A strain of *Yersinia lipolytica* that produces crocin and its application
By constructing a high-yield crocin-producing Yersinia lipolytica strain through genetic engineering, the problems of low efficiency and high cost in the production of crocin in existing technologies have been solved, realizing the industrialization potential of efficient synthesis of crocin-1.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- OCEAN UNIV OF CHINA
- Filing Date
- 2026-04-07
- Publication Date
- 2026-06-30
AI Technical Summary
Existing technologies are insufficient for the efficient production of crocin. Chemical synthesis is complex and polluting, plant extraction is costly and unstable, and microbial fermentation is difficult to construct. There are no reports of high-yield engineered Yersinia lipolyticis.
By constructing recombinant engineered bacteria through genetic engineering, a high-yield crocin-producing Yarrowia lipolytica strain, Yarrowia lipophila OUC-Cro13-EL8GGP, was screened out. Fermentation conditions were optimized and intermediate strains were modified multiple times to overexpress key enzymes and regulate metabolic nodes, thereby achieving efficient synthesis of crocin.
Crocin-1 was successfully synthesized efficiently in engineered Yersinia lipolytica, with a yield of 21.3 mg/L, demonstrating potential for industrial production and solving the problems of high production cost and low efficiency in existing technologies.
Smart Images
Figure CN122303058A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a strain of Yersinia lipolytica that produces crocin and its applications, belonging to the field of genetic engineering technology. Background Technology
[0002] Crocin is a natural carotenoid primarily found in the styles of saffron (Crocus sativus), giving it its vibrant color. Crocin possesses potent antioxidant properties, effectively scavenging free radicals in the body, protecting cells from oxidative damage, and slowing the aging process. It has significant protective effects on the skin, eyes, and nervous system, particularly in improving vision and slowing aging. Studies have also found that crocin has multiple health benefits, including anti-inflammatory, anti-tumor, and antidepressant effects, helping to alleviate chronic inflammation and promote mental well-being. Crocin has five derivatives based on the number of sugar moieties, with crocin-1 being the most abundant, having the most sugar moieties, and being the most water-soluble in plants. Since humans cannot synthesize crocin, it must be obtained through food or supplements. The safety of crocin has been confirmed by numerous studies, and it is currently widely used in pharmaceuticals, health products, cosmetics, and food additives, becoming an important natural health-promoting ingredient.
[0003] Currently, crocin is produced through two methods: chemical synthesis and plant extraction. Chemical synthesis involves complex reaction steps and requires stringent reaction conditions, resulting in high energy consumption and pollution. The synthesized product often contains trace byproducts, and its purity and safety may not meet natural standards. Furthermore, it may lack the synergistic active ingredients found in natural crocin, affecting its application efficacy. Plant extraction is similarly limited. Saffron plants have a long growth cycle, extremely low yields, and are dependent on specific climate and soil conditions, leading to unstable raw material supply and high costs. During extraction, crocin is easily oxidized and degraded, resulting in low extraction rates. It may also be accompanied by pesticide residues and heavy metal contamination. Large-scale production faces challenges such as high resource consumption and environmental pressure, making it difficult to meet large-scale market demand.
[0004] With the development of synthetic biology and genetic engineering, microbial fermentation has gradually become an effective method for producing natural products. *Yersinia lipolytica* is a generally recognized safe (GRAS) microorganism, rich in lipids and acetyl-CoA, making it an excellent substrate for carotenoid production. However, the difficulty in constructing recombinant engineered strains that produce crocin lies in the complexity of the crocin synthesis pathway. Recombining the expression genes of all enzymes involved in the pathway into the engineered strain requires multiple transformations, which is difficult and has a low success rate. Furthermore, after introducing heterologous genes to enable the engineered strain to synthesize the product, the intermediate strains need to undergo multiple modifications and screenings to obtain high-yield engineered strains. Currently, there are no reports on constructing engineered *Yersinia lipolytica* strains that produce crocin. Summary of the Invention
[0005] In response to the above-mentioned prior art, the present invention constructs recombinant engineered bacteria through genetic engineering and screens a strain of Yersinia lipolytica that produces high levels of crocin.
[0006] This invention is achieved through the following technical solution: A strain of *Yersinia lipolytica* that produces crocin was classified and named... Yarrowialipolytica OUC-Cro13-EL8GGP, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20252999, deposit date: December 24, 2025.
