Schizochytrium high-dha low-dpa oil fermentation regulation method and application

Abstract

The application discloses a kind of schizochytrium high DHA low DPA oil fermentation control method and application, through the time axis joint control strategy of "resource release-pathway bias-state reinforcement": in early and middle fermentation, small molecule of natural source is added in batches, and FAS competition path is moderately inhibited;In key time window, the programmed ventilation and stirring cascade control of "normal oxygen supply-low oxygen supply induction-recovery oxygen supply" is implemented to maintain target DO interval;And 28-34 ℃ temperature ladder is used to up-regulate and can be linked with feeding to strengthen metabolic state switching and fatty acid spectrum formation.After the above-mentioned joint regulation, DHA 67.5%, DPA 4.5% and oil yield 84.3 g / L can be stably obtained in 5 L fermenter;Scaling up to 500 L fermenter, DHA 68.3%, DPA 4.8% and oil yield 89.7 g / L can still be obtained.The method is safe and simple, and the parameters can be engineered to replicate, easy to scale up and migrate, can significantly reduce the burden of DHA / DPA separation and purification and improve the product specification stability, with good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and process enhancement technology, specifically relating to a fermentation regulation method and application of Schizochytrium high-DHA and low-DPA lipids. Background Technology

[0002] Schizochytrium sp. is a typical marine oil-producing microorganism characterized by rapid growth, short fermentation cycles, and shear resistance, making it suitable for the industrial production of polyunsaturated fatty acids such as docosahexaenoic acid (DHA). While continuous optimization of strain selection, culture medium formulation, and fermentation operating conditions has significantly improved oil yield from Schizochytrium sp., maintaining a stable high DHA content in industrial production remains challenging. After fermentation enters the high oil accumulation stage, significant changes in cell metabolic state occur, with competition for substrates and energy between different lipid synthesis pathways limiting the potential for increasing DHA content. Furthermore, variations in oxygen supply and mixing conditions within the fermentation tank at different locations can easily lead to batch-to-batch fluctuations in fatty acid composition, making it difficult to consistently achieve a high DHA content while maintaining high oil yield over the long term.

[0003] Furthermore, Schizochytrium primarily synthesizes long-chain polyunsaturated fatty acids via the polyketide synthase (PKS) pathway. In practice, it is frequently observed that when the proportion of DHA is increased through process intensification or regulation, the proportion of the byproduct docosapentaenoic acid (DPA) often increases accordingly, exhibiting a linked trend of DHA increase accompanied by DPA accumulation. Because DHA and DPA have the same carbon chain length and differ by only one double bond, their chemical structures are highly similar, leading to significant challenges and high costs in downstream purification and separation. In industrial scenarios requiring high-purity DHA products, an increased DPA proportion often directly increases purification burden and costs, affecting product specification stability and economic viability. Therefore, achieving a synergistic optimization of high DHA proportion and low DPA content without sacrificing oil yield is one of the key issues that urgently needs to be addressed in the industrialization of Schizochytrium.

[0004] Existing publicly available technologies primarily focus on improvements at the level of strain modification or fermentation regulation: one approach involves obtaining high-DHA strains through mutagenesis screening, while another uses metabolic engineering to directionally synthesize DHA. Both methods can be effective under certain conditions, but they generally suffer from long development cycles and uncertainties in scaling up across different production lines (e.g., CN120555215A). Adding chemical regulators to influence the metabolism of Schizochytrium and thus control the DHA / DPA ratio is also possible, but this approach cannot simultaneously and efficiently produce oils (e.g., CN120060388A). In summary, current technologies lack a process control scheme that is "directly controllable, replicable, and easily scaled up" in industrial fermentation, and which can significantly increase oil yield while stably achieving the simultaneous goals of high DHA and low DPA.

[0005] Therefore, there is an urgent need in this field to propose a process control scheme that can be directly implemented in industrial fermentation without changing the genetic background of the strain, so as to achieve synergistic optimization of high DHA content and low DPA content while ensuring increased oil yield. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a process control method that can be directly implemented in industrial fermentation without changing the genetic background of Schizochytrium. By inhibiting competitive pathways, programmatically controlling aeration and stirring intensity, and regulating temperature stepwise, a replicable and scalable metabolic guidance strategy is constructed, which can significantly improve oil yield while achieving synergistic optimization of high DHA content and low DPA content.

[0007] The present invention achieves the above objectives through the following technical solutions:

[0008] This invention provides a fermentation regulation method for high-DHA, low-DPA oil from Schizochytrium. Without altering the genetic background of Schizochytrium, a combined process control method is implemented sequentially along the time axis during fermentation, including competitive pathway inhibition, programmed oxygen supply and stirring control, and temperature stepwise regulation, to increase the proportion of DHA, decrease the proportion of DPA, and improve oil yield. The method includes at least the following steps: (1) inoculating Schizochytrium into a fermentation medium for fermentation culture; (2) adding naturally derived small molecules to the fermentation system in the early and / or middle stages of fermentation to moderately inhibit the fatty acid synthase (FAS) pathway; (3) implementing programmed oxygen supply and stirring cascade control with dissolved oxygen (DO) target range as the process command during fermentation, so that the fermentation process sequentially goes through a biomass accumulation stage, a low oxygen supply induction stage, and a recovery stage; (4) stepwise increasing the fermentation temperature to form a segmented enhancement of the fermentation process in conjunction with step (3); (5) obtaining Schizochytrium oil product after fermentation.

[0009] Furthermore, the Schizochytrium is identified as Schizochytrium sp. HX-308, with accession number CCTCC No. M209059.

[0010] Furthermore, the naturally derived small molecule is selected from one or more of curcumin, resveratrol, ellagic acid, and oleanolic acid, and the naturally derived small molecule is used to reduce the competitive consumption of acetyl / malonyl-CoA and reducing power in the FAS pathway, so as to improve the availability of polyunsaturated fatty acid synthesis resources.

[0011] Furthermore, the final concentration range of the natural-derived small molecules in the fermentation system when used alone is: curcumin 5–30 μM, resveratrol 10–60 μM, ellagic acid 10–80 μM, and oleanolic acid 5–60 μM.

