Vibrio cholerae x511 and its application in synthesizing tetrahydropyrimidine by using chitin

Abstract

This invention belongs to the field of microbial technology, specifically relating to a strain of Vibrio alginate ( Vibrio alginolyticus X511 and its application in the synthesis of tetrahydropyrimidine from chitin. The genome of *Vibrio alginate* X511 described in this invention contains a complete tetrahydropyrimidine synthesis gene cluster and chitin degradation-related genes, enabling direct fermentation production of tetrahydropyrimidine from colloidal chitin, crab shell flakes, or shrimp shell powder without the addition of chitin hydrolase. Fermentation using colloidal chitin as a carbon source and adding a nitrogen source yields high tetrahydropyrimidine production, comparable to or better than the yields of previously reported wild-type strains using soluble carbon sources. Characterization data analysis confirms the strain's ability to directly degrade highly crystalline chitin and natural shrimp shell powder. This invention provides a green and economical new approach for the resource utilization of chitin-based seafood waste, requiring no pretreatment or additional enzymes, and has promising industrial application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a strain of Vibrio alginate X511 and its application in the synthesis of tetrahydropyrimidine using chitin waste. Background Technology

[0002] Tetrahydropyrimidine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is a cyclic amino acid derivative widely found in halophilic bacteria as an osmoprotectant. It stabilizes the structure of biomolecules and mitigates the toxic effects of extreme conditions such as hyperosmolarity, high temperature, and freeze-thaw cycles on cells, biomembranes, proteins, enzymes, and nucleic acids. Due to this unique protective mechanism, tetrahydropyrimidine shows broad application prospects in biotechnology, cosmetics, and pharmaceuticals. Examples include protecting the skin, combating skin aging and cell damage, treating dementia, and treating inflammation. The annual market demand for tetrahydropyrimidine is approximately 15,000 tons, and its high price makes it a typical high-value-added product. The presence of a chiral carbon atom in the tetrahydropyrimidine molecule makes chemical synthesis difficult and costly; currently, it is mainly obtained through biosynthesis.

[0003] Currently, common carbon sources for tetrahydropyrimidine production include glucose, monosodium glutamate (MSG), and sucrose. Among these, metabolically engineered strains using glucose as a substrate have achieved significant breakthroughs in yield. However, the high cost of pure carbon sources remains a bottleneck restricting industrial production, necessitating the search for alternative, inexpensive carbon sources. In recent years, utilizing domestic, agricultural, and industrial waste as low-cost carbon and nitrogen sources for tetrahydropyrimidine production has become an important research direction balancing environmental governance and resource utilization. Shrimp shell waste, as an inexpensive natural renewable biomass resource, has potential value as a fermentation feedstock for tetrahydropyrimidine. However, research on the direct fermentation production of tetrahydropyrimidine using wild-type microorganisms with natural shrimp shells as the sole carbon source has not yet been reported in the literature.

[0004] Chitin is a high-molecular-weight linear polysaccharide composed of N-acetylglucosamine (GlcNAc) units linked by β-1,4 glycosidic bonds. It is the second most abundant biomass resource in nature, second only to cellulose. Only 20–40% of the total weight of shrimp and crab is edible, while the remaining 60–80% is discarded as waste. With the expansion of the global shrimp and crab market, the amount of crustacean waste generated by the food processing industry is increasing daily, with an annual output of approximately 6 to 8 million tons. Traditional physical methods of treating seafood waste, such as incineration, landfill, and ocean dumping, cause serious environmental impacts. Therefore, promoting the resource utilization and high-value transformation of seafood waste has become a research hotspot. Crustacean shells contain 20–40% protein, 20–50% calcium carbonate, and 15–40% chitin. Chitin waste has a huge global reserve, and using inexpensive chitin waste as a raw material to produce tetrahydropyrimidine has broad application prospects. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention first provides a strain of Vibrio alginate X511.

[0006] Furthermore, the present invention also provides the application of Vibrio alginate X511 in the synthesis of tetrahydropyrimidine using chitin (especially the resource utilization of chitin waste).

[0007] Chitin is the second most abundant renewable biomass resource in nature after cellulose. Naturally occurring chitin-rich seafood waste, characterized by its complex structure, high crystallinity, and insolubility, poses a significant barrier to direct microbial transformation, making single-strain systems particularly inefficient in chitin biotransformation. Tetrahydropyrimidine, as a highly compatible solute, has wide applications in the pharmaceutical and cosmetic fields; however, research reports on wild-type strains directly utilizing chitin waste to produce tetrahydropyrimidine are scarce. This application newly discovers that the genome of the marine-derived wild-type Vibrio X511 contains a complete tetrahydropyrimidine synthesis gene cluster (ectABC-ask) and genes related to chitin degradation, enabling direct fermentation synthesis of tetrahydropyrimidine from colloidal chitin, crab shell flakes, and shrimp shell powder without the need for additional chitin hydrolases. Using 50 g / L colloidal chitin as the carbon source and 5 g / L yeast extract as the nitrogen source, the tetrahydropyrimidine yield reached 2.172 g / L after 7 days of fermentation, which is comparable to, or even better than, the yield of previously reported wild-type strains utilizing soluble carbon sources. Furthermore, using highly crystalline crab shell flakes and shrimp shell powder as the sole carbon source, the tetrahydropyrimidine yields reached 0.259 g / L and 0.181 g / L, respectively, with weight losses of 60% and 43.47%, respectively. FESEM, XRD, and FTIR analyses further confirmed the degradation ability of strain X511 for highly crystalline chitin and raw shrimp shell powder. The wild-type Vibrio X511 provided by this invention possesses the ability to directly convert chitin waste into tetrahydropyrimidine, providing a new green pathway for the high-value utilization of chitin biomass resources.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A strain of Vibrio alginate ( Vibrio alginolyticus X511, the Vibrio alginate-depositing strain X511 is deposited at the China General Microbiological Culture Collection Center, with accession number GDMCC No: 59020.

