Method for improving yield of yarrowia lipolytica astaxanthin through metabolic regulation

Abstract

The invention relates to the technical field of microbial fermentation, and discloses a method for improving yield of yarrowia lipolytica astaxanthin through metabolism regulation, which comprises the following steps: firstly, preparing sebacic acid-beta-sitosterol monoester as a bifunctional metabolism regulation agent for subsequently releasing a metabolism precursor in a specific cell compartment; then carrying out growth culture on yarrowia lipolytica in a fermentation culture medium with a high carbon nitrogen ratio to obtain high-density thalli; after thalli grow into a plateau phase, adding the regulating agent into the culture medium until the final working concentration is 0.1-1.0 g / L; and finally, continuously carrying out induced fermentation culture, so that the yarrowia lipolytica efficiently accumulates astaxanthin in the cells. According to the method disclosed by the invention, a specific bifunctional regulating agent is combined with a staged culture strategy, so that fixed-point release of a metabolic precursor to a storage compartment is realized, and the technical contradiction between low utilization rate of the precursor and inhibition of cell growth is solved, so that the synthesis efficiency and the volume yield of astaxanthin are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a method for increasing the astaxanthin yield of Yersinia lipolytica through metabolic regulation. Background Technology

[0002] Astaxanthin is a high-value-added ketocarotenoid with broad application prospects in food, feed, and medicine due to its excellent antioxidant activity. Microbial fermentation is one of the main routes for obtaining natural astaxanthin. Among them, *Yarrowia lipolytica*, as a non-traditional yeast, is considered an ideal cell factory for astaxanthin production due to its high-density culture and strong lipid synthesis capabilities. Therefore, developing metabolic regulation strategies targeting *Yarrowia lipolytica* to increase astaxanthin yield is a current research focus in this field.

[0003] In existing fermentation processes of *Yarrowia lipolytica*, a high carbon-to-nitrogen ratio culture medium is typically used to promote lipid accumulation in cells, thus providing a basis for the synthesis and storage of lipid-soluble products such as astaxanthin. Simultaneously, to further enhance the synthesis of target products, researchers also attempt to directly add certain metabolic precursors (such as small-molecule organic acids) to the culture system, aiming to increase the carbon flux of specific metabolic pathways and thereby improve the final yield.

[0004] However, existing technologies still have shortcomings in application. When metabolic precursors are directly added to the fermentation medium, their metabolic flow within the cells after absorption is difficult to control. Most may be used to maintain basic cellular activities or enter other competitive metabolic pathways, leaving a limited proportion actually used for astaxanthin synthesis, resulting in low precursor utilization efficiency. Furthermore, adding these regulators or precursors in the early stages of fermentation can affect the normal growth and proliferation of the bacterial strain, thus limiting the total bacterial biomass available for astaxanthin synthesis. This makes it difficult to coordinate and optimize the two stages of cell growth and product synthesis, ultimately affecting the overall volumetric yield of astaxanthin.

[0005] Therefore, this invention proposes a method to increase the astaxanthin production of Yersinia lipolytica through metabolic regulation, in order to overcome the shortcomings of the prior art. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for increasing astaxanthin production in Yersinia lipolytica through metabolic regulation. This method solves the technical problem in existing technologies where the direct addition of metabolic precursors makes it difficult to achieve targeted enhancement of astaxanthin synthesis and effective synergy with the cell growth stage, resulting in low precursor utilization and limited final product yield.

[0007] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of this invention provides a method for increasing the astaxanthin production of Yersinia lipophila through metabolic regulation.

[0008] The method includes the following steps: First, a bifunctional metabolic regulator was prepared, the chemical structure of which is sebacic acid-β-sitosterol monoester.

[0009] Secondly, the *Yersinia lipolytica* strain capable of synthesizing astaxanthin was inoculated into a high carbon-to-nitrogen ratio fermentation medium for growth culture.

[0010] Then, after the culture of this growth stage is completed and the cell growth enters the plateau phase, the bifunctional metabolic regulator prepared in the aforementioned steps is added to the fermentation medium. After addition, the final working concentration of the bifunctional metabolic regulator in the fermentation medium is 0.1-1.0 g / L.

[0011] Finally, after adding this bifunctional metabolic regulator, induced fermentation culture was continued, thereby allowing Yersinia lipolyticis to accumulate astaxanthin in the cells.

