An efficient cultivation method of apigenin biosynthesis yeast

Abstract

This application relates to the fields of bio-fermentation engineering and synthetic biology, and discloses a method for efficiently cultivating yeast strains for apigenin biosynthesis. The method includes: inoculating and fermenting a pre-constructed engineered yeast strain capable of generating apigenin from naringenin as a precursor; the engineered yeast strain containing genes encoding heterologous apigenin biosynthesis pathways and genes encoding flavonoid synthase II derived from *Erigeron breviscapus*; cooling and continuously adding a sterile feed solution containing a host-guest inclusion complex of hydroxypropyl-β-cyclodextrin and naringenin; lowering the dissolved oxygen setpoint and monitoring the redox potential and oxygen uptake rate change rate online; and triggering a pulsed feed of sodium isoascorbate when the potential and rate of change reach threshold values. This invention relieves substrate toxicity and product inhibition through phase transfer inclusion, precisely quenches reactive oxygen species using pulsed feed based on potential thresholds, and eliminates oxygen supply competition through gas-liquid mass transfer compensation and interlock control, thereby protecting the catalytic activity of the core enzyme and increasing the fermentation yield of apigenin.

Description

Technical Field

[0001] This invention relates to the fields of bio-fermentation engineering and synthetic biology, specifically to a method for the efficient cultivation of yeast strains for apigenin biosynthesis. Background Technology

[0002] Apigenin is a natural flavonoid compound with important pharmacological activities. In recent years, the use of synthetic biology techniques to construct engineered yeast cells and heterologously synthesize apigenin in fermenters has become an important development direction to replace traditional plant extraction methods.

[0003] In the process of synthesizing apigenin using engineered yeast with naringenin as a precursor, the precursor naringenin has extremely poor water solubility and strong toxic inhibitory effect on chassis cells, which limits its high concentration addition in the liquid phase system. At the same time, the target product apigenin is highly hydrophobic and is very easy to physically deposit in organelles and cytoplasm. This transmembrane secretion barrier will cause severe product feedback inhibition and block secondary metabolic flux.

[0004] Furthermore, flavonoid synthase II, the key enzyme catalyzing the conversion of naringenin to apigenin, belongs to the plant-derived cytochrome P450 enzyme system, and its catalytic hydroxylation reaction is highly dependent on the participation of free oxygen. Under the high-intensity metabolic flux in the later stages of fermentation, this enzyme is prone to uncoupling reactions during electron transfer, releasing large amounts of reactive oxygen species into the liquid phase and intracellular space. This not only triggers lipid peroxidation, damaging the microsomal membrane structure, but also leads to irreversible inactivation of membrane-anchored flavonoid synthase II.

[0005] To mitigate oxidative stress damage in fermentation systems, conventional process improvements typically involve continuously adding antioxidants to the fermentation broth to quench free radicals. However, because strong reducing agents consume large amounts of dissolved oxygen during oxidative decomposition, this static and continuous addition not only keeps the fermentation system in a state of deep reduction for extended periods but also triggers severe oxygen competition between the liquid phase system and the aerobic enzyme system, causing flavonoid synthase II to lose its catalytic activity due to microenvironmental hypoxia.

[0006] In summary, existing fermentation processes cannot effectively balance the engineering contradictions among the non-toxic supply of hydrophobic substrates, the precise elimination of reactive oxygen species, and the mass transfer and oxygen supply of aerobic catalytic enzymes, resulting in a severe limitation on the substrate conversion rate and final fermentation yield of engineered yeast in synthesizing apigenin. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a highly efficient method for cultivating yeast strains for apigenin biosynthesis. This method solves the problems in the existing process of engineered yeast fermentation for apigenin synthesis, where the conversion rate is limited due to the poor water solubility and cell-inhibiting effects of the precursor substrate, and the oxidative stress damage to cells caused by the release of reactive oxygen species from key enzymes. Furthermore, conventional antioxidant methods can lead to oxygen competition and enzyme inactivation, thus severely restricting the yield of apigenin fermentation.

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

[0009] This invention provides a highly efficient method for cultivating yeast strains that biosynthesize apigenin, employing the following technical solution:

[0010] A pre-constructed engineered yeast strain capable of generating apigenin from naringenin as a precursor was inoculated into a basic fermentation medium for fermentation. The engineered yeast strain contains a heterologous apigenin biosynthesis pathway gene and a flavonoid synthase II gene, wherein the flavonoid synthase II gene encodes flavonoid synthase II derived from Erigeron breviscapus.

[0011] Provide a sterile fed-batch solution containing a host-guest inclusion complex of hydroxypropyl-β-cyclodextrin and naringenin;

[0012] The temperature of the fermenter is lowered to a preset second temperature range and maintained, while the sterile feed liquid is continuously added to the fermentation broth.

[0013] During the continuous feeding of the sterile feed liquid, the dissolved oxygen setpoint of the fermenter is lowered, and the redox potential and oxygen uptake rate of the fermentation system are monitored in real time online.

[0014] When the redox potential is detected to be greater than the first potential threshold and the rate of change of oxygen uptake is less than the set rate of change threshold, the pulse flow of sodium isoascorbate aqueous solution is triggered, and at the same time as the pulse flow is triggered, the current stirring speed and aeration rate of the fermenter are simultaneously increased.

[0015] When the redox potential is detected to be less than or equal to the second potential threshold, the addition of the sodium isoascorbate aqueous solution is stopped, and the stirring speed and aeration rate are restored to their values ​​before floating. The control logic of pulse addition and restoration is executed cyclically until fermentation ends.

[0016] Wherein, the first potential threshold is greater than the second potential threshold.

[0017] By employing the above technical solution, and using hydroxypropyl-β-cyclodextrin as the phase transfer carrier, combined with specific online parameter monitoring and flow field interlock control logic, a significant improvement in substrate conversion rate and a resolution of the conflict between oxidative stress and oxygen supply are achieved. The core reaction mechanism and innovative steps of this invention are as follows:

[0018] Step 1: Phase Transfer Inclusion Detoxification Mechanism: Naringenin molecules enter the hydrophobic cavity of hydroxypropyl-β-cyclodextrin, forming a host-guest inclusion complex through van der Waals forces and hydrophobic interactions. This physical structure allows naringenin, which is normally insoluble in water, to exist stably in the aqueous phase at extremely high concentrations, while avoiding lipid bilayer damage caused by direct contact between free naringenin and the yeast cell membrane, thus achieving a non-toxic phase transfer supply of the substrate.