[0010] The present invention also provides a method for producing tetrahydropyrimidine by fermentation of Vibrio alginate X511, which uses chitin as a carbon source, ferments Vibrio alginate X511 in a culture medium containing NaCl, collects the fermentation broth and extracts tetrahydropyrimidine.

[0011] Preferably, the chitinous substance is selected from one or more of colloidal chitin, α-chitin, crab shell, or shrimp shell.

[0012] Preferably, the carbon source is colloidal chitin, and its concentration in the fermentation broth is 10-60 g / L.

[0013] Preferably, the carbon source is α-chitin crab shell flakes or natural shrimp shell powder, and its concentration in the fermentation broth is 5-50 g / L.

[0014] Preferably, the NaCl concentration in the fermentation medium is 4% to 9%.

[0015] Preferably, the fermentation medium further comprises a nitrogen source selected from one or more of ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, yeast extract, or peptone.

[0016] Preferably, the fermentation medium comprises: 50 g / L colloidal chitin, 60 g / L NaCl, 5 g / L yeast extract, 9.7 g / L MgSO4·7H2O, 1 g / L K2HPO4, 2 g / L KCl, 0.2 g / L anhydrous CaCl2, 0.02 g / L FeSO4·7H2O, and pH 7.0.

[0017] Preferably, the fermentation medium comprises: colloidal chitin 10–60 g / L, NaCl 50–70 g / L, (NH4)2SO4 4–6 g / L, MgSO4·7H2O 8–11 g / L, K2HPO4 0.8–1.2 g / L, KCl 1.5–2.5 g / L, anhydrous CaCl2 0.1–0.3 g / L, FeSO4·7H2O 0.01–0.03 g / L, and pH 6.5–8.0.

[0018] Preferably, the fermentation medium comprises: 50 g / L colloidal chitin, 60 g / L NaCl, 5 g / L (NH4)2SO4, 9.7 g / L MgSO4·7H2O, 1 g / L K2HPO4, 2 g / L KCl, 0.2 g / L anhydrous CaCl2, 0.02 g / L FeSO4·7H2O, and pH 7.0.

[0019] Preferably, the carbon source is α-chitinous crab shell flakes, and its concentration in the fermentation broth is 10 g / L.

[0020] Further, the fermentation medium consists of: 10 g / L α-chitinous crab shell flakes, 50 g / L NaCl, 5 g / L (NH4)2SO4, 9.7 g / L MgSO4·7H2O, 1 g / L K2HPO4, 2 g / L KCl, 0.2 g / L anhydrous CaCl2, 0.02 g / L FeSO4·7H2O, and pH 7.0.

[0021] Preferably, the carbon source is natural shrimp shell powder, and its concentration in the fermentation broth is 10 g / L.

[0022] Furthermore, the fermentation medium consists of: 10 g / L natural shrimp shell powder, 50 g / L NaCl, 5 g / L (NH4)2SO4, 9.7 g / L MgSO4·7H2O, 1 g / L K2HPO4, 2 g / L KCl, 0.2 g / L anhydrous CaCl2, 0.02 g / L FeSO4·7H2O, and pH 7.0.

[0023] Preferably, the inoculum size for the fermentation culture is 1% to 5%, and the temperature is 28 to 32 °C.

[0024] This invention also protects the application of Vibrio alginate X511 or the method in the resource utilization of chitin waste or the production of tetrahydropyrimidine.

[0025] Furthermore, the chitinous waste is derived from shrimp or crab shells.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] 1. This invention utilizes Vibrio alginate X511 to produce tetrahydropyrimidine using colloidal chitin, crab shell flakes, and natural shrimp shell powder as carbon sources. Unlike traditional raw materials such as glucose, glutamate, and glycerol, shrimp shell waste is widely available and inexpensive, making this invention innovative in terms of raw materials.

[0028] 2. Better tetrahydropyrimidine yields can be obtained under low salinity (6% NaCl) conditions, with the highest yield of 2.172 g / L achieved in a single batch of shake-flask fermentation;

[0029] 3. Chitin mainly originates from the exoskeletons of marine crustaceans and is abundant in nature, making it an important non-food biomass resource. For example, the seafood aquaculture market in Guangxi has reached a scale of hundreds of billions of yuan, providing a sufficient and sustainable source of raw materials for the development and utilization of chitin. Compared with traditional biomass resources, chitin has significant advantages such as stable raw material supply, diverse sources, and no competition for land with food crops. Attached Figure Description

[0030] Figure 1The molecular basis for the utilization of shrimp shell chitin by strain X511. X511 produces various extracellular hydrolases: (a) chitinase; (b) protease; (c) esterase. (d) Domain composition of chitin-degrading enzymes: SP, signal peptide; GH, glycoside hydrolase family; GH20b, non-catalytic domain of GH20 family β-N-acetylglucosamine enzyme; Big_7, bacterial Ig-like domain; ChtBD3, chitin-binding domain; LPMO, cleaving polysaccharide monooxygenase. (e) Genetic organization of the GlcNAc-utilizing gene cluster: nagA, encoding N-acetylglucosamine-6-phosphate deacetylase; nagB, encoding glucosamine-6-phosphate deaminase; nagC, encoding ROK family transcriptional repressor, which controls the expression of this gene cluster in response to GlcNAc-6-P; nagE, encoding specific PTS permease (EIINag); nagK, encoding N-acetylglucosamine kinase.