[0012] Furthermore, the final concentration range of the naturally derived small molecules in the fermentation system when used in combination is: curcumin 3–15 μM, resveratrol 7–30 μM, ellagic acid 10–40 μM, and oleanolic acid 5–25 μM.

[0013] Furthermore, the naturally derived small molecules are added to the fermentation system in stages, with the first addition occurring 0–12 h after fermentation and the second addition occurring 20–30 h after fermentation.

[0014] Furthermore, the first addition was made 8 hours after fermentation, and the second addition was made 24 hours after fermentation, with the amount added at each time being 50% of the target final concentration.

[0015] Furthermore, the programmed oxygen supply and stirring cascade control includes three consecutive stages: (1) Biomass accumulation stage: fermentation 0–24 h, DO is 40–60%; (2) Low oxygen supply induction stage: fermentation 24–48 h, DO is 5–10%; (3) Recovery stage: after fermentation 48 h, DO is 15–25%.

[0016] Furthermore, in a 5 L fermenter, to achieve the target DO range for the three consecutive stages, the following typical control points were adopted: 0–24 h: aeration rate 3.0 L / min, stirring intensity 400 rpm; 24–48 h: aeration rate 1.0 L / min, stirring intensity 250 rpm; after 48 h: aeration rate 2.5 L / min, stirring intensity 450 rpm.

[0017] Furthermore, in a 500 L fermenter, to achieve the target DO range for the three consecutive stages, the following typical control points were adopted: 0–24 h: aeration rate 250 L / min, stirring intensity 200 rpm; 24–48 h: aeration rate 150 L / min, stirring intensity 150 rpm; after 48 h: aeration rate 200 L / min, stirring intensity 250 rpm.

[0018] Furthermore, the temperature step adjustment is a step-by-step increase within the range of 28–34℃, and the temperature step has two or three segments.

[0019] Furthermore, the temperature gradient is a three-stage gradient, with the following temperature program: 28℃ for 0–24 h of fermentation, 32℃ for 24–72 h of fermentation, and 34℃ after 72 h of fermentation until the end of fermentation.

[0020] Furthermore, the total fermentation cycle is 96–120 h.

[0021] Furthermore, the total fermentation cycle is 108 hours.

[0022] Furthermore, the fermentation regulation method also includes a feeding control step: the feeding carbon source is a combination of glucose and glycerol, with a mass ratio of 30:70 to 50:50, and the residual carbon source concentration is maintained at 5–15 g / L through feeding.

[0023] Furthermore, the mass ratio of glucose to glycerol is 40:60.

[0024] Furthermore, the combined process control adopts the following optimal combination: (1) The natural source small molecules are curcumin, resveratrol, ellagic acid and oleanolic acid in combination, with final concentrations of 10 μM curcumin, 20 μM resveratrol, 20 μM ellagic acid and 10 μM oleanolic acid, respectively, and are added in batches at 8 h and 24 h of fermentation, with each addition amount being 50% of the target final concentration of each component; (2) The target range of DO for the programmed oxygen supply and stirring cascade control is: DO 40–60% for 0–24 h, DO 5–10% for 24–48 h, and DO 15–25% after 48 h; (3) The temperature gradient is: 28℃ for 0–24 h, 32℃ for 24–72 h, and 34℃ after 72 h.

[0025] Furthermore, under the conditions of a 5 L fermenter and a fermentation cycle of 108 h, the optimal combination described above can achieve an oil yield of 84.3 g / L, with DHA accounting for 67.5% of the total fatty acids and DPA accounting for 4.5% of the total fatty acids in the obtained oil.

[0026] Furthermore, the fermentation control method, when scaled up to a 500 L fermenter, still yields an oil yield of 89.7 g / L, with DHA accounting for 68.3% of the total fatty acids and DPA accounting for 4.8% of the total fatty acids in the obtained oil.

[0027] This invention also provides an application of the above-mentioned fermentation regulation method in the preparation of high-DHA, low-DPA Schizochytrium oil products, which reduces the burden of DHA / DPA separation and purification and improves product specification stability through the fermentation regulation method.

[0028] The advantages and positive effects of this invention are as follows:

[0029] 1. This invention proposes a fermentation regulation method for high DHA and low DPA lipids in Schizochytrium, which guides cells to form a metabolic state that is conducive to DHA accumulation and inhibits DPA accumulation at different fermentation stages, thus solving the problem in the prior art that it is difficult to stably obtain high DHA and low DPA while ensuring high lipid yield.

[0030] 2. This invention introduces naturally derived small molecules to moderately inhibit the FAS competitive pathway, and provides feasible single / combination concentration ranges and preferred combination combinations (curcumin, resveratrol, ellagic acid and oleanolic acid). Without changing the genetic background of the strain, it reduces the competition between precursors and reducing power, providing more favorable resource conditions for the synthesis of DHA via the PKS pathway.

[0031] 3. This invention proposes a programmed oxygen supply strategy of "reducing oxygen supply and restoring oxygen supply", which is achieved through cascaded control of ventilation (L / min) and stirring intensity (rpm). The target range of DO is used as the process command. By taking advantage of the difference between the strong oxygen dependence of the ELO / DES pathway and the relative hypoxia dominance of the PKS pathway, metabolic pathway bias is achieved in the key time window, thereby increasing the proportion of DHA and inhibiting the accumulation trend of DPA.

[0032] 4. This invention enhances the fermentation process in stages by gradually increasing the temperature (28–34℃) and links it with the oxygen supply program and feed control to further stabilize the switching of metabolic states and the formation of fatty acid profiles. Under the optimal fermentation cycle of 108 h, this method can stably obtain 67.5% DHA and 4.5% DPA (as a percentage of total fatty acids) and 84.3 g / L of oil in a 5 L fermenter. After being scaled up to a 500 L fermenter, it can still stably obtain 68.3% DHA and 4.8% DPA (as a percentage of total fatty acids) and 89.7 g / L of oil, demonstrating significant feasibility and stability for engineering scale-up. Attached Figure Description

[0033] Figure 1This invention illustrates the changes in the relative expression intensity of key genes between the control group and the programmed oxygen supply group at 48h and 72h.