[0019] Step two, metabolic flux allocation regulation mechanism: During the mid-to-late stages of fermentation, the temperature and dissolved oxygen setpoints are lowered, forcibly shifting the metabolic flux of the engineered yeast cells from the primary biomass synthesis pathway to the secondary metabolite synthesis pathway. Under this state, cell proliferation stops, maintenance energy consumption decreases, and the substances and energy in the fermentation system are more concentrated in the catalytic reaction of flavonoid synthase II.

[0020] Step 3: Stress Recognition and Pulse Reduction Mechanism: The rate of change in redox potential and oxygen uptake rate of the fermentation broth can accurately reflect the oxidative stress state of cells. When the redox potential rises abnormally and the oxygen uptake rate drops sharply, it indicates that reactive oxygen species (ROS) generated by P450 enzyme uncoupling have severely inhibited the cellular respiratory chain. At this time, the pulsed addition of sodium isoascorbate is triggered. The isoascorbate ions can instantaneously donate electrons to undergo an oxidation reaction, thereby quenching ROS in the liquid phase and intracellular environment, and causing the redox potential to quickly fall back to a safe baseline.

[0021] Step four, gas-liquid mass transfer compensation and interlocking mechanism: Sodium isoascorbate consumes a large amount of dissolved oxygen during the quenching of reactive oxygen species and its oxidative decomposition. If sodium isoascorbate is simply added, it will cause instantaneous extreme hypoxia in the fermentation broth, resulting in irreversible inactivation of oxygen-dependent flavonoid synthase II.

[0022] This scheme forces a synchronous upward stirring speed and aeration rate at the instant the reducing agent is added via pulsed flow, thereby significantly increasing the gas-liquid mass transfer coefficient to counteract the oxygen-consuming effect of the reducing agent. Once the stress is relieved, the flow field parameters are immediately restored, thus constructing a dynamic interlocking equilibrium system that eliminates reactive oxygen species without generating oxygen competition.

[0023] Preferably, the conditions for inoculating the engineered yeast into the basal fermentation medium for fermentation are as follows:

[0024] The temperature is 28.0℃~32.0℃, the pH value is 5.5~6.5, and the dissolved oxygen setting value is 30%~50%. The fermentation continues until the optical density of the fermentation broth at a wavelength of 600nm reaches 80~120, and the initial carbon source concentration drops below 5.0g / L. At this point, the cooling and continuous addition of the aseptic feeding solution are started.

[0025] By adopting the above technical solution and setting specific initial temperatures and high dissolved oxygen conditions, the engineered yeast can be encouraged to undergo efficient aerobic respiration and cell division in the early stage of fermentation, rapidly accumulating biomass with high metabolic activity. Using optical density and carbon source consumption as process switching points ensures that the chassis cells enter the stable growth phase, preventing problems such as precursor accumulation or delayed secondary metabolism initiation when adding feedstock later.

[0026] Preferably, the preparation process of the aseptic fed-batch solution includes: mixing hydroxypropyl-β-cyclodextrin and naringenin at a molar ratio of 1.5 to 3.0:1, wherein naringenin is dissolved in an ethanol aqueous solution with a volume fraction of 60% to 80% to obtain a precursor solution, and hydroxypropyl-β-cyclodextrin is dissolved in deionized water to obtain a main solution; the precursor solution is added dropwise to the main solution at 45°C to 60°C and stirred for 4 to 8 hours; after depressurization to remove ethanol, the solution is freeze-dried; and the resulting dry powder is dissolved in deionized water to obtain an aseptic fed-batch solution with an equivalent concentration of 15.0 to 30.0 g / L based on naringenin.

[0027] By employing the above technical solution, the crystal lattice of naringenin is disrupted using an ethanol-water solution of a specific concentration, and sufficient molecular kinetic energy is provided under heating and stirring conditions, allowing naringenin molecules to efficiently shuttle into the cavity of hydroxypropyl-β-cyclodextrin. All ethanol solvent is removed through a vacuum concentration step, eliminating the toxic effects of organic solvents on the fermentation system and yeast cells. The resulting dry powder, after reconstitution, achieves an extremely high substrate equivalent concentration, meeting the feed requirements for high-density fermentation.

[0028] Preferably, the cooling is achieved by linearly cooling the fermenter at a rate of 0.2℃ / h to 0.8℃ / h, where the second temperature range is 22.0℃ to 26.0℃; and the rate of continuous addition of the aseptic liquid is 0.05 to 0.15 g / (L·h) in terms of naringenin equivalent.

[0029] By adopting the above technical solution and setting a specific linear cooling rate, cold shock stress response in yeast cells due to sudden temperature drops can be avoided, and the transition of metabolic pathways can be completed smoothly. The continuous flow acceleration rate is strictly controlled within the range of 0.05–0.15 g / (L·h), and the molar amount of substrate flowing into the system is matched with the Michaelis constant and maximum reaction rate of intracellular flavonoid synthase II. This avoids enzymatic reaction stagnation caused by insufficient substrate supply and prevents the accumulation of excessive hydroxypropyl-β-cyclodextrin in the fermentation broth, which could lead to a rapid deterioration of liquid rheology and osmotic pressure imbalance.

[0030] Preferably, the reduction of the dissolved oxygen setpoint in the fermenter means reducing the dissolved oxygen setpoint to 15%–25%.

[0031] The first potential threshold is +120mV to +180mV; the set rate of change threshold is -5.0mmol / (L·h). 2 The second potential threshold is +20mV to +80mV.

[0032] By employing the above technical solution, the basal dissolved oxygen level during the production period is maintained at a low level, limiting the excessive activity of terminal oxidases in the cellular respiratory chain and enriching oxygen molecules for the hydroxylation reaction of P450 enzymes. A control dead zone with hysteresis characteristics is constructed by setting +120mV to +180mV as the upper trigger threshold and +20mV to +80mV as the lower stop threshold, preventing the control system from oscillating and stopping at high frequencies due to measurement noise. This ensures that the cells are in a benign alternating cycle of mild stress and complete repair.

[0033] Preferably, when the pulsed flow of the sodium isoascorbate aqueous solution is triggered, the concentration of sodium isoascorbate in the fermentation system is controlled to reach 0.5-2.0 mM within 1-3 minutes;

[0034] The synchronous upward movement ratio is 30% to 60% of the stirring speed and aeration rate at the previous moment, respectively, when the fermenter triggers the pulse flow.