[0031] Figure 2 Genetic organization of the tetrahydropyrimidine biosynthesis and degradation gene cluster in strain X511. (a) Comparison of the tetrahydropyrimidine biosynthesis gene cluster. This gene cluster contains core biosynthesis genes: ectA (encoding L-2,4-diaminobutyric acid acetyltransferase), ectB (encoding diaminobutyric acid-2-ketoglutarate transaminase), ectC (encoding L-tetrahydropyrimidine synthase), and ask (encoding aspartate kinase); the betaine / carnitine / choline transporter BCCT is also shown in the figure. (b) Organizational structure of the tetrahydropyrimidine degradation gene cluster. This gene cluster includes the following genes: doeA, encoding tetrahydropyrimidine hydrolase; doeB, encoding N-α-acetyl-L-2,4-diaminobutyric acid deacetylase; doeC, encoding aspartate-4-semialdehyde dehydrogenase; doeD, encoding L-2,4-diaminobutyric acid transaminase; doeX, encoding an Lrp / AsnC family transcription factor (a tetrahydropyrimidine degradation gene regulator); ehuR, encoding a tetrahydropyrimidine / hydroxytetrahydropyrimidine utilization regulator; and TRAP-type transport system-related genes teaA, teaB, and teaC (encoding substrate-binding protein, small transmembrane protein, and large transmembrane protein, respectively).

[0032] Figure 3 The effect of NaCl concentration on the production of tetrahydropyrimidine by strain X511 using colloidal chitin. (a) Biomass and tetrahydropyrimidine yield of strain X511 using colloidal chitin as the sole carbon source under different NaCl concentrations; (b) HPLC chromatograms of tetrahydropyrimidine standard and tetrahydropyrimidine sample extracted from X511.

[0033] Figure 4Fermentation conditions for the production of tetrahydropyrimidine by strain X511 using colloidal chitin were optimized. (a) Time-varying curves of biomass, chitinase activity, and tetrahydropyrimidine yield during fermentation with colloidal chitin as the sole carbon source; (b) Effect of colloidal chitin concentration on tetrahydropyrimidine yield; (c) Effect of nitrogen source type on tetrahydropyrimidine yield.

[0034] Figure 5 The utilization effect of strain X511 on crab shell flakes. (a) Time-varying curves of biomass, chitinase activity, and tetrahydropyrimidine production of strain X511 during the degradation of crab shell flakes; (b) Weight loss rate of crab shell flakes after 7 days of fermentation; (c) FESEM images (5000x magnification) of untreated crab shell flakes and (d) crab shell flakes treated with strain X511 for 7 days; (e) FESEM images (20000x magnification) of untreated and (f) treated crab shell flakes for 7 days; (g) FTIR spectra of untreated (blue line) and treated (red line) crab shell flakes; (h) XRD patterns of untreated (blue line) and treated (red line) crab shell flakes.

[0035] Figure 6 The utilization effect of strain X511 on natural shrimp shell powder. (a) Time-varying curves of biomass, chitinase activity, and tetrahydropyrimidine production of strain X511 during the degradation of shrimp shell powder; (b) Weight loss rate of shrimp shell powder after 11 days of fermentation; (c) Untreated and (d) Shrimp shell flakes treated with strain X511 for 5 days; (e) FESEM images (20,000x magnification) of untreated and (f) shrimp shell powder treated with strain X511; (g) FTIR spectra of untreated (blue line) and shrimp shell powder treated for 11 days (red line); (h) XRD patterns of untreated (blue line) and shrimp shell powder treated for 11 days (red line). Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0038] Example 1: Analysis of Vibrio alginate X511 and its whole genome data

[0039] The applicant isolated a salt-tolerant alginate-degrading Vibrio X511 from decaying Sargassum and performed whole-genome sequencing on it.

[0040] The preservation information is as follows:

[0041] It was deposited with the China General Microbiological Culture Collection Center (CGMCC) on July 31, 2026.

[0042] The accession number is CGMCC No. 59020

[0043] The address for storage is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0044] 1. Strains Isolation

[0045] This strain was collected from Sargassum fusiforme from the waters surrounding Weizhou Island in Beihai, Guangxi. The Sargassum fusiforme samples were mixed thoroughly with a 2% (w / v) NaCl aqueous solution and left at room temperature (around 20℃) until the samples decomposed. 10 mL of the supernatant from the decomposed sample was mixed with 90 mL of the 2% (w / v) NaCl solution, and 2 mL was inoculated into an enrichment medium. The medium was then cultured in shake flasks at 30℃ for 3 days. The enriched bacterial solution was serially diluted and plated onto screening medium plates, incubated at 30℃ for 2 days, and the colony morphology was observed. Single colonies with good growth were selected for streak purification until a pure culture was obtained. The purified colonies were stained with 10% (w / v) hexadecylpyridine for initial screening, and the strain with the largest ratio of hydrolysis zone to colony diameter was selected and stored at low temperature for later use. The enrichment and separation media were both sodium alginate medium (1L): (NH4)2SO4 5g, K2HPO4 1g, NaCl 15g, MgSO4 1g, H2SO4 7H2O 0.01g, sodium alginate 6g, agar 17g, pH 7.5.

[0046] 2. Strain identification

[0047] Strain X511 forms regular, round colonies on solid selection plates. After growth exceeding 48 hours, the colonies change from milky white to pale yellow, with a raised center, smooth and moist surface, and neat edges, making them easy to pick up. 16S rDNA identification was performed according to literature. Bacterial DNA was extracted from cells cultured to the logarithmic growth phase according to the kit instructions, and the 16S rDNA was amplified using the following primers:

[0048] F-Primer: 5'-AGAGTT TGATCC TGG CTCAG-3';

[0049] R-Primer: 5'-GGTTACCTTGTTACGACT T-3'.

[0050] The recovered PCR products were sequenced by Beijing Aoke Dingsheng Biotechnology Co., Ltd. The obtained 16S rDNA fragment was 1574 bp in length (as shown in SEQ ID NO: 1), with a (G+C) content of 56%. The sequence was uploaded to NCBI for homology comparison. Strain X511 showed 99% similarity to multiple strains of the genus Vibrio. Based on the strain's morphological and physiological / biochemical characteristics, it was identified as *Vibrio alginate* and named... Vibrio alginolyticus X511.