[0007] The biological characteristics of the *Yarrowia lipolytica* strain that produces crocin are as follows: it is an aerobic yeast that, when cultured on YPD agar, produces red colonies with wrinkles and hairy edges. The cells exhibit three morphologies: yeast-like, hyphal-like, and pseudohyphae-like.
[0008] The application of *Yersinia lipolytica*, a crocin-producing yeast, in the preparation of crocin. Specifically, *Yersinia lipolytica* is cultured to extract crocin.
[0009] Furthermore, the specific method of cultivation can be as follows: the seed liquid of *Yarrowia lipolytica* producing crocin is inoculated into YPD medium at an inoculation amount of 2%, and inducing fermentation in a shaker at 30°C and 220 rpm for 60 hours, and then inducing fermentation in a shaker at 20°C and 220 rpm for 60 hours.
[0010] Furthermore, the specific extraction method can be as follows: take the fermentation broth of cultured Yeast lipolyticis, centrifuge and discard the supernatant, place it in a grinding tube, add the extractant, and then add zirconium oxide grinding beads for grinding; centrifuge, filter the supernatant to remove impurities, and obtain the extract, which contains crocin; the extractant is composed of methanol and dimethylformamide (DMF) in a volume ratio of 7:1.
[0011] The method for constructing the lipophilic yeast strain of the present invention, which produces crocin, is as follows: using *Yarrowia lipophilia*... Yarrowialipolytica OUC-Zea8-A7ZGL (this strain is a high-yielding zeaxanthin-producing Yersinia lipolytica strain constructed and screened in the inventor's laboratory in the early stage, its accession number is CCTCCNO: M 20222062, and its accession information, accession certificate and other details are recorded in CN 116396877 A) was used as the starting strain. Using pMT015 as the expression vector, it was sequentially transformed into the SUMO tag and carotenoid cleavage dioxygenase CsCCD gene fusion expression cassette and the phytoene synthase / lycopene cyclase bifunctional enzyme gene. carRP β-Carotene hydroxylase gene crtZSUMO tags with subcellular organelle localization signal peptides and carotenoid cleaving dioxygenases CsCCD Gene fusion expression cassette, primary glycosyltransferase gene UGT75L6 and secondary glycosyltransferase genes UGT94E5 After each transformation, the transformant with the highest carotenoid production is selected as the host for the next transformation.
[0012] This invention constructed a recombinant engineered bacterium, optimized fermentation conditions, and modified the intermediate strain multiple times. For example, it overexpressed key enzymes to enhance the rate-limiting step in the synthetic pathway, modified enzymes at key nodes to improve enzyme activity, and fused glycosyltransferases for expression. Ultimately, this invention successfully screened and obtained a genetically engineered bacterium that produces high levels of crocin.
[0013] This invention successfully obtained a genetically engineered *Yarrowia lipolytica* strain capable of high crocin production through strategies such as heterologous gene transfer, overexpression of key enzymes, regulation of key metabolic nodes, and modular enzyme assembly and expression. The crocin-1 yield reached 21.3 mg / L, achieving efficient synthesis of crocin in *Yarrowia lipolytica*. This crocin-producing *Yarrowia lipolytica* strain can be used for large-scale crocin production, possessing industrial production potential and promising application prospects.
[0014] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description
[0015] The *Yarrowia lipolytica* strain of this invention, which produces crocin, is classified and named as follows: Yarrowialipolytica OUC-Cro13-EL8GGP, deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20252999, deposited on December 24, 2025. Address: Wuhan University, Wuhan, China, 430072, China.
[0016] Figure 1 Synthetic pathway of crocin.
[0017] Figure 2 Schematic diagram of the optimal yield of crocin from key engineered bacteria (Cro9, Car10, Car11, Car12).
[0018] Figure 3 Schematic diagram of crocin production from transformant Car9.
[0019] Figure 4 Schematic diagram of crocin production from transformant Car10.
[0020] Figure 5 Schematic diagram of crocin production from transformant Car11.
[0021] Figure 6 Schematic diagram of crocin production from transformant Car12.
[0022] Figure 7 Schematic diagram of crocin-1 production in transformant Car13. Detailed Implementation
[0023] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.
[0024] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.