[0034] Figure 2 This invention illustrates the changes in the relative expression intensity of key genes between the control group and the temperature gradient group at 72h and 96h.

[0035] Figure 3 This is the gas phase spectrum of fatty acids after 108 hours under combined fermentation regulation in an embodiment of the present invention. Detailed Implementation

[0036] The following examples are used to further illustrate the present invention, but should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make equivalent substitutions or optimizations to the culture medium formulation, feeding strategy, detection methods, and process parameters without departing from the spirit of the present invention, and all such substitutions should fall within the scope of protection of the present invention.

[0037] This invention provides a fermentation regulation method and application for high-DHA, low-DPA lipids from Schizochytrium. The basic idea is as follows: without altering the genetic background of Schizochytrium, naturally derived small molecules are added to the system in the early and middle stages of fermentation to moderately inhibit the fatty acid synthase (FAS) competitive pathway, thereby reducing the competition between precursors and reducing power. Subsequently, a programmed control of "normal oxygen supply—low oxygen supply induction—restoration oxygen supply" is implemented during the critical fermentation time window. The target DO range for each stage is maintained through cascaded adjustments of aeration rate and stirring intensity, thereby guiding metabolic pathway bias and inhibiting the accumulation trend of DPA. Furthermore, the fermentation process is intensified in stages using a stepwise temperature adjustment method, and when necessary, it is linked with feed control to maintain the carbon source concentration within a set range, so as to stably achieve a high DHA ratio, low DPA content, and significantly increase lipid yield under fermenter conditions.

[0038] Preferably, the Schizochytrium sp. HX-308 is the accession number CCTCC No. M209059; other oil-producing strains of the Schizochytrium genus commonly used in the art may also be used.

[0039] The culture medium and fermentation conditions used in this invention are as follows:

[0040] The culture medium components used in each embodiment are as follows, but are not limited to these:

[0041] Seed culture medium (example formulation): glucose 50 g / L, yeast extract 2–4 g / L, sodium glutamate 15–25 g / L, MgSO4·7H2O 3–4 g / L, Na2SO4 16–20 g / L, KCl 1–2 g / L, NaCl 1–2 g / L, KH2PO4 3–5 g / L, with water as solvent and autoclaved at 121℃ for 15 min.

[0042] Fermentation medium (example formulation): glucose 80–120 g / L, yeast extract 3–6 g / L, sodium glutamate 20–30 g / L, MgSO4·7H2O 4–6 g / L, Na2SO4 18–22 g / L, KCl 1–2 g / L, NaCl 1–2 g / L, KH2PO4 3–5 g / L, (NH4)2SO4 2–6 g / L, sterilized in water at 121°C for 15 min by autoclaving.

[0043] It should be noted that small molecule regulators are not an inherent component of the culture medium, and their addition method and timing are described separately in each example.

[0044] To improve the reproducibility of the scale-up process, separate seed preparation procedures were described for 5 L and 500 L.

[0045] (1) Seed preparation and inoculation for 5 L fermenters

[0046] Schizochytrium was inoculated into seed culture medium and cultured at 28°C and 170 rpm for about 20 h to obtain primary seeds; then, it was transferred to fresh seed culture medium at an inoculation rate of 8–15% (v / v) and cultured under the same conditions for 24 h to obtain secondary seeds.

[0047] Preferably, secondary seeds are used for OD inoculation of 5 L fermenters. 600 The inoculation volume is 8–12, preferably 8–10; for a 5 L fermenter, the inoculation volume is 8–15% of the working volume, more preferably 10%. Microscopic examination can be performed before inoculation to confirm the absence of contamination.

[0048] (2) Seed preparation and inoculation for 500 L fermenters

[0049] The inoculum was first cultured in shake flasks for two generations, and microscopic examination confirmed that it was free of contamination. Then, it was inoculated into a 50 L seed tank at a 1% (v / v) inoculum to obtain seed culture for a 500 L fermenter. The initial stirring speed was 200 rpm, maintained for 16 h. The temperature was 28℃, and the aeration rate was 25 L / min. At h16, 18, 20, and 22, the stirring speed was increased by 10 rpm.

[0050] Microscopic examination was performed at 22 hours to confirm the absence of bacteria, and OD was measured. 600 When OD 600 When the OD value is >10, it is transferred to a 500 L fermenter for fermentation on the 24th hour; if the OD value is >10, it is transferred to a 500 L fermenter for fermentation. 600 If the requirements are not met, the seed tank culture time should be extended appropriately until the requirements for transfer to another tank are met.

[0051] The preferred carbon source for feeding is a combination of glucose and glycerol in a mass ratio of 30:70 to 50:50, preferably 40:60; the carbon source concentration is maintained at 5–15 g / L by feeding.

[0052] Oil and fat testing can be performed as follows:

[0053] After fermentation, a sample of the fermentation broth was taken. The system was first adjusted to alkalinity (pH approximately 11) using NaOH, and then 0.3% (w / w) of cell wall lysis enzyme was added. Enzymatic hydrolysis was carried out at approximately 50°C for approximately 4 hours to promote cell wall disruption and oil release. After enzymatic hydrolysis, hexane was added for extraction, and the mixture was allowed to stand for separation. The upper organic phase was collected. The extraction was repeated 2–4 times or until the color of the upper organic phase became significantly lighter. The organic phases were combined and the solvent was removed to obtain crude oil, which was used to calculate the total oil yield (g / L).

[0054] The obtained oil was subjected to methyl esterification: The obtained oil sample (approximately 60.0 mg) was placed in a stoppered test tube, and 4 mL of isooctane was added and dissolved completely. Then, 200 μL of 2 mol / L potassium hydroxide-methanol solution was added, the tube was sealed tightly, and the mixture was shaken vigorously for 30–60 s. The mixture was then allowed to stand at room temperature for 10–15 min. After the reaction was complete, approximately 1 g of sodium bisulfate was added. After standing and separating the layers, the upper organic phase was aspirated, filtered through a 0.22 μm organic phase filter membrane, and transferred to a sample vial. The proportions of each fatty acid were determined by gas chromatography.