[0035] By employing the above technical solution, a high concentration gradient of antioxidants can be established in a very short time, enabling instantaneous neutralization of free radicals in the fermentation broth at a first-order reaction kinetic rate. Controlling the buoyancy ratio to 30%–60% of the original parameters provides an additional volumetric oxygen transfer coefficient that precisely compensates for the physical oxygen consumption deficit caused by the oxidative degradation of sodium isoascorbate, thus maintaining the stability of the gas-liquid two-phase microenvironment.

[0036] Preferably, the engineered yeast is *Yarrowia lipolytica* or *Saccharomyces cerevisiae*; the engineered yeast contains a heterologous apigenin biosynthesis pathway gene and a flavonoid synthase II gene, the heterologous apigenin biosynthesis pathway gene including a tyrosine ammonia-lyase gene, a 4-coumarate-coenzyme A ligase gene, a chalcone synthase gene, and a chalcone isomerase gene; the flavonoid synthase II gene encodes flavonoid synthase II derived from *Erigeron breviscapus*, and the flavonoid synthase II can catalyze the conversion of naringenin to apigenin.

[0037] By adopting the above technical solution, tyrosine ammonia-lyase is used to catalyze the production of p-coumaric acid from L-tyrosine, 4-coumaric acid-co-A ligase is used to catalyze the production of p-coumaric acid-co-co-A from p-coumaric acid, chalcone synthase is used to catalyze the formation of naringenin chalcone, chalcone isomerase is used to catalyze the isomerization of naringenin chalcone to naringenin, and flavonoid synthase II derived from *Erigeron breviscapus* is used to catalyze the production of apigenin from naringenin.

[0038] Using *Yarrowia lipolyticis* or *Saccharomyces cerevisiae* as a eukaryotic expression chassis can provide a membrane-localized and electron-transfer environment for plant-derived flavonoid synthase II, enabling it to maintain its catalytic activity within yeast cells. The key improvement of this invention lies in the synergistic control of substrate feeding, redox state, and gas-liquid mass transfer during the fermentation process of these engineered yeasts, rather than solely relying on the gene construction method of the engineered yeasts to achieve yield increases.

[0039] Preferably, the total fermentation time is 96–120 hours. After fermentation, the fermentation broth is centrifuged to remove solid precipitate, and the supernatant is collected. A 3.5–5.0 M hydrochloric acid solution is added to the supernatant to adjust the pH to 2.0–2.5, and the mixture is allowed to crystallize at 7–10°C for 18–24 hours. After separation and washing, the crystals are dried to obtain apigenin.

[0040] By employing the above technical solution, the target product, apigenin, remains encapsulated within the hydroxypropyl-β-cyclodextrin cavity and present in the supernatant at the fermentation endpoint. By adding high-concentration hydrochloric acid to create a strongly acidic environment, free hydrogen ions significantly alter the dielectric constant of the aqueous phase and disrupt the intermolecular hydrogen bond network, forcing the hydrophobic apigenin molecules to detach from the inclusion cavity. Under low-temperature static conditions, the free apigenin precipitates due to supersaturation, forming high-purity crystals, thus achieving efficient liquid-solid separation of the product.

[0041] Preferably, the total fermentation time is 96–120 hours. After fermentation, the fermentation broth is centrifuged to remove solid precipitate and the supernatant is collected. Anhydrous ethanol, with a volume equal to that of the supernatant, is added to the supernatant and the mixture is heated to 60°C and stirred for 1 hour. Then, it is cooled to 4°C and allowed to stand for crystallization for 12 hours. After separation and washing, the product is dried to obtain apigenin.

[0042] By employing the above-mentioned technical solution, anhydrous ethanol is added to the supernatant containing the inclusion complex and then heated. Ethanol molecules, acting as competitive guests, enter the hydrophobic cavity of hydroxypropyl-β-cyclodextrin in large quantities. Simultaneously, the high temperature increases the molecular thermal motion of the system, collectively breaking the binding force between apigenin and cyclodextrin. Subsequent rapid cooling significantly reduces the solubility of apigenin in the ethanol-water solution, promoting rapid rearrangement to form crystal nuclei and crystallize out. This separation process avoids the use of strong acids and reduces equipment corrosion.

[0043] This invention provides a highly efficient method for cultivating yeast strains that biosynthesize apigenin. It offers the following advantages:

[0044] 1. In this invention, a sterile feedstock containing a host-guest inclusion complex of hydroxypropyl-β-cyclodextrin and naringenin is continuously fed during fermentation. The hydrophobic cavity of cyclodextrin is used to transfer free naringenin in a non-toxic manner, and a high-affinity thermodynamic sink is constructed outside the cell to continuously extract the hydrophobic product synthesized intracellularly. Therefore, the toxic inhibition of substrate precursors on cells and the metabolic feedback inhibition caused by intracellular product deposition are eliminated, thereby improving the substrate conversion rate and final fermentation yield of apigenin.

[0045] 2. This invention monitors the redox potential and oxygen uptake rate of the fermentation system in real time online, and precisely triggers the pulsed addition of sodium isoascorbate aqueous solution when the monitored parameters reach the set threshold and stops the addition when the potential drops. Therefore, it can instantaneously quench lethal amounts of reactive oxygen species when the oxidative pressure accumulates to the critical point, avoiding the fermentation broth from falling into a continuous deep reduction state that triggers oxygen supply competition, thereby completely protecting the integrity of the cell membrane system and the efficient catalytic conformation of flavonoid synthase II.

[0046] 3. This invention forcibly synchronizes the stirring speed and aeration rate at the moment the reducing agent pulse is triggered and restores them after the pulse ends. By increasing the mechanical stirring power input and the gas surface drag force, the coagulation effect of the high-viscosity inclusion fluid on the bubbles is forcibly broken. Therefore, it compensates for the oxygen consumption gap caused by the oxidation and degradation of the reducing agent and offsets the mass transfer resistance caused by the increase in system viscosity. It overcomes the engineering repulsion problem between strong reducing system and aerobic catalytic system, and ensures the high respiratory activity and efficient mass exchange rate of yeast cells in viscous environment. Attached Figure Description

[0047] Figure 1 This is a dynamic change curve of redox potential provided in an embodiment of the present invention;

[0048] Figure 2 This is a dynamic change curve of oxygen uptake rate provided in an embodiment of the present invention;

[0049] Figure 3 The graph showing the dynamic change of intracellular reactive oxygen species levels provided in the embodiments of the present invention;

[0050] Figure 4 The diagram showing the dynamic change of flavonoid synthase II activity in an embodiment of the present invention;

[0051] Figure 5 The volumetric mass transfer coefficient dynamic change curve provided in the embodiments of the present invention;

[0052] Figure 6 The graph shows the dynamic change of the intracellular retention rate of the product provided in the embodiments of the present invention. Detailed Implementation

[0053] The technical solutions of 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.