[0051] Plate enzyme activity assay results showed that strain X511 also has the ability to utilize chitin, protein, and fat. Figure 1 This lays the enzymatic foundation for the direct biotransformation of complex substrate shrimp shell waste. Based on whole-genome data analysis, nine chitin-degrading enzyme encoding genes were annotated in the X511 genome, including four GH18 family chitinases, one GH19 family chitinase, one GH20 and one GH3 family NAGase (β-N-acetylglucosaminidase), and two AA10 family cleaving polysaccharide monooxygenases LPMO (…). Figure 1 d). The amino acid sequences of five chitinases, one NAGase, and two LPMOs all contain one or two chitin-binding domains (ChtBD3). These domains facilitate the anchoring of enzyme proteins to the chitin surface and assist in the efficient degradation of insoluble chitin by catalytic domains. LPMOs are copper-dependent enzymes that oxidatively cleave the C1-H bonds in crystalline chitin, disrupting the substrate's surface topology and generating new chain ends, thus improving the accessibility of chitinases. Signal peptide prediction results showed that, except for N-acetylglucosidase (GM003638) of the GH3 family, the amino acid sequences of the remaining chitin-degrading enzymes encoded by X511 all contain a potential signal peptide. This finding indicates that these chitin-degrading enzymes are secretory proteins that can be secreted extracellularly to degrade chitin into soluble oligosaccharides or monosaccharides, which are then taken up and utilized by the cell. Strain X511 possesses a complete and synergistic chitin decomposition system, which can efficiently decompose highly crystalline chitin substrates that are difficult to degrade.

[0052] Several genes associated with N-acetylglucosamine (GlcNAc) utilization were identified in the X511 genome. The core genes nagC1, nagA, and nagE were tightly clustered together, while nagK, nagC2, and nagB were dispersed in different regions. Figure 1e). Unlike the compact structure of Nag regulation in *E. coli*, the genetic structure of GlcNAc catabolism in *Vibrio* often exhibits a more dispersed and complex pattern. This dispersed gene structure is similar to that of other *Vibrio* species, manifested in the presence of NagE-NagAC as operon 000, while NagB is not present in the same operon. These core genes constitute a complete GlcNAc utilization pathway, laying the metabolic foundation for the strain to utilize chitin and convert its carbon skeleton into high-value compounds.

[0053] Example 2: Identification of tetrahydropyrimidine synthesis and degradation gene clusters in strain X511

[0054] Analysis and identification confirmed that X511 is a halophilic Vibrio bacterium capable of surviving in high-salt environments, implying the presence of an osmotic regulatory system in its genome that enables microorganisms to adapt to high-salt conditions. We identified a complete tetrahydropyrimidine synthesis gene cluster in its genome, primarily including the core genes ectA, ectB, ectC, and ect-ask (…). Figure 2 a). Furthermore, a complete tetrahydropyrimidine catabolism gene cluster, including the doeABCDX, teaA, and eutRBC genes, was identified in a separate region of the X511 genome. Figure 2 b) These genes encode tetrahydropyrimidine hydrolase (DoeA), the TRAP-type transport system (TeaA), and regulatory factors (EutR / EutC), which play important roles in the catabolic metabolism and transport of tetrahydropyrimidine, together forming a complete tetrahydropyrimidine catabolic pathway.

[0055] Example 3: Effect of NaCl concentration in culture medium on the production of tetrahydropyrimidine by strain X511 using colloidal chitin

[0056] To evaluate the effect of NaCl concentration on tetrahydropyrimidine synthesis, fermentation media containing 1%, 3%, 5%, 6%, 7%, 8%, and 9% NaCl (w / v) were prepared using colloidal chitin (10 g / L) as the sole carbon source. The initial pH of the media was adjusted to 7.0 with 1 M NaOH or HCl, and fermentation was carried out for 7 days.

[0057] Detection of bacterial biomass: Colloidal chitin is an insoluble polymer, so to exclude its influence on OD... 600To mitigate interference, a two-step centrifugation method was used to process the samples. The fermentation broth and the uninoculated control medium were centrifuged at 500 rpm for 2 min to precipitate the insoluble colloidal chitin. The supernatant containing bacterial cells and the control supernatant were carefully transferred to new centrifuge tubes, respectively. The control supernatant served as a blank control. The supernatant of the experimental group was further centrifuged at 5000 rpm for 2 min, and the bacterial precipitate was collected. The bacterial cells were washed twice with 0.9% NaCl solution, resuspended in an equal volume of 0.9% NaCl solution, appropriately diluted, and the OD was measured. 600 value.

[0058] Tetrahydropyrimidine extraction: Intracellular tetrahydropyrimidine was released using a bacterial milking method. 1 mL of fermentation broth was centrifuged at 12000 rpm for 5 min at 4°C, the supernatant was discarded, and the cell pellet was collected. An equal volume of 1% NaCl solution was added to the cell pellet for hypotonic treatment, and the mixture was shaken at 200 rpm for 20 min at 30°C. The pellet was then centrifuged at 12000 rpm for 2 min at 4°C, and the supernatant was the released tetrahydropyrimidine sample.

[0059] Determination of tetrahydropyrimidine content:

[0060] HPLC (High Performance Liquid Chromatography) was used for detection and identification. A Waters e2695 HPLC system equipped with a 2998 PDA detector was used for detection under the following conditions:

[0061] a) Liquid chromatography column: Horizon AR C18 column (column length 250 mm, column inner diameter 4.6 mm, packing particle size 5 μm).

[0062] b) Mobile phase: 2% acetonitrile: 98% water;

[0063] c) Flow rate: 0.5 mL / min;

[0064] d) Column temperature: 30 ℃;

[0065] e) Injection volume: 10 μL;

[0066] f) Detection wavelength: 210 nm.

[0067] Example 4: Preliminary assessment of the potential of strain X511 to synthesize tetrahydropyrimidine using colloidal chitin.

