes (KfoF, GalU, GlmS, GlmU, and GlmM) are expressed in combination. Furthermore, by innovatively integrating multiple genomic loci to increase the McCS gene copy number, a series of genetically stable, plasmid-free Bacillus subtilis engineered strains have been successfully constructed.

[0377] Experimental data fully demonstrate the effectiveness of this invention: step-by-step scale-up verification from shake flasks to 5 L and 50 L fermenters shows that the preferred strain DH003 significantly improves the yield compared to existing Bacillus subtilis platforms at industrial-scale operations, significantly breaking through the yield bottleneck of this system. Furthermore, this strain exhibits excellent genetic stability (stable yield after 8 consecutive generations) and process scalability (no yield decline from 5 L to 50 L). Product analysis confirms that the produced chondroitin has the correct structure and uniform molecular weight distribution, meeting quality standards.

[0378] Comparative Example 1: Fermentation of chondroitin by engineered Bacillus subtilis strain DH003 in a 3 L fermenter.

[0379] Chondroitin was produced by fermentation using the engineered strain DH003 of Bacillus subtilis, following the culture medium and fermentation conditions described in patent CN106497845B (A recombinant Bacillus subtilis strain with high chondroitin production and its application).

[0380] A single colony of the engineered Bacillus subtilis strain DH003 was inoculated into 150 mL of the seed culture medium shown in Table 12 and cultured on a shaker at 37°C and 200 rpm for 16 h. The seed culture was then transferred at a volume ratio of 10% to a 3 L fermenter containing 1.35 L of the fermentation medium shown in Table 13, and fermented for 70 h according to the control process shown in Table 14. This process was repeated three times. After fermentation, the fermentation broth was collected, and the chondroitin yield was determined using the Bitter-Muir carbazole sulfate colorimetric method employed in patent CN106497845B (A high-yield recombinant Bacillus subtilis with high chondroitin production and its application). The results are shown in Table 15, and the fermentation curve is shown in Table 15. Figure 9 As shown.

[0381] Table 12 Seed Culture Medium Formulation

[0382]

[0383] Table 13 Fermentation medium formulation

[0384]

[0385] Table 14.3 L-tank fermentation control process

[0386]

[0387] Table 15 Results of Chondroitin 3 L Tank Fermentation

[0388]

[0389] Compared with the chondroitin production of 6.06 g / L by the engineered strain of patent CN106497845B (a high-chondroitin-producing recombinant Bacillus subtilis and its application), the Bacillus subtilis engineered strain DH003 of the present invention, fermented for 70 h using the same fermentation medium and controlled process, and with the same detection method, achieved a chondroitin yield of 9.86 g / L, which is significantly higher than the chondroitin yield of the recombinant Bacillus subtilis of the present invention.

[0390] In summary, this invention not only provides a set of specific high-yield engineered bacterial strains and their defined construction methods, but more importantly, it establishes a technical pathway for the efficient, stable, and large-scale fermentation production of chondroitin using the Generally Recognized As Safe (GRAS) Bacillus subtilis as a substrate. This technology combines the advantages of high yield, process safety, environmental friendliness, and controllable costs, providing reliable technical support with significant industrialization prospects for the widespread application of chondroitin and its sulfated derivatives in the food, health product, and pharmaceutical fields.

[0391] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An engineered bacterial strain, characterized in that, Using Bacillus subtilis as the chassis strain, and expressing enzymes including UDP-N-acetylglucosamine C4 isomerase, chondroitin synthase, and combinations of enzymes selected from one or more of the following: (a) UDP-glucose dehydrogenase; (b) UTP-glucose-1-phosphate uridine transferase; (c) Glutamine-fructose-6-phosphate aminotransferase; (d) UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase; (e) Phosphoglucose mutase.

2. The engineered strain as described in claim 1, characterized in that, The chassis strain expresses: Enzyme combination 1 containing UDP-N-acetylglucosamine C4 isomerase, chondroitin synthase, and UDP-glucose dehydrogenase; or Enzyme combination 2 comprising UDP-N-acetylglucosamine C4 isomerase, chondroitin synthase, UDP-glucose dehydrogenase, and UTP-glucose-1-phosphate uridine transferase; or Enzyme combination 3 containing UDP-N-acetylglucosamine C4 isomerase, chondroitin synthase, UDP-glucose dehydrogenase, UTP-glucose-1-phosphate uridine transferase, and glutamine-fructose-6-phosphate aminotransferase; or Enzyme combination 4 containing UDP-N-acetylglucosamine C4 isomerase, chondroitin synthase, UDP-glucose dehydrogenase, UTP-glucose-1-phosphate uridine transferase, glutamine-fructose-6-phosphate aminotransferase, and UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase; or Enzyme combination 5 containing UDP-N-acetylglucosamine C4 isomerase, chondroitin synthase, UDP-glucose dehydrogenase, UTP-glucose-1-phosphate uridine transferase, glutamine-fructose-6-phosphate aminotransferase, UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase and phosphoglucose mutase.

3. The engineered strain as described in claim 2, characterized in that: The amino acid sequence of the UDP-N-acetylglucosamine C4 isomerase is shown in SEQ ID NO:1; The amino acid sequence of the chondroitin synthase is shown in SEQ ID NO:2; The amino acid sequence of the UDP-glucose dehydrogenase is shown in SEQ ID NO:3; The amino acid sequence of the UTP-glucose-1-phosphate uridine transferase is shown in SEQ ID NO:4; The amino acid sequence of the glutamine-fructose-6-phosphate aminotransferase is shown in SEQ ID NO:5; The amino acid sequence of the UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase is shown in SEQ ID NO:6; The amino acid sequence of the glucose-phosphotransformase is shown in SEQ ID NO:

7.

4. The engineered strain according to any one of claims 1 to 3, characterized in that, The chassis strain expresses 1 to 4 copies of chondroitin synthase.

5. The engineered strain as described in claim 4, characterized in that, The preservation number of the engineered strain is: CGMCC No.36960.

6. An engineered bacterial strain, characterized in that, The preservation number of the engineered strain is: CGMCC No. 36960.

7. The method for constructing the engineered strain according to any one of claims 1 to 6, characterized in that, The enzyme combination was expressed on the chassis strain to obtain the engineered strain.

8. A nucleic acid molecule encoding the enzyme combination in the engineered strain as described in any one of claims 1 to 6 or the engineered strain obtained by the construction method as described in claim 7.

9. The use of the engineered strain as described in any one of claims 1 to 6, the engineered strain obtained by the construction method as described in claim 7, and / or the nucleic acid molecule as described in claim 8 in the preparation of chondroitin.

10. A method for preparing chondroitin, characterized in that, The chondroitin is obtained by inoculating the engineered strain as described in any one of claims 1 to 6 or the engineered strain obtained by the construction method as described in claim 7, fermenting and culturing, collecting the fermentation broth, and obtaining the chondroitin.

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