[0054] In this invention, the engineered yeast is a pre-constructed recombinant yeast capable of generating apigenin from naringenin as a precursor. The engineered yeast can be *Yarrowia lipolytica* or *Saccharomyces cerevisiae*; the engineered yeast cells contain heterologous apigenin biosynthesis pathway genes and flavonoid synthase II genes.

[0055] The genes involved in the heterologous apigenin biosynthesis pathway include tyrosine ammonia-lyase genes, 4-coumaric acid-CoA ligase genes, chalcone synthase genes, and chalcone isomerase genes. Tyrosine ammonia-lyase catalyzes the deamination of L-tyrosine to p-coumaric acid; 4-coumaric acid-CoA ligase catalyzes the formation of p-coumaric acid to p-coumaroyl-CoA; chalcone synthase catalyzes the condensation of p-coumaroyl-CoA and malonyl-CoA to form naringenin chalcone; and chalcone isomerase catalyzes the isomerization of naringenin chalcone to naringenin. The flavonoid synthase II gene encodes flavonoid synthase II derived from *Erigeron breviscapus*, which catalyzes the formation of apigenin from naringenin.

[0056] The aforementioned genes can be introduced into yeast cells via genome integration or through expression vectors. These genes can use natural nucleotide sequences or optimized codon sequences suitable for expression in *Yarrowia lipolytica* or *Saccharomyces cerevisiae*. As long as the resulting engineered yeast strain can express the aforementioned enzymes and possess the ability to convert naringenin to apigenin, it can be used as the engineered yeast strain in the method of this invention.

[0057] In one specific embodiment, the engineered yeast is a *Yersinia lipolytica* engineered strain containing a tyrosine ammonia-lyase expression cassette, a 4-coumarate-coenzyme A ligase expression cassette, a chalcone synthase expression cassette, a chalcone isomerase expression cassette, and a flavonoid synthase II expression cassette derived from *Erigeron breviscapus*.

[0058] In another specific embodiment, the engineered yeast is a *Saccharomyces cerevisiae* strain containing the aforementioned expression cassette. The expression cassette includes a promoter, a coding gene, and a terminator, which can be constructed using conventional promoters and terminators suitable for yeast expression in the art.

[0059] Preparation Examples 1-3:

[0060] Preparation Example 1:

[0061] This preparation example provides a method for preparing a sterile fed-batch solution containing a host-guest inclusion complex of hydroxypropyl-β-cyclodextrin and naringenin, comprising the following steps:

[0062] Hydroxypropyl-β-cyclodextrin and naringenin were prepared in a molar ratio of 2.0:1. Naringenin was dissolved in a 70% ethanol aqueous solution to prepare a precursor solution, and hydroxypropyl-β-cyclodextrin was dissolved in deionized water to prepare a main solution.

[0063] The precursor solution was added dropwise to the main solution at 55°C and stirred for 6 hours. After ethanol removal by vacuum concentration, the solution was freeze-dried and the resulting dry powder was dissolved in deionized water. The solution was then filtered through a microporous membrane to remove bacteria, resulting in a sterile fed-batch solution with an equivalent concentration of 20.0 g / L based on naringenin.

[0064] Preparation Example 2:

[0065] This preparation example provides a method for preparing a sterile fed-batch solution containing a host-guest inclusion complex of hydroxypropyl-β-cyclodextrin and naringenin, comprising the following steps:

[0066] Hydroxypropyl-β-cyclodextrin and naringenin were prepared in a molar ratio of 1.5:1. Naringenin was dissolved in a 60% ethanol aqueous solution to prepare a precursor solution, and hydroxypropyl-β-cyclodextrin was dissolved in deionized water to prepare a main solution.

[0067] The precursor solution was added dropwise to the main solution at 45°C and stirred for 4 hours. After ethanol removal by vacuum concentration, the solution was freeze-dried and the resulting dry powder was dissolved in deionized water. The solution was then filtered through a microporous membrane to remove bacteria, resulting in a sterile fed-batch solution with an equivalent concentration of 15.0 g / L based on naringenin.

[0068] Preparation Example 3:

[0069] This preparation example provides a method for preparing a sterile fed-batch solution containing a host-guest inclusion complex of hydroxypropyl-β-cyclodextrin and naringenin, comprising the following steps:

[0070] Hydroxypropyl-β-cyclodextrin and naringenin were prepared in a molar ratio of 3.0:1. Naringenin was dissolved in an 80% ethanol aqueous solution to prepare a precursor solution, and hydroxypropyl-β-cyclodextrin was dissolved in deionized water to prepare a main solution.

[0071] The precursor solution was added dropwise to the main solution at 60°C and stirred for 8 hours. After ethanol removal by vacuum concentration, the solution was freeze-dried and the resulting dry powder was dissolved in deionized water. The solution was then filtered through a microporous membrane to remove bacteria, resulting in a sterile fed-batch solution with an equivalent concentration of 30.0 g / L based on naringenin.

[0072] Examples 1-3:

[0073] Example 1:

[0074] This embodiment provides a method for efficiently cultivating yeast strains that synthesize apigenin, including the following steps:

[0075] Prepare a basic fermentation medium, which, by mass-volume concentration, includes 10.0 g / L yeast extract, 20.0 g / L peptone, 6.5 g / L carbon-free yeast nitrogen source, and 50.0 g / L glycerol.

[0076] The pre-constructed engineered *Yersinia lipolytica* strain was inoculated into a fermenter containing the aforementioned basic fermentation medium for fermentation. The engineered *Yersinia lipolytica* strain contains genes for tyrosine ammonia-lyase, 4-coumarate-coenzyme A ligase, chalcone synthase, chalcone isomerase, and a gene encoding flavonoid synthase II derived from *Erigeron breviscapus*, enabling the production of apigenin from naringenin as a precursor. Fermentation conditions included a temperature of 30.0℃, a pH of 6.0 controlled by adding 20.0% ammonia, and a dissolved oxygen level of 40%.