[0068] The seed culture preparation and fermentation culture method were the same as in Example 1. The fermentation culture medium components were: colloidal chitin 10 g / L, (NH4)2SO4 5 g / L, NaCl 50 g / L, K2HPO4 1 g / L, MgSO4·7H2O 9.7 g / L, KCl 2 g / L, and anhydrous CaCl2 0.2 g / L. Samples were taken every day, and fermentation was carried out for 11 days. The aim was to reveal the dynamic mechanism of tetrahydropyrimidine synthesis by strain X511 using colloidal chitin.

[0069] Different concentrations of colloidal chitin (10, 30, 50, and 60 g / L) were added to the fermentation medium to determine the optimal concentration for tetrahydropyrimidine synthesis. Based on this concentration, 5 g / L of different nitrogen sources (ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium oxalate, ammonium carbonate, yeast extract, and peptone) were added to evaluate the effect of these different nitrogen sources on tetrahydropyrimidine synthesis by strain X511. The culture period for all experiments was 7 days. Cell biomass detection, tetrahydropyrimidine release from cells, and detection methods were the same as in Example 1.

[0070] Example 5: Preliminary investigation into the ability of strain X511 to synthesize tetrahydropyrimidine directly from highly crystalline α-chitin.

[0071] The seed culture preparation and fermentation culture method were the same as in Example 1. Fermentation medium components: crab shell flakes 10 g / L, (NH4)2SO4 5 g / L, NaCl 50 g / L, K2HPO4 1 g / L, MgSO4·7H2O 9.7 g / L, KCl 2 g / L, anhydrous CaCl2 0.2 g / L. Samples were taken every 2 days, and fermentation was carried out for 7 days. The cell biomass detection, tetrahydropyrimidine release from cells, and detection methods were the same as in Example 1.

[0072] After fermentation, crab shell residues inoculated with X511 and those not inoculated with X511 were collected separately. The residues were resuspended in distilled water, centrifuged at 500 rpm for 5 min, the supernatant was removed, and the residues were collected. This process was repeated three times. The washed residues were then freeze-dried, and their dry weight was determined.

[0073] Weight loss rate (%) = [(W0 - W1) / W0] × 100%, where W0 is the dry weight of the substrate without inoculum and W1 is the dry weight of the fermentation substrate residue.

[0074] Chitinase activity assay: Extracellular chitinase activity was determined using the DNS method. The fermentation supernatant (after removing cell pellet) was used as the crude enzyme solution, and 50 mM PBS (pH 6.0) buffer containing 1% colloidal chitin was used as the substrate. 200 μL of crude enzyme solution was mixed with 500 μL of substrate and reacted in a 40 ℃ water bath for 30 min. The reaction was terminated by adding 750 μL of DNS, followed by boiling in a 100 ℃ water bath for 10 min, centrifugation at 5000 rpm for 2 min, and the supernatant was used for OD measurement. 540nm The inactivated crude enzyme solution was used as a blank control. The amount of enzyme required to release 1 μmol of β-N-acetylglucosamine per minute is defined as one enzyme activity unit (U).

[0075] Field emission scanning electron microscopy (FESEM) analysis: freeze-dried substrate residue was sputtered with gold at 40 mA for 60 s and then photographed by FESEM. Microscopic images were acquired at magnifications of 5000× and 10000× under accelerating voltages of 10.0–20.0 kV.

[0076] Fourier Transform Infrared (FTIR) Analysis: Spectral analysis was performed using a Fourier Transform Infrared Spectrometer. Spectra were acquired in the wavenumber range of 4000–400 cm⁻¹, with a resolution of 2 cm⁻¹. -1 .

[0077] X-ray diffraction (XRD) patterns: X-ray diffraction (XRD) patterns of the substrate residue were obtained using an X-ray diffractometer (ULTIMA IV, Rigaku, Japan) at an operating voltage of 40 kV and a current of 40 mA. Patterns were acquired within a diffraction angle 2θ range of 5°–60°, with a step size of 0.02° and a scan rate of 5° / min.

[0078] Example 6: Investigating the ability of X511 to directly produce tetrahydropyrimidine from shrimp shell waste.

[0079] The seed culture preparation and fermentation culture method were the same as in Example 1. Fermentation culture medium components: 10 g / L natural shrimp shell powder, 5 g / L (NH4)2SO4, 50 g / L NaCl, 1 g / L K2HPO4, 9.7 g / L MgSO4·7H2O, 2 g / L KCl, and 0.2 g / L anhydrous CaCl2. Samples were taken every 2 days, and fermentation was carried out for 11 days.

[0080] The methods for detecting bacterial biomass, cellular release of tetrahydropyrimidine, and their detection are the same as in Example 1. The methods for detecting chitinase activity, FESEM, FTIR, and XRD are the same as in Example 5.

[0081] Effect verification

[0082] Experiment Example 1: Effect of NaCl concentration in culture medium on the production of tetrahydropyrimidine by strain X511 using colloidal chitin

[0083] Strain X511 used colloidal chitin (10 g / L) as its sole carbon source, and its tetrahydropyrimidine production exhibited a salinity-dependent pattern. Under low salinity conditions (1-3% (m / v) NaCl), tetrahydropyrimidine was almost undetectable in the samples. The yield increased sharply at a NaCl concentration of 5%, reaching its highest level of 0.489 g / L at a NaCl concentration of 6%. At higher NaCl concentrations (9%), the yield decreased sharply to only 0.107 g / L. Figure 3 a). HPLC results showed that the retention time of the main peak in the sample was consistent with that of the tetrahydropyrimidine standard, confirming that the final product synthesized by X511 under high salt stress was indeed tetrahydropyrimidine (a). Figure 3 (b) This result is consistent with genomic analysis. No ectD homolog was detected in the X511 genome. The absence of this gene indicates that the synthetic pathway of strain X511 terminates at tetrahydropyrimidine, rather than its hydroxylated derivative. In summary, the optimal NaCl concentration for inducing tetrahydropyrimidine synthesis in strain X511 is 6%.