[0012] In one specific embodiment, the above-mentioned bifunctional metabolic regulator, namely sebacic acid-β-sitosterol monoester, is prepared as follows: β-sitosterol and sebacic acid are used as raw materials, and an esterification reaction is carried out in the presence of a catalyst. The molar ratio of β-sitosterol to sebacic acid is 1:(1.5-2.5). The catalyst is p-toluenesulfonic acid.

[0013] In one specific embodiment, the carbon-nitrogen ratio of the above-mentioned high carbon-nitrogen ratio fermentation medium ranges from 80 to 120.

[0014] In one specific implementation, the plateau phase of bacterial growth refers to a culture time of 48-72 hours during the growth phase.

[0015] In one specific embodiment, the process conditions for the above-mentioned induced fermentation culture include: temperature controlled at 28-30℃ and pH value controlled at 5.5-6.5.

[0016] The technical solution provided by this invention has the following effect: After the added bifunctional metabolic regulator (sepiacetic acid-β-sitosterol monoester) is absorbed by Yersinia lipolytica cells, during the induced fermentation culture, the β-sitosterol end of its molecular structure is anchored in lipid droplets within the cells due to its hydrophobicity. Simultaneously, the other end of the molecule, the sebacic acid end, is exposed on the surface of the lipid droplets. This structure creates a polar region on the surface of the lipid droplets composed of numerous sebacic acid ends. This region can capture and immobilize the newly synthesized lipid-soluble product astaxanthin, reducing the free concentration of astaxanthin at synthesis sites (such as the endoplasmic reticulum), thereby alleviating product feedback inhibition.

[0017] Furthermore, during induced fermentation, the ester bonds attached to the sebacic acid termini exposed on the lipid droplet surface can be hydrolyzed by endogenous esterases, releasing free sebacic acid near the lipid droplet-endoplasmic reticulum interface. The released sebacic acid can enter the cell's β-oxidation pathway, generating reducing power (NADH and FADH2). This reducing power provides energy for the ketation and hydroxylation reactions at the end of the astaxanthin synthesis pathway, improving the efficiency of the conversion from precursors to astaxanthin.

[0018] This invention provides a method for increasing astaxanthin production in *Yersinia lipolyticis* through metabolic regulation. It has the following beneficial effects: 1. This invention achieves site-directed enhancement of product synthesis by designing and applying a bifunctional metabolic regulator with a specific structure of sebacic acid-β-sitosterol monoester. This regulator utilizes the physicochemical properties of its β-sitosterol terminus to enter and anchor into lipid droplets within *Yarrowia lipolytica* cells. Subsequently, its ester bond is hydrolyzed, releasing sebacic acid near the astaxanthin storage compartment. This molecular design, which directly replenishes the precursor at a critical location, effectively guides metabolic resources to the target pathway, providing the structural basis for achieving the technical effect of increasing astaxanthin production through metabolic regulation.

[0019] 2. This invention optimizes both cell growth and product accumulation stages by constructing a phased sequential fermentation process. The method first utilizes a high C / N ratio culture medium to bring the biomass and lipids of *Yersinia lipolytica* to a state suitable for product synthesis; this is the first stage. Subsequently, when cell growth enters the plateau phase, the aforementioned bifunctional metabolic regulator is added to initiate the second stage of induced fermentation. This sequential arrangement avoids the potential impact of the regulator on the early stages of cell growth, ensuring a sufficient cell factory for subsequent production, thereby effectively increasing the final astaxanthin yield from *Yersinia lipolytica*.

[0020] 3. This invention achieves a synergistic technical effect by combining specific culture conditions with subsequent metabolic regulation. A high carbon-to-nitrogen ratio culture environment provides the necessary physiological basis for the intracellular synthesis and storage of astaxanthin, namely, sufficient lipid droplets. Based on this, the added bifunctional metabolic regulator can precisely act on these pre-formed lipid droplet structures and release precursors. This mutual matching and promotion between culture conditions and molecular function works synergistically to ultimately achieve the goal of increasing astaxanthin production in *Yersinia lipophilia* through metabolic regulation. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the method flow of the present invention. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0024] β-sitosterol, CAS No.: 83-46-5; Sebacic acid, CAS No.: 111-20-6; p-Toluenesulfonic acid, CAS: 104-15-4; Toluene, CAS No.: 108-88-3; Reference Figure 1 .