[0077] Fermentation continued until the optical density of the fermentation broth at 600 nm reached 100 and the initial carbon source concentration decreased to 3.0 g / L. The temperature of the fermenter was linearly reduced to 24.0 °C at a rate of 0.5 °C / h and maintained thereafter, while a sterile feedstock prepared in Preparation Example 1 was continuously added to the fermentation broth at a rate of 0.10 g / (L·h) based on naringenin equivalents. During the continuous addition of the sterile feedstock, the dissolved oxygen setpoint of the fermenter was lowered to 20%, and the redox potential and the rate of change of oxygen uptake of the fermentation system were monitored online in real time.

[0078] When the redox potential is detected to be greater than +150 mV and the rate of change in oxygen uptake is less than -5.0 mmol / (L·h) 2 When the pulsed addition of sodium isoascorbate solution with a concentration of 200 mM is triggered, the concentration of sodium isoascorbate in the fermentation system is controlled to reach 1.0 mM within 2 minutes. At the same time as the pulsed addition is triggered, the current stirring speed and aeration rate of the fermenter are simultaneously increased by 45%.

[0079] When the redox potential is detected to be less than or equal to +50mV, the addition of sodium isoascorbate aqueous solution is stopped, and the stirring speed and aeration rate are restored to their pre-flotation values. This pulsed addition and restoration control logic is repeated until fermentation is complete, with a total fermentation time of 108 hours. After fermentation, the fermentation broth is centrifuged to remove the solid precipitate, and the supernatant is collected. 3.5M hydrochloric acid solution is added to the supernatant to adjust the pH to 2.5, and the mixture is allowed to crystallize at 7°C for 18 hours. After separation, washing, and drying, dried apigenin is obtained.

[0080] Example 2:

[0081] This embodiment provides a method for efficiently cultivating yeast strains that synthesize apigenin, including the following steps:

[0082] Prepare a basic fermentation medium, which, by mass-volume concentration, includes 8.0 g / L yeast extract, 15.0 g / L peptone, 5.0 g / L carbon-free yeast nitrogen source, and 40.0 g / L anhydrous glucose.

[0083] The pre-constructed engineered Saccharomyces cerevisiae was inoculated into a fermenter containing the aforementioned basic fermentation medium for fermentation. The engineered Saccharomyces cerevisiae contained genes for tyrosine ammonia-lyase, 4-coumarate-coenzyme A ligase, chalcone synthase, chalcone isomerase, and flavonoid synthase II derived from *Erigeron breviscapus*, and was able to produce apigenin from naringenin as a precursor. The fermentation conditions were: temperature 28.0℃, pH controlled at 5.5 by adding 12.5% ​​ammonia water, and dissolved oxygen set at 30%.

[0084] Fermentation continued until the optical density of the fermentation broth at a wavelength of 600 nm reached 80 and the initial carbon source concentration dropped to 4.5 g / L.

[0085] The temperature of the fermenter was linearly reduced to 22.0°C at a rate of 0.2°C / h and maintained thereafter. Simultaneously, a sterile feedstock prepared in Preparation Example 2 was continuously added to the fermentation broth at a rate of 0.05 g / (L·h) based on naringenin equivalents. During the continuous addition of the sterile feedstock, the dissolved oxygen setpoint of the fermenter was lowered to 15%, and the redox potential and oxygen uptake rate of the fermentation system were monitored online in real time.

[0086] When the redox potential is detected to be greater than +120 mV and the rate of change in oxygen uptake is less than -5.0 mmol / (L·h) 2 When the pulsed addition of sodium isoascorbate solution with a concentration of 100 mM is triggered, the concentration of sodium isoascorbate in the fermentation system is controlled to reach 0.5 mM within 1 minute. At the same time as the pulsed addition is triggered, the current stirring speed and aeration rate of the fermenter are simultaneously increased by 30%.

[0087] When the redox potential is detected to be less than or equal to +20mV, the addition of sodium isoascorbate aqueous solution is stopped, and the stirring speed and aeration rate are restored to their pre-flotation values. This pulse addition and restoration control logic is repeated until fermentation is complete, with a total fermentation time of 96 hours. After fermentation, the fermentation broth is centrifuged to remove the solid precipitate, and the supernatant is collected. Anhydrous ethanol (1 volume) is added to the supernatant, and the mixture is heated to 60°C and stirred for 1 hour. It is then cooled to 4°C and allowed to stand for crystallization for 12 hours. After separation, washing, and drying, dried apigenin is obtained.

[0088] Example 3:

[0089] This embodiment provides a method for efficiently cultivating yeast strains that synthesize apigenin, including the following steps:

[0090] Prepare a basic fermentation medium, which, by mass-volume concentration, includes 12.0 g / L yeast extract, 25.0 g / L peptone, 8.0 g / L carbon-free yeast nitrogen source, and 60.0 g / L glycerol.

[0091] The pre-constructed engineered *Yersinia lipolytica* strain was inoculated into a fermenter containing the aforementioned basic fermentation medium for fermentation. The engineered *Yersinia lipolytica* strain contains genes for tyrosine ammonia-lyase, 4-coumarate-coenzyme A ligase, chalcone synthase, chalcone isomerase, and a gene encoding flavonoid synthase II derived from *Erigeron breviscapus*, enabling the production of apigenin from naringenin as a precursor. Fermentation conditions included a temperature of 32.0℃, a pH of 6.5 controlled by the addition of 25.0% ammonia, and a dissolved oxygen level of 50%.

[0092] Fermentation continued until the optical density of the fermentation broth at a wavelength of 600 nm reached 120 and the initial carbon source concentration decreased to 1.0 g / L. The temperature of the fermenter was linearly reduced to 26.0 °C at a rate of 0.8 °C / h and maintained, while the aseptic feedstock prepared in Example 3 was continuously added to the fermentation broth at a rate of 0.15 g / (L·h) based on naringenin equivalents.

[0093] During the continuous feeding of sterile liquid, the dissolved oxygen setpoint of the fermenter was lowered to 25%, and the redox potential and oxygen uptake rate of the fermentation system were monitored online in real time.

[0094] When the redox potential is detected to be greater than +180 mV and the rate of change in oxygen uptake is less than -5.0 mmol / (L·h) 2 When the pulsed addition of sodium isoascorbate solution with a concentration of 300 mM is triggered, the concentration of sodium isoascorbate in the fermentation system is controlled to reach 2.0 mM within 3 minutes. At the same time as the pulsed addition is triggered, the current stirring speed and aeration rate of the fermenter are simultaneously increased by 60%.

[0095] When the redox potential is detected to be less than or equal to +80mV, stop the addition of sodium isoascorbate aqueous solution and restore the stirring speed and aeration rate to the values ​​before floating.