[0025] The shake-flask fermentation method used in this invention is as follows: 50 mL of YPD medium is prepared in a 250 mL shake flask, and the genetically engineered bacterial seed culture is inoculated at a 2% inoculum. The culture is then carried out at 30℃ and 220 rpm for 60 h, followed by a second inoculum at 20℃ and 220 rpm for 60 h. The engineered strain (… Figures 3-7 (As shown) During the screening process, the amount of glucose added to the fermentation broth was 2% (mass-volume ratio, unit g / ml); the optimal yield was determined ( Figure 2 The amount of glucose added to the fermentation broth was 3% (as shown in the figure).
[0026] The extraction method for crocin or crocin acid used in this invention is as follows: (1) Processing of bacterial cells: Take 1 mL of fermentation broth and place it in a 2 mL grinding tube. Centrifuge at 10000 g for 2 min, discard the supernatant, add a small amount of 0.52 mm zirconia grinding beads and 1 mL of extraction solvent (methanol:DMF=7:1) to the grinding tube, and use a fully automated rapid sample grinder to break and extract the engineered bacteria. The grinding conditions are as follows: the frequency is set to 65 Hz, the running time is 2 min, the interruption time after each run is 10 s, and the number of runs is 8. After grinding, centrifuge the grinding tube at 10000 g for 2 min, draw up the supernatant with a syringe, filter it with a 0.22 μm nylon filter membrane to remove impurities, and obtain the extract.
[0027] (2) Treatment of supernatant: Take 2 ml of fermentation broth and centrifuge at 10000 g for 10 min; take 1 ml of supernatant, freeze dry, add 1 ml of extraction solvent, vortex for 1 min to reconstitute, let stand for 10 min, use a syringe to draw up the supernatant, filter it with a 0.22 μm nylon filter membrane to remove impurities, and obtain the extract.
[0028] The method for UPLC analysis of the extract in this invention is as follows: C18 column (2.1×100 mm, 1.6 μm, CORTECS@UPLC, waters), gradient elution, ultraviolet detection analysis, injection volume of 2 μL, and column temperature set to 25℃.
[0029] The detection conditions for crocin were as follows: total flow rate 0.325 mL / min; detection wavelength 430 nm; mobile phase A was a formic acid solution (concentration of formic acid 0.1%, volume percentage), and mobile phase B was a formic acid-acetonitrile mixed solution (concentration of formic acid 0.1%, volume percentage). The gradient of mobile phase A was: 0 min, 95%; 0.07 min, 95%; 3.5 min, 25%; 4 min, 25%; 4.5 min, 95%; 5 min, 95%.
[0030] The detection conditions for other carotenoids were as follows: total flow rate 0.3 mL / min; detection wavelength 450 nm; mobile phase A was water, mobile phase B was acetonitrile, and mobile phase C was isopropanol. The ratio gradient of ABC was: 0 min, 20-80-0; 8 min, 5-20-75; 12 min, 20-80-0; 14 min, 20-80-0.
[0031] In this invention, the synthetic pathway of crocin is as follows: Figure 1 As shown.
[0032] The meanings of the English abbreviations used in this invention are as follows: CarRP: phytoene synthase / lycopene cyclase.
[0033] CCD2: Carotenoid lysin dioxygenase.
[0034] ALD: Aldehyde dehydrogenase.
[0035] CrtZ: β-carotene hydroxylase.
[0036] UGT75L6: Primary glycosyltransferase.
[0037] UGT94E5: Secondary glycosyltransferase.
[0038] Example 1: Construction of a genetically engineered *Yarrowia lipolytica* strain producing crocin The steps are as follows: (1) CsCCD2 The gene is derived from saffron and has been optimized with codons. CsCCD2 The nucleotide sequence of the gene is shown in SEQ ID NO.1. The SUMO tag is derived from a small ubiquitin-related modified protein in nature, and after codon optimization, the nucleotide sequence of the SUMO tag is shown in SEQ ID NO.2.