[0025] Example 1: This embodiment illustrates a method for increasing the astaxanthin production of Yersinia lipolytica through metabolic regulation.

[0026] S1. Prepare a bifunctional metabolic regulator, wherein the bifunctional metabolic regulator is sebacic acid-β-sitosterol monoester.

[0027] To a 500 mL three-necked flask equipped with a magnetic stirrer, reflux condenser, and Dean-Stark water separator, 41.4 g (0.1 mol) of β-sitosterol, 30.3 g (0.15 mol) of sebacic acid, and 1.9 g (0.01 mol) of p-toluenesulfonic acid monohydrate were added as a catalyst. The molar ratio of β-sitosterol to sebacic acid in this step was 1:1.5. 250 mL of toluene was added to the flask. The reaction mixture was heated to 130 °C and maintained under reflux, and the generated water was removed using a Dean-Stark water separator. After 20 hours of reaction, heating was stopped and the mixture was cooled. The reaction solution was washed with saturated sodium bicarbonate aqueous solution and saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluting with a gradient of n-hexane / ethyl acetate) to obtain pure sebacic acid-β-sitosterol monoester.

[0028] S2. Yersinia lipolyticis was inoculated into a high carbon-to-nitrogen ratio fermentation medium for the growth stage.

[0029] The seed culture of *Yarrowia lipolyticis* strain was inoculated at a rate of 10% (v / v) into a 5L fermenter containing 3L of fermentation medium. The fermentation medium was formulated (per liter): 80g D-glucose, 2.0g yeast extract, 1.0g ammonium sulfate, 2.0g potassium dihydrogen phosphate, and 1.5g magnesium sulfate heptahydrate. Based on this formulation, the carbon-to-nitrogen ratio of the medium was calculated to be 80. The culture conditions for the growth phase were set as follows: temperature 28℃, pH 5.5, and dissolved oxygen level maintained above 20%.

[0030] S3. After the growth stage is completed, that is, after the cell growth enters the plateau phase, add the bifunctional metabolic regulator prepared in step S1 to the fermentation medium so that the final working concentration of the bifunctional metabolic regulator in the fermentation medium is 0.1-1.0 g / L.

[0031] After 48 hours of growth in step S2, the cell growth enters the plateau phase. At this time, the anhydrous ethanol stock solution of sebacic acid-β-sitosterol monoester prepared in step S1 is aseptically added by a peristaltic pump to make its final working concentration in the fermentation medium 0.1 g / L.

[0032] S4. After adding the bifunctional metabolic regulator, continue the induced fermentation culture to allow the Yersinia lipolyticis to accumulate astaxanthin in the cells.

[0033] After adding the bifunctional metabolic regulator in step S3, induced fermentation was continued for 84 hours. The culture conditions during induced fermentation were controlled as follows: temperature maintained at 28℃, and pH maintained at 5.5 by automatic addition of acid and alkali solutions. Samples were taken after fermentation for subsequent analysis.

[0034] Example 2: This embodiment illustrates a method for increasing the astaxanthin production of Yersinia lipolytica through metabolic regulation.

[0035] S1. Prepare a bifunctional metabolic regulator, wherein the bifunctional metabolic regulator is sebacic acid-β-sitosterol monoester.

[0036] To a 500 mL three-necked flask equipped with a magnetic stirrer, reflux condenser, and Dean-Stark water separator, 41.4 g (0.1 mol) of β-sitosterol, 40.4 g (0.2 mol) of sebacic acid, and 1.9 g (0.01 mol) of p-toluenesulfonic acid monohydrate were added as a catalyst. The molar ratio of β-sitosterol to sebacic acid in this step was 1:2.0. 250 mL of toluene was added to the flask. The reaction mixture was heated to 130 °C and maintained under reflux, and the generated water was removed using a Dean-Stark water separator. After 20 hours of reaction, heating was stopped and the mixture was cooled. The reaction solution was washed with saturated sodium bicarbonate aqueous solution and saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluting with a gradient of n-hexane / ethyl acetate) to obtain pure sebacic acid-β-sitosterol monoester.

[0037] S2. Yersinia lipolyticis was inoculated into a high carbon-to-nitrogen ratio fermentation medium for the growth stage.