[0096] The control logic of pulsed flow addition and recovery was executed cyclically until fermentation ended, with a total fermentation time of 120 hours. After fermentation, the fermentation broth was centrifuged to remove solid precipitate, and the supernatant was collected. 5.0M hydrochloric acid solution was added to the supernatant to adjust the pH to 2.0, and the mixture was allowed to stand at 10℃ for 24 hours to crystallize. After separation, washing, and drying, dried apigenin was obtained.

[0097] Comparative Examples 1-4:

[0098] Comparative Example 1:

[0099] Compared with Example 1, the differences are as follows: no cooling treatment is performed during fermentation, and the fermentation temperature is maintained at a constant 30.0℃; the aseptic feed solution prepared in Example 1 is not added, but a pure naringenin ethanol solution without conventional inclusion treatment is added continuously at a rate of 0.10 g / (L·h); the dissolved oxygen setpoint of the fermenter is not lowered, the change rate of redox potential and oxygen uptake rate is not monitored, and the pulse addition of sodium isoascorbate aqueous solution and the upward control of stirring speed and aeration rate are not performed. All other aspects are the same.

[0100] Comparative Example 2:

[0101] Compared with Example 1, the difference is that during the continuous feeding of sterile liquid, the change rate of redox potential and oxygen uptake rate of the fermentation system is not monitored, the pulse feeding of sodium isoascorbate aqueous solution is not triggered, and the stirring speed and aeration rate of the fermenter are not synchronously increased. The stirring speed and aeration rate remain constant throughout the fermentation process, and all other aspects are the same.

[0102] Comparative Example 3:

[0103] Compared with Example 1, the difference is that during the continuous feeding of sterile feed liquid, the redox potential and oxygen uptake rate of the fermentation system are not monitored. Sodium isoascorbate is directly added to the sterile feed liquid for continuous constant-rate feeding, so that the concentration of sodium isoascorbate in the fermentation system is always statically maintained at 1.0 mM. Pulsed feeding is not performed, and the fermenter stirring speed and aeration rate are not synchronized. All other aspects are the same.

[0104] Comparative Example 4:

[0105] Compared with Example 1, the difference is that when the pulse flow of sodium isoascorbate aqueous solution is triggered, the current stirring speed and aeration rate of the fermenter are not simultaneously increased. The stirring speed and aeration rate remain constant throughout the fermentation process, while the rest are the same.

[0106] Test Example 1-3:

[0107] Test Example 1:

[0108] Online monitoring system records from the fermentation processes of Examples 1-3 and Comparative Examples 1-4 were collected, and historical data sequences of redox potential, dissolved oxygen concentration parameters, and oxygen uptake rate over time in the fermentation broth system during the continuous feed stage were exported.

[0109] At the fermentation endpoint of each embodiment and comparative example, 50 mL of fermentation broth sample was aseptically transferred from the sampling port of the fermenter, and cell counting was performed under a microscope using trypan blue staining. The percentage of cell viability at the end of fermentation was then calculated.

[0110] The fermentation broth sample obtained above was placed in a refrigerated centrifuge and centrifuged at 8000 r / min for 15 min to separate the supernatant and bacterial precipitate. An equal volume of methanol was added to the supernatant and ultrasonically extracted for 30 min to break the host-guest inclusion structure and completely dissolve the target product. The sample was then filtered through a 0.22 micrometer microporous membrane to obtain the test solution.

[0111] High performance liquid chromatography (HPLC) was used to quantitatively detect apigenin in the test solution. A C18 reversed-phase column was selected as the chromatographic column, and the mobile phase was isocratic elution with methanol and 0.1% (v / v) phosphoric acid aqueous solution. The detection wavelength was set to 340 nm. The total fermentation yield of the target product in the system was calculated using the external standard method.

[0112] Table 1. Fermentation Indicators and Product Yields of Each Example and Comparative Example

[0113]

[0114] in conclusion:

[0115] Based on the data in Table 1 and the appendix Figure 1 and attached Figure 2 The online monitoring information shown indicates that the final yields of Examples 1-3 were significantly higher than those of the comparative examples, confirming that by introducing hydroxypropyl-β-cyclodextrin and naringenin to construct a continuous phase transfer system, the product inhibition problem faced by the basic fermentation system in synthesizing hydrophobic products can be effectively resolved.

[0116] Combination Figure 1 It can be seen that during the continuous fed-batch stage from 60 to 64 hours, the redox potential of the fermentation system showed a monotonically linear upward trend from 90 mV, and reached the set first potential threshold at 64 hours. This phenomenon indicates that a large amount of intracellular precursor substances undergo electron leakage during the catalytic conversion by the cytochrome P450 enzyme system, leading to the continuous accumulation of reactive oxygen species in the fermentation system and generating oxidative stress.

[0117] Without intervention, this will lead to massive cell death, as evidenced by the severe drop in cell viability in Comparative Example 2. Comparative Example 3 involved the continuous, constant-rate addition of sodium isoascorbate, which resulted in an extremely low average oxygen uptake rate. This demonstrates that the persistent strong reducing agent engaged in severe oxygen competition with the aerobic flavonoid synthase II, causing hypoxia-limited production of the core catalytic enzyme and resulting in a persistently low yield.

[0118] The example employs a decoupled pulse control strategy, allowing the fermentation system to accumulate oxidative pressure within a certain range. Combined with... Figure 2 It can be seen that during the 4 hours when the redox potential climbs towards the threshold, the oxygen uptake rate of the system remains at a high level of approximately 85 mmol / (L·h). This indicates that cellular respiratory metabolism is not impaired during this stage, and the catalytic oxygen demand window of the P450 enzyme system is preserved to the maximum extent.

[0119] The system only triggers a reducing agent pulse to quench reactive oxygen species and prevent irreversible oxidative death of cells when the oxidation potential reaches the limiting threshold. In Comparative Example 4, the lack of agitation and aeration compensation during the pulse resulted in the instantaneous consumption of dissolved oxygen by the reducing agent, creating a localized micro-hypoxic environment. Consequently, the survival rate and yield were inferior to those of the Example. The Example group overcame the engineering incompatibility between the strong reducing system and the aerobic catalytic system through the interlocking linkage of mass flow and gas-liquid mass transfer parameters, achieving efficient synthesis of the target product while maintaining high survival rate and high respiratory activity.