[0055] It should be noted that the above steps are examples of commonly used testing procedures. Those skilled in the art can make equivalent adjustments to the cell disruption method, number of extractions, and methyl esterification conditions without affecting the comparability of the test results.

[0056] Example 1: Addition scheme for naturally derived small molecules to inhibit the FAS competitive pathway

[0057] This embodiment aims to illustrate that, without altering the genetic background of Schizochytrium, by adding naturally derived small molecules to the system in the early and middle stages of fermentation, the FAS pathway can be moderately inhibited, thereby reducing the competitive consumption of precursors and reducing power. This provides the foundation for subsequently increasing the proportion of DHA and decreasing the proportion of DPA, and determines the recommended final concentration and the method of adding the small molecules in stages.

[0058] A 5 L fermenter fermentation system was established according to the above culture medium and fermentation conditions for small molecule screening. The working volume of the 5 L fermenter was 3.0 L, the inoculum size was 10% (v / v), and the seed OD at inoculation was [not specified]. 600 The concentration was 8–10, and microscopic examination was performed before inoculation to confirm the absence of contamination. In this embodiment, normal fermentation conditions were used for screening in this part: aeration rate of 3.0 L / min, temperature of 28℃, and stirring speed of 450 rpm. The screening fermentation time in this embodiment was 120 h (which can also be adjusted within the range of 96–120 h); except for the small molecule addition scheme, the other culture media remained consistent with the fermentation process.

[0059] The naturally derived small molecules are curcumin, resveratrol, ellagic acid, and oleanolic acid. Each small molecule was prepared as a stock solution (200 mM) using DMSO, sterilized by 0.22 μm filtration, and then used for later use. When added to the fermentation system, the final solvent volume fraction was controlled to be ≤0.5% (v / v), and an equal volume solvent control group was set up. To cover the critical metabolic transition period, the small molecules were added in stages: the first addition was made at 8 h of fermentation to reach a final concentration of 50%; the second addition of the same amount was made at 24 h of fermentation to reach a final concentration of 100%.

[0060] To determine the concentration window for single use, this embodiment selected three representative final concentrations for comparison for each small molecule: curcumin 5, 10, and 15 μM; resveratrol 20, 40, and 60 μM; ellagic acid 10, 20, and 40 μM; and oleanolic acid 5, 10, and 20 μM. In addition to single use, four small molecule combination groups were also set up, with the final concentrations of each component in combination being: curcumin 10 μM, resveratrol 20 μM, ellagic acid 20 μM, and oleanolic acid 10 μM, added in portions at 8 h and 24 h of fermentation. After fermentation, the total oil yield (g / L) and fatty acid composition were determined according to general oil and fatty acid detection methods, with particular attention to C14:0, C16:0, DPA, and DHA.

[0061] The experimental results are shown in Table 1: Compared with the control group, the addition of the four natural small molecules individually can reduce the ratio of C14:0 to C16:0 and increase the proportion of DHA to varying degrees. Further comparison of the effects of each monomer at different final concentrations revealed that the optimal final concentrations for each of the four monomers were: curcumin 10 μM (C14: 0.46%, C16: 0.172%, DPA 16.6%, DHA 54.1%, total oil 63.5 g / L); resveratrol 20 μM (C14: 0.48%, C16: 0.175%, DPA 16.4%, DHA 54.0%, total oil 64.5 g / L); ellagic acid 20 μM (C14: 0.45%, C16: 0.170%, DPA 16.2%, DHA 54.5%, total oil 64.9 g / L); oleanolic acid 10 μM (C14: 0.47%, C16: 0.174%, DPA 16.3%, DHA 54.5%, total oil 64.9 g / L); and oleanolic acid 10 μM (C14: 0.47%, C16: 0.174%, DPA 16.3%, DHA 54.5%).

[0062] (54.2%, total fat 65.7 g / L). The above results indicate that the monomer can achieve a clear improvement in the fatty acid profile at its optimal final concentration, but the improvement is still limited.

[0063] Based on this, the four small molecules were combined at their optimal final concentrations (curcumin 10 μM, resveratrol 20 μM, ellagic acid 20 μM, and oleanolic acid 10 μM), and added in portions at 8 h and 24 h of fermentation. The combined group showed the most significant improvement: C14:0 further decreased to 3.2%, C16:0 decreased to 15.7%, DPA decreased to 15.3%, DHA increased to 57.6%, and total oil increased to 68.3 g / L, which was better than any single-monomer optimal group, indicating that the four have an additive or synergistic effect in inhibiting the FAS competitive pathway. Therefore, in subsequent examples, the combined use of curcumin 10 μM, resveratrol 20 μM, ellagic acid 20 μM, and oleanolic acid 10 μM is preferred, and added in portions at 8 h and 24 h of fermentation.

[0064] Table 1. Effects of the addition of four small molecules on fatty acid ratio and total fat content.

[0065]

[0066] Example 2: Programmed control of aeration rate and stirring intensity

[0067] This embodiment aims to illustrate that, without altering the genetic background of *Schizochytrium*, the difference between its oxygen-dependent elongation / desaturation enzyme pathway and the relatively hypoxic-dominant PKS pathway can be utilized. Simultaneously, changes in oxygen levels can easily alter the relative flux of the chain elongation / double bond introduction pathway, ultimately favoring the DHA branch over the DPA branch. Maintaining the target DO range through cascaded regulation of aeration rate and stirring intensity can increase the DHA proportion and suppress the DPA accumulation trend under fermenter conditions.

[0068] Establish a 5 L fermenter fermentation system according to the culture medium and fermentation conditions described above.