[0038] The seed culture of *Yarrowia lipolyticis* strain was inoculated at a rate of 10% (v / v) into a 5L fermenter containing 3L of fermentation medium. The fermentation medium was formulated (per liter): 100g D-glucose, 2.0g yeast extract, 1.0g ammonium sulfate, 2.0g potassium dihydrogen phosphate, and 1.5g magnesium sulfate heptahydrate. Based on this formulation, the carbon-to-nitrogen ratio of the medium was approximately 100. The growth conditions were set as follows: temperature 29℃, pH 6.0, and dissolved oxygen level maintained above 20%.

[0039] S3. After the growth stage is completed, that is, after the cell growth enters the plateau phase, add the bifunctional metabolic regulator prepared in step S1 to the fermentation medium so that the final working concentration of the bifunctional metabolic regulator in the fermentation medium is 0.1-1.0 g / L.

[0040] After 60 hours of growth in step S2, the cell growth enters a plateau phase. At this point, the anhydrous ethanol stock solution of sebacic acid-β-sitosterol monoester prepared in step S1 is aseptically added using a peristaltic pump to bring the final working concentration in the fermentation medium to 0.5 g / L.

[0041] S4. After adding the bifunctional metabolic regulator, continue the induced fermentation culture to allow the Yersinia lipolyticis to accumulate astaxanthin in the cells.

[0042] After adding the bifunctional metabolic regulator in step S3, induced fermentation was continued for 84 hours. The culture conditions during induced fermentation were controlled as follows: temperature maintained at 29℃, and pH maintained at 6.0 by automatic addition of acid and alkali solutions. Samples were taken after fermentation for subsequent analysis.

[0043] Example 3: This embodiment illustrates a method for increasing the astaxanthin production of Yersinia lipolytica through metabolic regulation.

[0044] S1. Prepare a bifunctional metabolic regulator, wherein the bifunctional metabolic regulator is sebacic acid-β-sitosterol monoester.

[0045] To a 500 mL three-necked flask equipped with a magnetic stirrer, reflux condenser, and Dean-Stark water separator, 41.4 g (0.1 mol) of β-sitosterol, 50.5 g (0.25 mol) of sebacic acid, and 1.9 g (0.01 mol) of p-toluenesulfonic acid monohydrate were added as a catalyst. The molar ratio of β-sitosterol to sebacic acid in this step was 1:2.5. 250 mL of toluene was added to the flask. The reaction mixture was heated to 130 °C and maintained under reflux, and the generated water was removed using a Dean-Stark water separator. After 20 hours of reaction, heating was stopped and the mixture was cooled. The reaction solution was washed with saturated sodium bicarbonate aqueous solution and saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluting with a gradient of n-hexane / ethyl acetate) to obtain pure sebacic acid-β-sitosterol monoester.

[0046] S2. Yersinia lipolyticis was inoculated into a high carbon-to-nitrogen ratio fermentation medium for the growth stage.

[0047] The seed culture of *Yarrowia lipolyticis* strain was inoculated at a rate of 10% (v / v) into a 5L fermenter containing 3L of fermentation medium. The fermentation medium was formulated (per liter): 120g D-glucose, 2.0g yeast extract, 1.0g ammonium sulfate, 2.0g potassium dihydrogen phosphate, and 1.5g magnesium sulfate heptahydrate. Based on this formulation, the carbon-to-nitrogen ratio of the medium was approximately 120. The growth conditions were set as follows: temperature 30℃, pH 6.5, and dissolved oxygen level maintained above 20%.

[0048] S3. After the growth stage is completed, that is, after the cell growth enters the plateau phase, add the bifunctional metabolic regulator prepared in step S1 to the fermentation medium so that the final working concentration of the bifunctional metabolic regulator in the fermentation medium is 0.1-1.0 g / L.

[0049] After 72 hours of growth in step S2, the cell growth enters the plateau phase. At this time, the anhydrous ethanol stock solution of sebacic acid-β-sitosterol monoester prepared in step S1 is aseptically added by a peristaltic pump to make its final working concentration in the fermentation medium 1.0 g / L.

[0050] S4. After adding the bifunctional metabolic regulator, continue the induced fermentation culture to allow the Yersinia lipolyticis to accumulate astaxanthin in the cells.