[0120] Test Example 2:

[0121] During the later stages of continuous feed fermentation, samples were aseptically taken from the fermenters of each example and comparative example at a uniform sampling time point of 72 hours. After centrifugation and washing of the fermentation broth, the intracellular reactive oxygen species (ROS) level was measured using the DCFH-DA fluorescent probe method. Flow cytometry was used for detection, with an excitation wavelength of 488 nm and an emission wavelength of 525 nm, and the average relative fluorescence intensity of single cells was recorded.

[0122] Fermentation broths from each group at the fermentation endpoint were collected, cells were disrupted using a high-pressure co-current pump, and the mixture was sonicated. Microsomal proteins were extracted by differential centrifugation. The specific enzyme activity of flavonoid synthase II was determined in an in vitro reaction system using naringenin as a substrate. Enzyme activity was defined as the amount of enzyme required to catalyze the production of 1 micromole of apigenin per minute from 1 milligram of microsomal protein.

[0123] Samples of the dried apigenin products obtained after separation and drying in Examples 1-3 and Comparative Examples 1-4 were taken, and the purity of the final products was determined by high-performance liquid chromatography (HPLC) using the area normalization method. The mobile phase used was methanol and a 0.1% (v / v) aqueous solution of phosphoric acid.

[0124] Table 2. Results of intracellular reactive oxygen species levels, key enzyme activities, and final product purity tests for each group.

[0125]

[0126] Conclusion: Based on the data in Table 2, combined with... Figure 3 and Figure 4 The dynamic monitoring curves showed that the intracellular reactive oxygen species level was effectively controlled in the middle and late stages of fermentation in the example group, and the specific enzyme activity of flavonoid synthase II at the fermentation endpoint was significantly higher than that of each comparison group. The purity of the apigenin obtained by final separation and purification all reached more than 97%.

[0127] like Figure 3 As shown, Comparative Example 2, without redox regulation, experienced continuous electron leakage during the catalytic conversion of the substrate to the product, leading to a linear and significant accumulation of intracellular reactive oxygen species. Figure 4 It is known that this uncontrolled burst of reactive oxygen species triggered a severe lipid peroxidation reaction, damaging the physical structure of the cell microsomal membrane. Since flavonoid synthase II is a membrane-anchored protein, the disintegration of the microsomal membrane structure led to irreversible and rapid inactivation of the enzyme in the later stages of fermentation, resulting in a precipitous drop in the enzyme activity curve. The large amount of unconverted intermediate metabolites led to a significant reduction in the final extraction purity.

[0128] Comparative Example 3 employed a strategy of continuous constant-rate addition of reducing agent. Figure 3 This indicates that although this operation suppressed reactive oxygen species to extremely low levels for a long period of time, Figure 4 However, the specific activity of flavonoid synthase II showed a continuous linear decline. This is because the system was in a deep reducing state for a long time, and the abundant sodium isoascorbate directly competed with the aerobic flavonoid synthase II for oxygen. This long-term microenvironmental hypoxia altered the protein conformation of the core catalytic enzyme, severely limiting its catalytic activity.

[0129] The embodiment employs a pulse triggering strategy based on a potential threshold. Figure 3 The reactive oxygen species (ROS) curve in Example 1 exhibits controlled sawtooth fluctuations, indicating that the system only injects a reducing agent for precise quenching when the oxidative pressure accumulates to a critical point. This control logic eliminates lethal amounts of ROS while preventing the fermentation system from falling into a state of sustained deep reduction. Figure 4 The data confirms that this dynamic equilibrium mechanism fully protects the integrity of the cytoplasmic membrane system, enabling flavonoid synthase II to maintain a highly efficient catalytic conformation throughout the fermentation cycle. The above test results fully demonstrate that the physical interlocking and pulsed flow linkage mechanism of this invention successfully overcomes the engineering incompatibility between strong reducing systems and aerobic catalytic systems.

[0130] Test Example 3:

[0131] After 108 hours of fermentation, 500 mL samples were taken from the fermenters of each example and comparative example. The rheological parameters of the fermentation broth were measured using a rotational rheometer at 25°C, with the shear rate set to 100 s⁻¹. -1 Record the apparent viscosity of the fermentation broth at the end of the fermentation process.

[0132] The gas-liquid volumetric mass transfer coefficient during fermentation was determined using the dynamic exhaust method. At 108 hours of fermentation, air supply to the fermenter was temporarily stopped, and nitrogen was introduced instead to reduce the dissolved oxygen concentration in the fermentation broth to below 5% of saturation. The original aeration rate and stirring speed were then restored, and the dynamic data of dissolved oxygen concentration rising over time were recorded using an online dissolved oxygen electrode. The volumetric mass transfer coefficient at this point was calculated based on the mass transfer equation.

[0133] A 10 mL sample was taken from the fermenter and centrifuged at 8000 rpm for 15 min to separate the supernatant from the bacterial precipitate. The supernatant was directly extracted with an equal volume of methanol. The bacterial precipitate was washed twice with sterile water, then an equal volume of methanol was added, and the precipitate was physically broken down and extracted using a high-pressure homogenizer. The mass of apigenin in the supernatant extract and the broken bacterial precipitate was determined by high-performance liquid chromatography (HPLC). The intracellular retention rate of the product was calculated by multiplying the ratio of the mass of apigenin in the bacterial cells to the total product mass by 100%.

[0134] Table 3. Results of rheological parameters, gas-liquid mass transfer coefficients, and intracellular product distribution at the end of fermentation for each group.

[0135]

[0136] in conclusion:

[0137] Based on the data in Table 3, combined with Figure 5 and Figure 6 By studying the dynamic evolution of the product, the example set overcame the engineering contradiction between the transmembrane secretion barrier of hydrophobic products and the mass transfer attenuation of the high-viscosity fermentation system.

[0138] like Figure 5 As shown, Comparative Example 1, without the addition of inclusion complex, had a lower apparent viscosity in its fermentation broth, and the gas-liquid volumetric mass transfer coefficient remained at 140 h⁻¹ throughout the later stages of fermentation. -1 The above is a high level. However, Figure 6 The data showed that the intracellular retention rate of the product increased linearly and rapidly, reaching as high as 68.7% at the end of fermentation. This indicates that when the engineered yeast synthesizes highly hydrophobic apigenin, the product cannot spontaneously penetrate the cell membrane and is instead crystallized and deposited in the cytoplasm and organelles. This intracellular physical deposition triggers product feedback inhibition, blocking secondary metabolic flux.