[0069] Except for the adjustment of stirring speed and aeration rate, the fermentation parameters are consistent with those in Example 1. This example adopts a cascaded control method with the DO target range as the process command, and maintains the set DO by adjusting the aeration rate (L / min) and stirring speed (rpm). Specifically, the oxygen supply process is divided into three continuous stages: the biomass accumulation stage is 0–24 h of fermentation, with a target DO of 40–60%; the induction stage is 24–48 h of fermentation, with a target DO of 5–10%; and the recovery stage is after 48 h of fermentation, with a target DO of 15–25%. In a 5 L fermenter, to achieve the above-mentioned DO target range, the following typical control points (example) can be used: 0–24 h: aeration rate of 3.0 L / min and stirring speed of 400 rpm to stably maintain DO 40–60%; 24–48 h: aeration rate of 1.0 L / min and stirring speed of 250 rpm to stably maintain DO 5–10%; after 48 h: aeration rate of 2.5 L / min and stirring speed of 450 rpm to stably maintain DO 15–25%. In actual operation, stirring speed adjustment can be prioritized to meet the DO setting. When stirring reaches the upper or lower limit of the set value, the aeration rate can be adjusted accordingly to improve control stability and repeatability between different fermenter types. The results showed that, compared with the control group, the programmed oxygen supply group obtained a fatty acid profile more conducive to DHA accumulation: C14:0 decreased to 5.2%, C16:0 decreased to 14.3%, DPA decreased to 12.7%, and DHA increased to 61.3%. The results above indicate that reducing DO to 5-10% during the critical fermentation window of 24–48 h and restoring it to 15-25% after 48 h can effectively guide cells to form a metabolic state that favors the accumulation of PKS pathway products, thereby significantly increasing the proportion of DHA and inhibiting the accumulation trend of DPA (Table 2).

[0070] Preferably, to verify the effect of programmed oxygen supply switching on the fermentation process at the molecular level, samples were taken at 24 h, 48 h, and 72 h of fermentation to determine the transcriptional level of key genes. The specific operation is as follows: at each time point, 1 mL of fermentation broth was taken from the fermenter, centrifuged at 4°C and 8000×g for 5 min to collect the cell pellet; after washing once with RNase-free water, it was immediately flash-frozen in liquid nitrogen and stored at -80°C for later use. For RNA extraction, the bacterial cell pellet was added to a centrifuge tube containing Trizol or equivalent lysis buffer, along with an equal volume of 0.5 mm glass beads. Lysis was performed at 4°C using a shaking method (example: 60 Hz, 60 s × 3 times, with a 60 s interval on ice in between). Total RNA extraction was then performed according to the kit instructions. Genomic DNA contamination was removed using DNase I (example: 37°C for 15 min), and RNA integrity and purity were assessed by 1% agarose gel electrophoresis or spectrophotometry (A260 / A280 = 1.8–2.1). For reverse transcription, 1 μg of total RNA was used to synthesize cDNA using a commercial reverse transcription kit (example: 42°C for 15–30 min, followed by a 5 min incubation at 85°C). The resulting cDNA was diluted to a uniform fold with RNase-free water and used as a qPCR template. The qPCR reaction system, using 20 μL as an example, consisted of: 10 μL of 2×SYBR Green qPCR Mix, 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 1.0 μL of cDNA template, and RNase-free water to a final volume of 20 μL. An example amplification program was: 95℃ for 30 s pre-denaturation; 95℃ for 5 s, 60℃ for 30 s, for 35 cycles. Melting curve analysis was then performed to confirm amplification specificity. Normalization was performed using the internal reference gene (β-actin in this example), and the relative expression level was calculated using the 2^-ΔΔCt method. The relative fold change of the programmed oxygen supply group was obtained by using the expression level of the control group at the corresponding time point as a baseline (set as unit 1). Specific primer sequences are shown in Table 3.

[0071] Table 3 Primer sequence listing

[0072]

[0073] like Figure 1 As shown, Figure 1 The graph shows the relative expression intensity changes of key genes between the control group and the programmed oxygen supply group at 48h and 72h, where the horizontal axis represents key genes (orfA, orfB, orfC, FAS, Δ8DE, Δ15DE).

[0074] The vertical axis represents the relative expression level of genes. Results: Gene expression in the programmed oxygen supply group was significantly upregulated, while FAS and desaturase gene expression was significantly downregulated. Key genes included PKS pathway-related genes orfA, orfB, and orfC, as well as representative genes of the elongation-desaturase pathway, such as FAS, Δ8 desaturase (Δ8DE), and Δ15 desaturase (Δ15DE). The results showed that at the end of the low oxygen induction phase (48 h, DO 5–10% window), orfA, orfB, and orfC were upregulated by 7.3-fold, 4.5-fold, and 5.1-fold, respectively, while FAS was significantly downregulated to 23.7% of the control group. Δ8 desaturase and Δ15 desaturase were 58.3% and 67.4% of the control group, respectively. During the recovery phase (72 h, DO 15–25%), the above trend continued, with orfA, orfB, and orfC remaining at 5.9-fold, 3.6-fold, and 4.2-fold of the control group, respectively. FAS was 31.4% of the control group, and Δ8 desaturase and Δ15 desaturase were 64.8% and 72.6% of the control group, respectively. The aforementioned changes in transcriptional levels are consistent with the results of a significant increase in DHA and a decrease in DPA in the fatty acid composition, further demonstrating that the three-stage programmed oxygen supply strategy can trigger the enhancement of the PKS pathway and simultaneously inhibit FAS and related desaturation reactions in the critical window, thereby achieving the effect of "increasing DHA and inhibiting DPA".

[0075] Table 2. Effects of programmed control of aeration rate and stirring intensity on fatty acid profiles and total oil content.

[0076]

[0077] Example 3: Temperature Step Control

[0078] This embodiment aims to illustrate that Schizochytrium can induce a metabolic state more conducive to the accumulation of PKS pathway products in cells at different stages by stepwise increasing the fermentation temperature, thereby improving the ability of lipid synthesis and accumulation. Under fermentation tank conditions, it can further increase the proportion of DHA, decrease the proportion of DPA, and promote lipid accumulation, thus determining the temperature stepwise program and its reproducible implementation method.