[0051] After adding the bifunctional metabolic regulator in step S3, induced fermentation was continued for 84 hours. The culture conditions during induced fermentation were controlled as follows: temperature maintained at 30℃, and pH maintained at 6.5 by automatic addition of acid and alkali solutions. Samples were taken after fermentation for subsequent analysis.

[0052] Comparative Example 1: Compared with Example 2, the difference is that no metabolic regulator was added in step S3, only an equal volume of anhydrous ethanol solvent was added, and all other conditions were the same.

[0053] Comparative Example 2: Compared with Example 2, the difference is that in step S3, sebacic acid-β-sitosterol monoester is not added, but a physical mixture of β-sitosterol and sebacic acid is added in a molar amount equivalent to 0.5 g / L sebacic acid-β-sitosterol monoester, while the other conditions are the same.

[0054] Comparative Example 3: Compared with Example 2, the difference is that the addition of sebacic acid-β-sitosterol monoester in step S3 was carried out at the beginning of fermentation (0 hours) instead of at the plateau period (60 hours), while all other conditions were the same.

[0055] Comparative Example 4: Compared with Example 2, the difference is that the fermentation medium in step S2 is a low carbon-to-nitrogen ratio medium (e.g., D-glucose 20 g / L, yeast extract 10 g / L, peptone 20 g / L; carbon-to-nitrogen ratio of about 10), while all other conditions are the same.

[0056] Test Example 1: Detection of fermentation products.

[0057] To verify the technical effectiveness of the aforementioned method, the products of Examples 1-3 and Comparative Examples 1-4 were tested after fermentation (144 hours).

[0058] 1. Procedure for determining cell dry weight: Take 50.0 mL of fermentation broth from the fermenter and place it into a pre-recorded centrifuge tube. Centrifuge at 8000 rpm for 10 minutes and discard the supernatant. Add 20 mL of deionized water to the centrifuge tube to resuspend the cell pellet, and repeat the centrifugation and supernatant discarding process. Repeat this washing step twice. Place the centrifuge tube containing the cell pellet in an 80°C oven to constant weight. After cooling to room temperature in a desiccator, weigh the tube. The cell dry weight (g / L) is obtained using the following formula: ; 2. Astaxanthin Yield Determination Procedure: Approximately 50 mg of freeze-dried bacterial powder was accurately weighed and placed in a 2 mL centrifuge tube. 0.5 mL of 2M hydrochloric acid solution was added, and the mixture was treated at 50°C for 30 minutes. After treatment, 1.0 mL of acetone was added to the tube, and the mixture was vortexed for 5 minutes. The tube was centrifuged at 8000 rpm for 5 minutes, and the supernatant was collected. The acetone extraction procedure was repeated on the precipitate until it turned grayish-white. All collected supernatants were combined. The combined extracts were analyzed using high-performance liquid chromatography (HPLC).

[0059] The chromatographic conditions were set as follows: C18 reversed-phase column (4.6 mm × 250 mm, 5 µm), mobile phase: acetonitrile, methanol, dichloromethane (v / v 80:15:5), flow rate: 1.0 mL / min, column temperature: 35 °C, detection wavelength: 474 nm. Quantification was performed using a standard curve established with astaxanthin standard (CAS: 472-61-7). Astaxanthin content (mg / gDCW) and astaxanthin volume yield (mg / L) were obtained using the following formulas: ; ; Table 1: Fermentation results data for each example and comparative example.

[0060] Analysis of the data in Table 1 leads to the following conclusions: The volumetric astaxanthin yields of Examples 1, 2, and 3 are significantly higher than those of Comparative Examples 1, 2, 3, and 4. Comparing the results of Example 2 with Comparative Example 1 shows that adding sebacic acid-β-sitosterol monoester to the fermentation medium increases the final volumetric astaxanthin yield. Comparing the results of Example 2 with Comparative Example 2 shows that using sebacic acid-β-sitosterol monoester molecules results in a higher volumetric astaxanthin yield compared to using a physical mixture of β-sitosterol and sebacic acid, indicating that linking the two components via ester bonds is a necessary structure to achieve the desired effect.