[0084] Experimental Example 2: Preliminary assessment of the potential of strain X511 to synthesize tetrahydropyrimidine using colloidal chitin.

[0085] To reveal the dynamics of tetrahydropyrimidine synthesis by strain X511 using colloidal chitin and to explore the temporal coupling relationship between chitin degradation and product synthesis, time-series analysis was performed on key indicators during fermentation. The results showed that the tetrahydropyrimidine yield reached a maximum of 0.423 g / L at 3 days, decreased to 0.364 g / L at 5 days, and gradually decreased with increasing fermentation time. Figure 4 a) This phenomenon may be related to the tetrahydropyrimidine degradation gene cluster present in the genome of strain X511. We speculate that after 3 days of fermentation, colloidal chitin is gradually consumed, leading to insufficient carbon source supply. To maintain basal cellular metabolism, the strain may initiate a degradation pathway, decomposing and utilizing the previously accumulated tetrahydropyrimidine as a backup carbon or nitrogen source, thus resulting in a decrease in intracellular tetrahydropyrimidine content. Meanwhile, chitinase activity remains relatively stable, within the range of 0.70–0.79 U / mL. Interestingly, although tetrahydropyrimidine production decreases, cell biomass (OD) remains relatively stable. 600 However, it continues to increase ( Figure 4a) The results indicate that, under the test conditions, the biosynthesis of tetrahydropyrimidine was not tightly coupled with cell growth. It is speculated that the rapid accumulation of tetrahydropyrimidine in the early stages of fermentation is an immediate strategy employed by the strain to cope with osmotic stress. However, in the later stages of fermentation, carbon source depletion may reverse metabolic regulation and activate the tetrahydropyrimidine degradation pathway, leading to a shift in carbon flow from product synthesis to biomass accumulation. These results demonstrate that strain X511 possesses the ability to efficiently coordinate substrate degradation, carbon source conversion, and osmotic adaptation, exhibiting unique metabolic advantages in utilizing complex carbon sources.

[0086] The types and balance of carbon and nitrogen sources are crucial for the synthesis of tetrahydropyrimidine. To further explore the potential of X511 in synthesizing tetrahydropyrimidine using colloidal chitin, we optimized the concentration of colloidal chitin and the types of nitrogen sources in the culture medium. Figure 4 bc). The concentration of colloidal chitin in the culture medium was 10-50 g / L. The yield of tetrahydropyrimidine was positively correlated with the carbon source concentration, gradually increasing with increasing carbon source concentration. When the carbon source concentration was 50 g / L, the yield of tetrahydropyrimidine reached its peak at 0.760 g / L, at which point the biomass also reached its maximum. Therefore, the optimal colloidal chitin concentration was determined to be 50 g / L. When the carbon source concentration reached 60 g / L, the growth of strain X511 began to be inhibited, the biomass decreased, and the yield of tetrahydropyrimidine decreased to 0.697 g / L. Figure 4 b).

[0087] Furthermore, the fermentation effects of seven nitrogen sources (ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium oxalate, ammonium carbonate, yeast extract, and peptone) were investigated to determine the nitrogen source most favorable for strain X511 to synthesize tetrahydropyrimidine using colloidal chitin. Figure 4 c). The results showed that the highest tetrahydropyrimidine yield (2.172 g / L) was achieved when yeast extract was used as the nitrogen source, significantly higher than other nitrogen sources, and the biomass was also significantly higher than other treatment groups. This indicates that adding yeast extract to the culture medium is beneficial for tetrahydropyrimidine synthesis. Among inorganic nitrogen sources, ammonium phosphate was the most effective, with a tetrahydropyrimidine yield of 1.001 g / L; ammonium sulfate and ammonium chloride yields were 0.802 g / L and 0.826 g / L, respectively, with no significant difference between the two. These results indicate that the optimal colloidal chitin concentration for tetrahydropyrimidine synthesis by strain X511 is 50 g / L, and the optimal nitrogen source is yeast extract.

[0088] Experiment Example 3: Investigating the ability of strain X511 to synthesize tetrahydropyrimidine directly from highly crystalline α-chitin.

[0089] Alpha-chitin is the most common and widely studied form, characterized by its close-packed crystal structure formed by the antiparallel alignment of its molecular chains. Unlike colloidal chitin, which has lower crystallinity and a looser morphology, commercially available crab shell flakes, although deproteinized and decalcified, still retain their original highly crystalline α-chitin crystal form and dense, sheet-like morphology, making them a more difficult carbon source for microbial decomposition and utilization. Using commercially available crab shell flakes as the sole carbon source, strain X511 achieved a maximum tetrahydropyrimidine yield of 0.259 g / L after 5 days of fermentation. After 7 days, the tetrahydropyrimidine yield decreased slightly to 0.242 g / L. After 5 days of fermentation, the biomass of X511 entered a plateau phase, with OD... 600 It reached its maximum value, consistent with the yield change trend. Chitinase activity remained at 0.539-0.603 U / mL during the 3-7 day fermentation cycle. Figure 5 a) indicates that the strain maintained stable chitin degradation activity throughout the fermentation process. After fermentation, the weight loss of the crab shell flakes reached 60% ( Figure 5 (b) This further confirms the strain's effective degradation of crab shell flakes. Although commercial crab shell flakes have high crystallinity and are difficult to degrade, strain X511 can still accumulate tetrahydropyrimidine within 5 days, indicating that it possesses a highly efficient chitin-degrading enzyme secretion system and a strong ability to attack highly crystalline chitin. This characteristic demonstrates the good potential of X511 to directly utilize chitin waste to produce high-value products.

[0090] Field emission scanning electron microscopy (FESEM) was used to observe the changes in its microstructure. Untreated α-chitin exhibited a dense and ordered stacked layered structure with a relatively intact and smooth surface. Figure 5 c, e), and after 7 days of X511 treatment, the originally highly crystalline and dense structure developed cracks, the surface broke and formed fragments, and obvious erosion holes could be observed, forming a rough area (c, e). Figure 5 d, f).