[0079] A 5 L fermenter fermentation system was established according to the above-described culture medium and fermentation conditions. The working volume of the 5 L fermenter was 3.0 L, the inoculum size was 10% (v / v), and the seed OD at inoculation was... 600The concentration was 8–10, and microscopic examination was performed before inoculation to confirm the absence of contamination. Except for temperature gradient control, the other process parameters were consistent with Example 1. To avoid interference from carbon source fluctuations on the fatty acid profile, this example used a fed-batch method to control the residual carbon source: the fed-batch carbon source was a combination of glucose and glycerol at a mass ratio of 40:60, and the residual carbon source was maintained at 5–15 g / L through feeding. To ensure comparability, oxygen supply and stirring were maintained under normal fermentation conditions (aeration rate 3.0 L / min, stirring speed 450 rpm), and the fermentation cycle was preferably 108 h (which could also be adjusted within the range of 96–120 h).

[0080] This embodiment sets up a constant-temperature control group and a temperature-stepped group. The constant-temperature control group was maintained at 28℃ throughout the fermentation process; the temperature-stepped group used a three-stage step-up program: 28℃ for 0–24 h of fermentation, 32℃ for 24–72 h of fermentation, and 34℃ after 72 h of fermentation until the end of fermentation (108 h in the example). Except for the temperature control method, the two groups maintained the same culture medium, inoculum size, feeding strategy, aeration rate, and stirring intensity. After fermentation, the total oil yield (g / L) and fatty acid composition (expressed as a percentage of total fatty acids) were determined according to general oil and fatty acid detection methods, with particular attention to C14:0, C16:0, DPA, and DHA.

[0081] The results showed that, compared with the isothermal control group, the fatty acid profile of the temperature-stepped group shifted significantly towards higher DHA and lower DPA, accompanied by an increase in oil yield. The C14:0 ratio significantly decreased to 2.1%, the C16:0 ratio significantly decreased to 12.8%, the DPA ratio significantly decreased to 10.9%, the DHA ratio significantly increased to 61.3%, and the total oil content significantly increased to 78.2 g / L (Table 5). These results indicate that by adjusting the temperature stepwise from 28℃ to 32℃ to 34℃, the formation of the target fatty acid profile can be enhanced in the mid-to-late stages, and the oil accumulation capacity can be improved.

[0082] Preferably, to verify the effect of temperature stepwise upregulation on pathway bias at the molecular level, samples were taken at 72 h (after completing the 32℃ stage) and 96 h (after entering the 34℃ stage) to measure changes in the transcriptional level of key genes. The specific procedures were as follows: at each time point, 1 mL of fermentation broth was taken from the fermenter, centrifuged at 4℃ and 8000×g for 5 min to collect the cell pellet; after washing once with RNase-free water, it was immediately flash-frozen in liquid nitrogen and stored at -80℃ for later use. For RNA extraction, the bacterial cell pellet was added to a centrifuge tube containing Trizol or equivalent lysis buffer, along with an equal volume of 0.5 mm glass beads. Lysis was performed at 4°C using a shaking method (example: 60 Hz, 60 s × 3 times, with a 60 s interval on ice in between). Total RNA extraction was then performed according to the kit instructions. Genomic DNA contamination was removed using DNase I (example: 37°C for 15 min), and RNA integrity and purity were assessed by 1% agarose gel electrophoresis or spectrophotometry (A260 / A280 = 1.8–2.1). For reverse transcription, 1 μg of total RNA was used to synthesize cDNA using a commercial reverse transcription kit (example: 42°C for 15–30 min, followed by a 5 min incubation period at 85°C). The resulting cDNA was diluted to a uniform fold with RNase-free water and used as a qPCR template. The qPCR reaction system, using 20 μL as an example, consisted of: 10 μL of 2×SYBR Green qPCR Mix, 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 1.0 μL of cDNA template, and RNase-free water to a final volume of 20 μL. An example amplification program was: 95℃ for 30 s pre-denaturation; 95℃ for 5 s, 60℃ for 30 s, for 35 cycles. Melting curve analysis was then performed to confirm amplification specificity. Normalization was performed using the internal reference gene (β-actin in this example), and the relative expression level was calculated using the 2^-ΔΔCt method. The relative fold change of the temperature gradient group was obtained by using the expression level of the control group at the corresponding time point as a baseline (set to 1). Specific primer sequences are shown in Table 4.

[0083] Table 4 Primer sequence listing

[0084]

[0085] Changes in the relative expression intensity of key genes between the 72h and 96h control groups and the temperature gradient group are as follows: Figure 2As shown, the horizontal axis represents key genes (orfA, orfB, orfC, FAS, Δ8DE, Δ15DE, ACC1, DGAT, etc.); the vertical axis represents the relative expression level of genes. The key genes include PKS pathway-related genes orfA, orfB, and orfC; competitive pathway representative genes FAS, Δ8 desaturase (Δ8DE), and Δ15 desaturase (Δ15DE); and key genes related to lipid synthesis and accumulation, ACC1, DGAT, LPAT, and GPAT. The results showed that, compared to the isothermal control group, after completing the 32℃ stage (72 h), the expression of PKS pathway-related genes was significantly enhanced, with orfA, orfB, and orfC upregulated by 3.8-fold, 2.6-fold, and 3.1-fold, respectively. Simultaneously, competitive pathway-related genes were suppressed, with FAS downregulated to 46.5% of the control group, and Δ8 desaturase and Δ15 desaturase at 72.4% and 78.6% of the control group, respectively. Furthermore, genes related to lipid synthesis and accumulation showed an upregulation trend during this stage. ACC1, DGAT, LPAT, and GPAT increased to 3.2, 4.8, 3.9, and 3.4 times that of the control group, respectively, indicating that temperature-induced upregulation not only promoted PKS pathway bias but also facilitated the enhancement of acetyl-CoA carboxylation and triglyceride assembly. Upon entering the 34℃ stage (96 h), this trend was further consolidated: orfA, orfB, and orfC remained at 4.6, 3.1, and 3.8 times that of the control group, respectively; FAS further decreased to 38.2% of the control group; and Δ8 desaturase and Δ15 desaturase were 66.9% and 73.5% of the control group, respectively. Simultaneously, ACC1, DGAT, LPAT, and GPAT remained at 4.6, 6.3, 3.2, and 4.8 times that of the control group, respectively. The changes in transcriptional levels mentioned above are consistent with the results of increased DHA, decreased DPA, and increased total lipid content in fatty acid composition. This indicates that the upregulation of the temperature gradient can, in the mid-to-late stages, continuously enhance the expression of PKS pathway-related genes and relatively inhibit competitive reactions, while also enhancing lipid synthesis and assembly capabilities, thereby achieving the effect of "increasing DHA, decreasing DPA, and increasing lipid yield".