[0061] The sebacic acid-β-sitosterol monoester molecule provided in this technical solution comprises a lipophilic β-sitosterol terminus and a sebacic acid terminus. When this molecule is added to a fermentation medium and absorbed by *Yarrowia lipolytica* cells, the physicochemical properties of its β-sitosterol terminus allow it to enter and localize within lipid droplets, the main storage compartments for astaxanthin in cells. This localization exposes the other end of the molecule, the sebacic acid terminus, to the surface of the lipid droplet or its surrounding cytoplasm. During induced fermentation, the ester bond connecting sebacic acid and β-sitosterol undergoes hydrolysis, releasing sebacic acid from the vicinity of the lipid droplet.

[0062] The overall technical effectiveness of this method stems from the synergistic effect of multiple step conditions. First, comparing the results of Example 2 and Comparative Example 4 shows that culturing in a fermentation medium with a carbon-to-nitrogen ratio of 80-120 yields higher cell dry weight and lipid accumulation, providing a foundation for subsequent astaxanthin synthesis and storage. Second, comparing the results of Example 2 and Comparative Example 3 shows that adding this bifunctional metabolic regulator after cell growth enters the plateau phase is more beneficial for the final accumulation of astaxanthin than adding it at the beginning of fermentation, avoiding interference with the cell growth stage. Finally, sebacic acid released near the lipid droplets can serve as a carbon source to enter the central metabolic pathway of the cell, supplementing the large amount of precursors (such as acetyl-CoA) required for astaxanthin synthesis, thereby increasing the astaxanthin synthesis throughput.

Claims

1. A method for increasing astaxanthin production in Yersinia lipolyticis through metabolic regulation, characterized in that, Includes the following steps: S1. Prepare a bifunctional metabolic regulator, wherein the bifunctional metabolic regulator is sebacic acid-β-sitosterol monoester; S2. Yersinia lipolyticis was inoculated into a high carbon-to-nitrogen ratio fermentation medium for the growth stage. S3. After the growth stage is completed, that is, after the cell growth enters the plateau phase, add the bifunctional metabolic regulator prepared in step S1 to the fermentation medium so that the final working concentration of the bifunctional metabolic regulator in the fermentation medium is 0.1-1.0 g / L. S4. After adding the bifunctional metabolic regulator, continue the induced fermentation culture to allow the Yersinia lipolyticis to accumulate astaxanthin in the cells.

2. The method for increasing astaxanthin production in Yersinia lipolyticis according to claim 1, characterized in that, In step S1, the sebacic acid-β-sitosterol monoester is prepared by esterification reaction using β-sitosterol and sebacic acid as raw materials in the presence of a catalyst.

3. The method for increasing astaxanthin production in Yersinia lipolyticis through metabolic regulation according to claim 2, characterized in that, In the esterification reaction, the molar ratio of β-sitosterol to sebacic acid is 1:(1.5-2.5).

4. The method for increasing astaxanthin production in Yersinia lipolyticis according to claim 3, characterized in that, The catalyst is p-toluenesulfonic acid.

5. The method for increasing astaxanthin production in Yersinia lipolyticis via metabolic regulation according to claim 1, characterized in that, In step S2, the carbon-to-nitrogen ratio of the high carbon-to-nitrogen fermentation medium is 80-120.

6. The method for increasing astaxanthin production in Yersinia lipolyticis according to claim 1, characterized in that, In step S3, the plateau phase of bacterial growth refers to a culture period of 48-72 hours.

7. The method for increasing astaxanthin production in Yersinia lipolyticis according to claim 1, characterized in that, In step S4, the temperature of the induced fermentation culture is controlled at 28-30℃.

8. The method for increasing astaxanthin production in Yersinia lipolyticis according to claim 1, characterized in that, In step S4, the pH value of the induced fermentation culture is controlled at 5.5-6.

5.

9. The method for increasing astaxanthin production in Yersinia lipolyticis via metabolic regulation according to claim 1, characterized in that, During the induced fermentation culture in step S4, the β-sitosterol end of the bifunctional metabolic regulator is anchored in the lipid droplets of the Yersinia lipolytica, and the sebacic acid end of the bifunctional metabolic regulator is exposed on the surface of the lipid droplets.

10. A method for increasing astaxanthin production in Yersinia lipolyticis according to claim 9, characterized in that, The ester bonds connected to the sebacic acid ends exposed on the surface of the lipid droplets are hydrolyzed during the induced fermentation process, releasing sebacic acid.

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