[0091] The FTIR spectra of X511-treated and untreated crab shell slices showed the same characteristic peaks ( Figure 5 g), typical absorption peaks of α-chitin (897, 1318, 1560, 1659 cm⁻¹) can be observed. -1 (etc.), indicating that the treatment with this strain did not alter the main chemical composition of the crab shell slices. In the FTIR spectrum of the crab shell slices, at 3440 cm⁻¹... -1 The characteristic peaks at 2929 cm⁻¹ correspond to the O–H and C–H stretching vibrations, respectively, in the FTIR spectrum of the crab shell. At 3440 cm⁻¹... -1 The characteristic peaks at 2929 cm⁻¹ correspond to O–H and C–H stretching vibrations, respectively. 1700–1600 cm⁻¹ -1The nearby absorption peaks are attributed to the stretching vibrations of the C=O band of amide I and intramolecular and intermolecular hydrogen bonds, while the 1025 cm⁻¹ peak... -1 The peak at 3261 cm⁻¹ corresponds to the asymmetric stretching vibration of C–O–C on the sugar ring. Furthermore, the peak at 3261 cm⁻¹... -1 The peak at 3105 cm⁻¹ is attributed to the N–H stretching vibration constrained by the intermolecular C(2)-NH⋯O=C(7) hydrogen bond, and the peak at 3105 cm⁻¹ is attributed to the O–H stretching vibration constrained by the intermolecular C(6)-OH⋯H–O–C(6′) hydrogen bond. After treatment with X511 for 7 days, the peak at 3105 cm⁻¹ is associated with the O–H stretching vibration (~3440 cm⁻¹). -1 ), C–H (2929 cm) -1 ) and N–H (~3261 cm -1 The peak intensities related to stretching vibrations were significantly increased, while the C=O band of amide I (~1659 cm⁻¹) was also significantly enhanced. -1 ) and glycosidic bonds C–O–C (~1026 cm) -1 The peak intensity of the chitin was also significantly enhanced. These changes indicate that the dense hydrogen bond network between chitin molecules (including types such as C(2)-NH⋯O=C(7) and C(6)-OH⋯H–O–C(6′)) is disrupted, exposing free hydroxyl (-OH) and amino (-NH) groups and increasing vibrational freedom. This spectral feature of significantly enhanced infrared absorption peaks corresponding to hydroxyl and carbonyl groups is consistent with the spectral response of BpLPMO10 to chitin substrates, suggesting that strain X511 may employ a similar LPMO-mediated oxidative cleavage strategy.

[0092] The crystallinity of chitin affects its degradation efficiency by glycoside hydrolases. Therefore, reducing crystallinity through physical, chemical, and microbial pretreatment methods is a key mechanism for improving chitin accessibility and hydrolysis efficiency. XRD spectroscopy shows strong diffraction peaks in crab shell chitin around 9° and 20°. Figure 5 h), which is a characteristic peak of α-chitin. Compared with the control group, after 7 days of X511 treatment, the XRD diffraction peak signal intensity of crab shell chitin decreased significantly, indicating that the crystalline region of crab shell chitin was destroyed and the crystallinity decreased. Figure 5 h). FESEM, FTIR, and XRD analyses collectively indicate that X511 can effectively act on the crystalline region of α-chitin, causing its ordered structure to be destroyed, exposing more amorphous regions, and improving the degradation efficiency of glycoside hydrolases.

[0093] Experiment Example 4: Preliminary Investigation into the Ability of X511 to Produce Tetrahydropyrimidine Directly from Shrimp Shell Waste

[0094] Chitin in shrimp and crab shells can be completely degraded by microorganisms through the synergistic action of chitinase, chitosanase, and lysin-degrading polysaccharide monooxygenase (LPMO). However, the complex structure and chemical composition of shrimp shell waste directly limit the efficiency of microbial biotransformation. Studies have shown that the shell of *Fenneropenaeus chinensis* (Chinese prawn, giant prawn) contains 40.64 ± 0.52% protein, 31.44 ± 0.01% ash, and 15.15 ± 0.69% chitin. Strain X511 exhibits chitinase, protease, and esterase activities. Figure 1 This invention utilizes strain X511 with untreated natural shrimp shell powder as the sole carbon source. After 3 days of fermentation, the tetrahydropyrimidine yield reached a peak of 0.181 g / L. However, after 5 days of fermentation, the yield sharply decreased to 0.06 g / L, and gradually decreased with increasing fermentation time. Chitinase activity remained relatively stable at 0.467-0.522 U / mL during the 3-11 day fermentation cycle. The strain biomass (OD) is also shown. 600 The value reaches its maximum of 1.78 after 7 days of fermentation. Figure 6 a) Bacterial reproduction and growth lag behind the biosynthesis of tetrahydropyrimidine, which is consistent with the timing characteristics of X511's synthesis of tetrahydropyrimidine using colloidal chitin as a substrate. After fermentation, the shrimp shell powder showed a weight loss of 43.47% ( Figure 6 (b) Meanwhile, after 5 days of fermentation, most of the shrimp shells were observed to disintegrate into fragments. Figure 6 (cd), these pieces of evidence confirm that this strain effectively degrades natural shrimp shell biomass. In conclusion, strain X511 can directly synthesize tetrahydropyrimidine from natural shrimp shell powder, with a yield of 0.181 g / L after 3 days of fermentation.