[0086] Table 5. Effects of temperature gradient regulation on fatty acid profiles and total oil content.

[0087]

[0088] Example 4: Verification of Fermentation Co-regulation

[0089] This embodiment aims to illustrate that by combining the optimal conditions determined in the above three embodiments, a higher level of DHA ratio and low DPA content can be achieved under 5 L fermentation tank conditions, and the total oil yield can be significantly increased, forming a synergistic effect.

[0090] A 5 L fermenter system was established according to the above-described culture medium and fermentation conditions, with a working volume of 3.0 L and a fermentation cycle of 108 h. Except for the combined control conditions explicitly described in this embodiment, the other culture medium formulations, seed preparation and inoculation, feeding and residual carbon source control, detection methods, etc. are all consistent with the above-described culture medium and fermentation conditions.

[0091] The following combined regulation of optimal conditions was adopted in this embodiment: (1) Small molecule inhibition of FAS competitive pathway (combined use + partial addition): curcumin 10 μM, resveratrol 20 μM, ellagic acid 20 μM, oleanolic acid 10 μM; each small molecule was prepared with DMSO to prepare a 200mM mother liquor and filtered with 0.22 μm for sterilization; it was added in partial addition: the first addition was made at 8 h of fermentation so that each component reached 50% of the target final concentration; the same amount was added again at 24 h of fermentation so that each component reached 100% of the target final concentration. (2) Programmed oxygen supply / stirring cascade control (DO target range): 0–24 h maintain DO 40–60% (aeration rate 3.0 L / min, stirring 400 rpm); 24–48 h maintain DO 5–10% (aeration rate 1.0 L / min, stirring 250 rpm); after 48 h maintain DO 15–25% (aeration rate 2.5 L / min, stirring 450 rpm). (3) Temperature stepwise adjustment: 0–24 h 28℃, 24–72 h 32℃, after 72 h 34℃ until fermentation ends.

[0092] After 108 hours of fermentation, the combined control group consistently achieved the following results: total oil yield of 84.3 g / L, DHA of 67.5%, and DPA of 4.5%. (Gas chromatogram shown below.) Figure 3 This indicates that combining competitive pathway inhibition, programmed oxygen supply control, and temperature gradient enhancement can effectively further improve the simultaneous optimization of high DHA, low DPA, and high oil yield.

[0093] Comparative Example 500 L: Scale-up Verification Comparison with and without Combined Control

[0094] To verify the reproducibility of the combined control strategy under scale-up conditions, fermentation was conducted in both the comparative and example groups under the same 500 L fermenter conditions. The 500 L fermenter had a working volume of approximately 300 L. Seed preparation and inoculation followed the 50 L seed tank process described in the general section, resulting in seed culture which was then transferred to the 500 L fermenter. The comparative group used conventional fermentation methods: no of the four small molecules were added (if the process required solvent addition, an equal volume of DMSO was added as a control). Oxygen supply and stirring did not involve the three-stage program switching (maintaining conventional stable oxygen supply control). The temperature was maintained at 28°C throughout. The carbon source addition and control methods were consistent with the example group to ensure comparability. The example group fully implemented the combined control scheme under 500 L conditions: the four small molecules were added in portions at 8 h and 24 h to the aforementioned final concentrations; oxygen supply / stirring was cascaded and controlled within the target ranges of 0–24 h DO 40–60%, 24–48 h DO 5–10%, and after 48 h DO 15–25%, achieved through aeration rate and stirring intensity. In the example group, during 500 L scale-up, the aeration rate and stirring intensity were adjusted to typical control points matching the tank type to achieve the above-mentioned DO range: 0–24 h, aeration rate of 250 L / min, stirring at 200 rpm to maintain DO 40–60%; 24–48 h, aeration rate of 150 L / min, stirring at 150 rpm to maintain DO 5–10%; after 48 h, aeration rate of 200 L / min, stirring at 250 rpm to maintain DO 15–25%; temperature was increased in a stepwise manner from 28℃ (0–24 h) → 32℃ (24–72 h) → 34℃ (after 72 h); and the feed was maintained at a carbon source concentration of 5–15 g / L. After fermentation in both groups, the total oil yield and fatty acid composition were measured using the same method.

[0095] The test results showed that the comparative example (500 L conventional fermentation) had an oil yield of 65.3 g / L, DHA of 46.5%, and DPA of 17.8%; while the example group (500 L combined regulation) saw an increase in oil yield to 89.7 g / L, with DHA remaining stable at 68.3% and DPA decreasing to 4.8%. These scale-up comparisons demonstrate that the combined regulation scheme of this invention can still stably achieve simultaneous optimization of high DHA, low DPA, and high oil yield at a 500 L scale, exhibiting good feasibility for engineering scale-up and batch stability.

[0096] In summary, the fermentation process control method for high DHA, low DPA and high oil yield of Schizochytrium provided by this invention has the advantages of not changing the genetic background, safety and simplicity, engineerable parameter replication, easy scale-up and migration and strong applicability. It can reduce the burden of DHA / DPA separation and purification and improve product specification stability, and has good prospects for industrial application.