[0039] (2) Using pMT015 plasmid as the expression vector, the two expression cassettes containing the promoter and terminator are P hp4d -T LIP1 and P TEF -T XPR2 The promoter is followed by the kozak sequence "GCCACC". The selection marker for this pMT015 plasmid is URA3, and the promoter and terminator are P... LEU2 and T LEU2 It has LoxP sites at both ends. CsCCD2 The gene was constructed into plasmid pMT015 using a seamless cloning method. TEF -T XPR2 The pMT015-CsCCD2 plasmid was obtained from the expression cassette. The SUMO tag was then constructed into the pMT015-CsCCD2 plasmid using a seamless cloning method. TEF -CsCCD2-T XPR2 The pMT015-SUMO-CsCCD2 plasmid was obtained from the expression cassette.
[0040] (3) The CsCCD2-SUMO-URA expression cassette in the pMT015-SUMO-CsCCD2 plasmid was linearized by PCR and transformed into Yersinia lipolytica by PEG / LiAc chemical transformation. Yarrowialipolytica In OUC-Zea8-A7ZGL, a genetically engineered *Yarrowia lipolytica* strain producing crocin was obtained. Ten transformants with the highest yields were selected (named Car9), and the crocin production of each transformant was compared. The crocin production of transformant Car9 is shown below. Figure 3 As shown, the genetically engineered bacterium OUC-Cro9-2GGP with the highest yield was selected.
[0041] (4) The obtained genetically engineered bacteria OUC-Cro9S-2GGP were subjected to optimal conditions for shake-flask fermentation, extraction, and UPLC analysis. The optimal yield results are as follows: Figure 2 As shown, the crocin yield of the genetically engineered bacterium OUC-Cro9S-2GGP was 56.9 mg / L.
[0042] Example 2: Metabolic engineering strategy to enhance crocin production in genetically engineered Yersinia lipophila. The steps are as follows: (1) carRP The gene originates from Mucor truncatula and has been codon-optimized. carRP The nucleotide sequence of the gene is shown in SEQ ID NO.3. crtZ The gene originated from Pantotheca pineapple and underwent codon optimization. crtZ The nucleotide sequence of the gene is shown in SEQ ID NO. 4. The peroxisome localization signal peptide is SKL, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO. 5; the endoplasmic reticulum localization signal peptide is KDEL, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO. 6.
[0043] (2) Using pMT015 plasmid as the expression vector, the expression vector was... CarRP The gene was constructed into plasmid pMT015 using a seamless cloning method. hp4d -T LIP1 The pMT015-CarRP plasmid was obtained from the expression cassette. Based on the pMT015-CarRP plasmid, a seamless cloning method was used to... CrtZ Gene construction into the P plasmid TEF -T XPR2 The pMT015-CarRP-CrtZ plasmid was obtained on the expression cassette. Based on the pMT015-SUMO-CsCCD2 plasmid (constructed in Example 1), the gene fragments corresponding to the signal peptides SKL and KDEL (as shown in SEQ ID NO. 5 and 6) were constructed into the PTEF-SUMO-CsCCD2-TXPR2 expression cassette using a seamless cloning method, resulting in pMT015-SUMO-CsCCD2-SKL and pMT015-Lip-SUMO-CsCCD2-KDEL.
[0044] (3) The pUB4-CRE plasmid was transformed into the genetically engineered bacteria OUC-Cro9S-2GGP using the PEG / LiAc chemical transformation method to remove the URA3 selection marker so that the marker could be reused repeatedly.
[0045] (4) The CarRP-CrtZ-URA expression cassette in the pMT015-CarRP-CrtZ plasmid was linearized by PCR and transformed into the genetically engineered bacteria obtained in step (3) using PEG / LiAc chemical transformation. Ten transformants with the highest yield were selected (named Car10), and the crocin yield of each transformant was compared. The crocin yield of transformant Car10 was as follows: Figure 4 As shown, the genetically engineered bacterium OUC-Cro10-2GGP, which has a higher yield of crocin, was obtained.
[0046] (5) Transform the pMT015-SUMO-CsCCD2-SKL plasmid into the genetically engineered bacteria OUC-Cro10-2GGP obtained in step (4), following the procedures in steps (3) and (4) above. Select the 10 transformants with the highest yield (named Car11), and compare the crocin yield of each transformant. The crocin yield of transformant Car11 is as follows: Figure 5 As shown, the genetically engineered bacterium OUC-Cro11-7GGP, which has a higher yield of crocin, was obtained.