[0095] To further elucidate its degradation mechanism on shrimp shell waste, the morphological changes of shrimp shell powder before and after treatment were observed using FESEM. The results showed that the surface of shrimp shells untreated with strain X511 was smooth and even. Figure 6 e) The shrimp shells treated with strain X511 showed severe erosion, numerous pores, and observed aggregation of spherical particles and flower-like crystal structures. Figure 6 f). This flower-like crystal structure is remarkably similar to the three-dimensional flower-like structure reported in the literature, which is formed by the self-assembly of hydroxyapatite nanosheets under microwave hydrothermal conditions, suggesting that it may be biogenic hydroxyapatite. FESEM results indicate that strain X511 can effectively disrupt the crystal structure of chitin, leading to the exposure of more disordered regions. This process also exposes or recombines inorganic mineral crystals and may induce the formation of new mineral crystals.

[0096] The FTIR spectra of X511-treated and untreated shrimp shell powder showed significant differences, with several characteristic peaks observed in the control group, such as 1152 cm⁻¹. -1 1205 cm -1 1316 cm -1 1379 cm -1 The presence of this substance in the X511-treated samples indicates a substantial change in the structure of the shrimp shell powder. Figure 6 g). FTIR and XRD analyses further revealed the changes in organic and inorganic matter during shrimp shell degradation. FTIR results showed that after shrimp shell powder was treated with strain X511 for 11 days, the COC stretching vibration peak attributable to chitin glycosidic bonds (1026 cm⁻¹) was observed. -1 ), Amide I band C=O stretching vibration peak (1659 cm⁻¹) -1 ), C–H (2929cm) -1 The absorption intensities of the stretching vibration peaks were significantly reduced, and the 1540 cm⁻¹ absorption peak associated with proteins in α-chitin disappeared, indicating that both chitin and proteins in the shrimp shell were effectively degraded. Simultaneously, the absorption peaks at 1409 cm⁻¹, attributed to carbonate CO vibration, and calcite CO₃²⁻ also showed significant reductions. 2- 874 cm -1 The absorption peak intensity was significantly enhanced. Figure 6 The result (g) reflects the relative enrichment of inorganic salts in shrimp shell powder after the removal of organic matter by strain X511. XRD analysis further verified this characteristic, observing characteristic chitin diffraction peaks at 9.13° and 19.19° in untreated shrimp shell powder, and diffraction peaks attributed to calcite at 39.59°, 43.34°, and 47.69°. Figure 6 h). After 11 days of X511 treatment, the chitin diffraction peak disappeared, while the calcite diffraction peak narrowed. The diffraction peak signal at 29.53°, attributed to quartz (SiO2, ICDD No. 00-070-3755), was significantly enhanced, indicating that the strain effectively degraded the organic components in the shrimp shell, leading to a relative enrichment of crystalline inorganic matter (calcite and quartz). Notably, the shrimp shell powder treated with X511 showed new narrow and sharp diffraction peaks at 31.81° and 45.48°, which coincided with the characteristic peaks of the hydroxyapatite phase, suggesting that biogenic hydroxyapatite may have formed in situ during the degradation process.

[0097] Table 1. Comparison of tetrahydropyrimidine production with other wild-type bacteria

[0098] In recent years, the production of tetrahydropyrimidine from inexpensive biomass hydrolysates has attracted widespread attention, but the yield of wild-type strains is usually low. As shown in Table 1, reported high-yielding wild-type strains typically use soluble carbon sources such as glucose, glutamate, and biomass hydrolysates. Most wild-type microorganisms can effectively utilize soluble carbon sources such as glucose to synthesize tetrahydropyrimidine, but their ability to utilize polysaccharide substrates such as chitin and cellulose is poor. To date, there are few research reports on the direct production of tetrahydropyrimidine from chitin waste by wild-type strains. This invention uses insoluble polymer colloidal chitin, crab shell (highly crystalline α-chitin), and shrimp shell powder as substrates, and can directly convert these three chitin substrates into tetrahydropyrimidine without the addition of chitin hydrolase. Among them, X511, using colloidal chitin as substrate and 5 g / L yeast powder as nitrogen source, achieved a tetrahydropyrimidine yield of up to 2.172 g / L after 7 days of fermentation. This yield not only validates the feasibility of this pathway but also matches the yield levels reported in most wild-type fungi utilizing soluble carbon sources such as glucose and sucrose, and even surpasses some previously reported wild-type strains (Table 1). Furthermore, X511 can directly synthesize tetrahydropyrimidine from highly crystalline α-chitin and untreated shrimp shell powder, demonstrating the unique advantages of this strain in the high-value utilization of chitin waste. These characteristics indicate that strain X511 has the potential to serve as a microbial cell factory for the production of tetrahydropyrimidine from chitin waste.

[0099] Obviously, the above embodiments of the present invention are merely examples to clearly illustrate the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. A strain of Vibrio alginate ( Vibrio alginolyticus X511, characterized in that, The Vibrio alginate X511 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No: 59020.

2. A method for producing tetrahydropyrimidine by fermentation using Vibrio alginate X511 as described in claim 1, characterized in that, Using chitin as a carbon source, Vibrio alginate X511 was fermented in a culture medium containing NaCl. The fermentation broth was collected and tetrahydropyrimidine was extracted.

3. The method according to claim 2, characterized in that, The chitinous substance is selected from one or more of colloidal chitin, α-chitin, crab shell, or shrimp shell.

4. The method according to claim 2, characterized in that, The carbon source is colloidal chitin, α-chitin crab shell flakes, or natural shrimp shell powder, and the concentration of the carbon source in the fermentation broth is 10–60 g / L.

5. The method according to claim 2, characterized in that, The NaCl concentration in the fermentation medium is 4% to 9%.

6. The method according to claim 2, characterized in that, The fermentation medium also contains a nitrogen source selected from one or more of ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, yeast extract, or peptone.

7. The method according to claim 2, characterized in that, The inoculum size for the fermentation culture is 1%–5%, and the temperature is 28–32 °C.

8. The application of Vibrio alginate X511 as described in claim 1 or the method as described in any one of claims 2-7 in the resource utilization of chitin waste or the production of tetrahydropyrimidine.

9. The application according to claim 8, characterized in that, The chitinous waste is derived from shrimp or crab shells.

Images