[0097] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for regulating the fermentation of Schizochytrium bacteria with high DHA and low DPA lipids, characterized in that, Without altering the genetic background of Schizochytrium, a combined process control method is implemented sequentially along the time axis during fermentation, including competitive pathway inhibition, programmed oxygen supply and stirring control, and temperature stepwise regulation, to increase the proportion of DHA, decrease the proportion of DPA, and improve oil yield. The method includes at least the following steps: (1) Schizochytrium is inoculated into a fermentation medium for fermentation culture; (2) Naturally derived small molecules are added to the fermentation system in the early and / or middle stages of fermentation to moderately inhibit the fatty acid synthase (FAS) pathway; (3) Programmed oxygen supply and stirring cascade control with dissolved oxygen (DO) target range as the process command are implemented during fermentation, so that the fermentation process sequentially goes through the biomass accumulation stage, the low oxygen supply induction stage, and the recovery stage; (4) The fermentation temperature is stepped up to form a segmented enhancement that complements step (3); (5) Schizochytrium oil product is obtained after fermentation.

2. The fermentation regulation method according to claim 1, characterized in that, The Schizochytrium sp. HX-308 was identified as having the accession number CCTCC No. M209059.

3. The fermentation regulation method according to claim 1, characterized in that, The naturally derived small molecules are selected from one or more of curcumin, resveratrol, ellagic acid, and oleanolic acid, and are used to reduce the competitive consumption of acetyl / malonyl-CoA and reducing power in the FAS pathway, thereby improving the availability of polyunsaturated fatty acid synthesis resources.

4. The fermentation regulation method according to claim 3, characterized in that, The final concentration range of the naturally derived small molecules in the fermentation system when used alone is: curcumin 5–30 μM, resveratrol 10–60 μM, ellagic acid 10–80 μM, and oleanolic acid 5–60 μM.

5. The fermentation regulation method according to claim 3, characterized in that, The final concentration range of the naturally derived small molecules in the fermentation system is as follows: curcumin 3–15 μM, resveratrol 7–30 μM, ellagic acid 10–40 μM, and oleanolic acid 5–25 μM.

6. The fermentation control method according to any one of claims 3-5, characterized in that, The naturally derived small molecules are added to the fermentation system in stages, with the first addition occurring 0–12 h into the fermentation process and the second addition occurring 20–30 h into the fermentation process.

7. The fermentation control method according to claim 6, characterized in that, The first addition was made after 8 hours of fermentation, and the second addition was made after 24 hours of fermentation. The amount added each time was 50% of the target final concentration.

8. The fermentation control method according to any one of claims 1-5, characterized in that, The programmed oxygen supply and stirring cascade control includes three consecutive stages: (1) Biomass accumulation stage: fermentation 0–24 h, DO is 40–60%; (2) Low oxygen supply induction stage: fermentation 24–48 h, DO is 5–10%; (3) Recovery stage: after fermentation 48 h, DO is 15–25%.

9. The fermentation regulation method according to claim 8, characterized in that, In a 5 L fermenter, to achieve the target DO range for the three consecutive stages, the following typical control points were used: 0–24 h: aeration rate 3.0 L / min, stirring intensity 400 rpm; 24–48 h: aeration rate 1.0 L / min, stirring intensity 250 rpm; after 48 h: aeration rate 2.5 L / min, stirring intensity 450 rpm.

10. The fermentation control method according to claim 8, characterized in that, In a 500 L fermenter, to achieve the target DO range for the three consecutive stages, the following typical control points were used: 0–24 h: aeration rate 250 L / min, stirring intensity 200 rpm; 24–48 h: aeration rate 150 L / min, stirring intensity 150 rpm; after 48 h: aeration rate 200 L / min, stirring intensity 250 rpm.

11. The fermentation control method according to claim 1, characterized in that, The temperature step adjustment is a step-by-step increase within the range of 28–34℃, and the temperature step has two or three segments.

12. The fermentation control method according to claim 11, characterized in that, The temperature gradient consists of three steps, with the following temperature program: 28℃ for 0–24 h of fermentation, 32℃ for 24–72 h of fermentation, and 34℃ after 72 h of fermentation until the end of fermentation.

13. The fermentation control method according to claim 1, characterized in that, The total fermentation cycle is 96–120 h.

14. The fermentation control method according to claim 13, characterized in that, The total fermentation cycle is 108 hours.

15. The fermentation regulation method according to claim 1, characterized in that, The fermentation regulation method also includes a feeding control step: the feeding carbon source is a combination of glucose and glycerol, with a mass ratio of 30:70 to 50:50, and the residual carbon source concentration is maintained at 5–15 g / L through feeding.

16. The fermentation control method according to claim 15, characterized in that, The mass ratio of glucose to glycerol is 40:

60.

17. The fermentation control method according to any one of claims 9-16, characterized in that, The combined process control adopts the following optimal combination: (1) The natural source small molecules are curcumin, resveratrol, ellagic acid and oleanolic acid in combination, with final concentrations of 10 μM curcumin, 20 μM resveratrol, 20 μM ellagic acid and 10 μM oleanolic acid, respectively, and are added in batches at 8 h and 24 h of fermentation, with each addition amount being 50% of the target final concentration of each component; (2) The target range of DO for the programmed oxygen supply and stirring cascade control is: DO 40–60% for 0–24 h, DO 5–10% for 24–48 h, and DO 15–25% after 48 h; (3) The temperature gradient is: 28℃ for 0–24 h, 32℃ for 24–72 h, and 34℃ after 72 h.

18. The fermentation control method according to claim 17, characterized in that, Under the conditions of a 5 L fermenter and a fermentation cycle of 108 h, the optimal combination described above can yield an oil yield of 84.3 g / L, with DHA accounting for 67.5% of the total fatty acids and DPA accounting for 4.5% of the total fatty acids in the obtained oil.

19. The fermentation regulation method according to claim 17, characterized in that, The fermentation control method, when scaled up to a 500L fermenter, still yields an oil yield of 89.7 g / L, with DHA accounting for 68.3% of the total fatty acids and DPA accounting for 4.8% of the total fatty acids in the obtained oil.

20. The application of the fermentation regulation method according to any one of claims 1-19 in the preparation of high-DHA, low-DPA Schizochytrium oil products, characterized in that, The fermentation regulation method described above reduces the burden of DHA / DPA separation and purification and improves product specification stability.

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