[0047] (6) Transform the pMT015-Lip-SUMO-CsCCD2-KDEL plasmid into the genetically engineered bacteria OUC-Cro11-7GGP obtained in step (5), following the procedures in steps (3) and (4) above. Select the 10 transformants with the highest yield (named Car12), and compare the crocin yield of each transformant. The crocin yield of transformant Car12 is as follows: Figure 6 As shown, the genetically engineered bacterium OUC-Cro12-6GGP, which has a higher yield of crocin, was obtained.
[0048] (7) The obtained genetically engineered bacteria OUC-Cro10-2GGP, OUC-Cro11-7GGP, and OUC-Cro12-6GGP were subjected to optimal conditions for shake-flask fermentation, extraction, and UPLC analysis. The optimal yield results are as follows: Figure 2 As shown, the crocin yield of the genetically engineered strain OUC-Cro10-2GGP was 86.57 mg / L, the crocin yield of the genetically engineered strain OUC-Cro11-7GGP was 188.26 mg / L, and the crocin yield of the genetically engineered strain OUC-Cro12-6GGP was 196.5 mg / L.
[0049] Example 3 Construction of a genetically engineered *Yarrowia lipolytica* strain producing crocin The steps are as follows: (1) UGT75L6 Genes and UGT94E5 The genes all come from gardenia ( Gardenia jasminoides After codon optimization, UGT75L6 The nucleotide sequence of the gene is shown in SEQ ID NO.7. UGT94E5 The nucleotide sequence of the gene is shown in SEQ ID NO.8.
[0050] (2) Using pMT015 plasmid as the expression vector, the expression vector was... UGT75L6 The gene was constructed into plasmid pMT015 using a seamless cloning method. TEF -T XPR2The pMT015-UGT75L6 plasmid was obtained from the expression cassette. Based on the pMT015-UGT75L6 plasmid, a seamless cloning method was used to... UGT94E5 Gene construction into the P plasmid TEF -UGT75L6-T XPR2 On the expression cassette, the UGT75L6 and UGT94E5 gene sequences are linked together by a linker to obtain the pMT015-UGT94E5-UGT75L6 plasmid.
[0051] (3) The UGT94E5-UGT75L6-URA gene fragment was transferred into the genetically engineered strain OUC-Cro12-6GGP. The operation process was the same as steps (3) and (4) in Example 2. The resulting Yersinia lipolytica genetically engineered strain produced crocin. Ten transformants with the highest yield were selected (named Car13). The crocin yield (represented by crocin-1) of each transformant was compared. The crocin-1 yield of transformant Car13 was as follows: Figure 7 As shown, the genetically engineered bacterium OUC-Cro13-8GGP, which produced the highest yield of crocin-1, reached a yield of 21.3 mg / L.
[0052] This invention constructed a recombinant engineered strain producing crocin. The intermediate strain underwent five modifications and screenings, ultimately yielding a high-crocin-producing *Yarrowia lipolytica* engineered strain—OUC-Cro13-8GGP. This transformant exhibited a crocin-1 yield as high as 21.3 mg / L, significantly superior to other transformants. The transformant OUC-Cro13-8GGP was deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20252999, deposit date: December 24, 2025, address: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China.
[0053] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.
Claims
1. A strain of *Yarrowia lipolytica* that produces crocin, characterized by: Category naming Yarrowialipolytica OUC-Cro13-EL8GGP, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20252999, deposit date: December 24, 2025.
2. The application of the crocin-producing Yersinia lipolytica strain according to claim 1 in the preparation of crocin.
3. The application according to claim 2, characterized in that: Crocin was extracted from Yersinia lipolytica, a yeast strain that produces crocin.
4. The application according to claim 3, characterized in that, The specific cultivation method is as follows: the seed liquid of *Yarrowia lipolytica*, which produces crocin, is inoculated into YPD medium at an inoculation amount of 2%, and incubated on a shaker at 30°C and 220 rpm for 60 hours, and then incubated on a shaker at 20°C and 220 rpm for another 60 hours.
5. The application according to claim 3, characterized in that: The specific extraction method is as follows: take the fermentation broth of cultured Yersinia lipophila, centrifuge and discard the supernatant, place it in a grinding tube, add the extractant, and then add zirconium oxide grinding beads for grinding; centrifuge, filter the supernatant to remove impurities, and obtain the extract, which contains crocin; the extractant is composed of methanol and dimethylformamide, and the volume ratio of the two is 7:1.