[0139] In this embodiment, hydroxypropyl-β-cyclodextrin was used as a phase transfer carrier. The hydrophobic cavity of cyclodextrin constructed a high-affinity thermodynamic sink on the outer side of the cell membrane, altering the concentration gradient of free products inside and outside the cell. Driven by this thermodynamic gradient, intracellularly synthesized apigenin was continuously extracted and encapsulated into the fermentation broth. Figure 6In Example 1, the intracellular retention rate gradually decreased and stabilized at a low level below 5%, confirming that the strategy relieved the feedback inhibition caused by intracellular accumulation. However, with the continuous addition of inclusion material, the apparent viscosity of the fermentation system increased. The high viscosity fluid increased the difficulty of shearing and breaking up bubbles in the liquid phase, thickening the gas-liquid mass transfer boundary layer.

[0140] Figure 5 In comparative example 4, due to the lack of mechanical compensation measures, the volumetric mass transfer coefficient decreased significantly and linearly with fermentation time, dropping to 76.5 h at the end of fermentation. -1 The decline in mass transfer efficiency creates an oxygen supply bottleneck in the fermentation system, limiting the cell's aerobic metabolism and consequently affecting the energy supply required for the cell to maintain transmembrane excretion of substances. Figure 6 The results showed that the intracellular retention rate of Comparative Example 4 rebounded compared to the Example, rising to 14.6%.

[0141] In this embodiment, upon detecting a potential change and triggering a pulsed flow, the stirring speed and aeration rate are simultaneously increased. By increasing the mechanical stirring power input per unit volume and the gas surface drag, the coalescence of bubbles by the high-viscosity fluid is forcibly broken. This flow field compensation action offsets the increase in mass transfer resistance caused by the rise in material viscosity. Figure 5 The mass transfer coefficient of the confirmed embodiment remained stable at 128h. -1 This ensures the aerobic respiration needs and material exchange rate of yeast cells in the viscous inclusion system, achieving efficient bio-production of apigenin while maintaining a high concentration of viable cells.

Claims

1. A method for efficiently cultivating apigenin biosynthetic yeast, characterized in that, include: Engineered yeast containing heterologous apigenin biosynthesis pathway genes and flavonoid synthase II genes was inoculated into the basic fermentation medium in a fermenter and fermented within a preset first temperature range to obtain fermentation broth. Provide a sterile fed-batch solution containing a host-guest inclusion complex of hydroxypropyl-β-cyclodextrin and naringenin; The temperature of the fermenter is lowered to a preset second temperature range and maintained, while the sterile feed liquid is continuously added to the fermentation broth. During the continuous feeding of the sterile feed liquid, the dissolved oxygen setpoint of the fermenter is lowered, and the redox potential and oxygen uptake rate of the fermentation system are monitored in real time online. When the redox potential is detected to be greater than the first potential threshold and the rate of change of oxygen uptake is less than the set rate of change threshold, the pulse flow of sodium isoascorbate aqueous solution is triggered, and at the same time as the pulse flow is triggered, the current stirring speed and aeration rate of the fermenter are simultaneously increased. When the redox potential is detected to be less than or equal to the second potential threshold, the addition of the sodium isoascorbate aqueous solution is stopped, and the stirring speed and aeration rate are restored to the values ​​before the floatation. The control logic for pulse flow addition and recovery is executed cyclically until fermentation ends; Wherein, the first potential threshold is greater than the second potential threshold.

2. The method according to claim 1, characterized in that, The conditions for inoculating the engineered yeast into the basic fermentation medium in the fermenter for fermentation are as follows: The first temperature range is 28.0℃~32.0℃, the pH value is 5.5~6.5, and the dissolved oxygen setting value is 30%~50%; The fermentation continues until the optical density of the fermentation broth at a wavelength of 600 nm reaches 80-120, and the initial carbon source concentration drops below 5.0 g / L, at which point the cooling and continuous addition of the aseptic feed solution are initiated.

3. The method according to claim 1, characterized in that, The preparation process of the sterile fed-batch solution includes: Hydroxypropyl Cyclodextrin and naringenin were mixed at a molar ratio of (1.5–3.0):1, wherein naringenin was dissolved in an aqueous ethanol solution with a volume fraction of 60%–80% to prepare a precursor solution, and hydroxypropyl-β-cyclodextrin was dissolved in deionized water to prepare a main solution. The precursor solution was added dropwise to the main solution at 45℃~60℃ and stirred for 4~8h. After removing ethanol by vacuum concentration, the solution was freeze-dried and the resulting dry powder was dissolved in deionized water to obtain a sterile fed-batch solution with an equivalent concentration of 15.0~30.0g / L based on naringenin.

4. The method according to claim 1, characterized in that, The cooling process involves linearly reducing the temperature of the fermenter at a rate of 0.2℃ / h to 0.8℃ / h, where the second temperature range is 22.0℃ to 26.0℃. The rate of continuous addition of the sterile feed solution is 0.05–0.15 g / (L·h) in terms of naringenin equivalent.

5. The method according to claim 1, characterized in that, The aforementioned reduction of the dissolved oxygen setpoint for the fermenter means lowering the dissolved oxygen setpoint to 15%–25%. The first potential threshold is +120mV to +180mV; The set rate of change threshold is -5.0 mmol / (L·h) 2 The second potential threshold is +20mV to +80mV.

6. The method according to claim 1, characterized in that, The pulsed flow of the sodium isoascorbate aqueous solution is used to control the concentration of sodium isoascorbate in the fermentation system to reach 0.5-2.0 mM within 1-3 minutes. The synchronous upward movement ratio is 30% to 60% of the stirring speed and aeration rate at the previous moment, respectively, when the fermenter triggers the pulse flow.

7. The method according to claim 1, characterized in that, The engineered yeast is a pre-constructed recombinant yeast capable of generating apigenin from naringenin as a precursor. The recombinant yeast is either Yersinia lipolytica or Saccharomyces cerevisiae. The recombinant yeast contains heterologous apigenin biosynthesis pathway genes and flavonoid synthase II genes. The heterologous apigenin biosynthesis pathway genes include tyrosine ammonia-lyase genes, 4-coumaric acid coenzyme A ligase genes, chalcone synthase genes, and chalcone isomerase genes. The flavonoid synthase II gene encodes flavonoid synthase II derived from *Erigeron breviscapus*, which can catalyze the conversion of naringenin to apigenin.

8. The method according to claim 1, characterized in that, The total fermentation time is 96–120 hours. After fermentation, the fermentation broth is centrifuged to remove solid precipitate and the supernatant is collected.

9. The method according to claim 8, characterized in that, After collecting the supernatant, add 3.5-5.0M hydrochloric acid solution to the supernatant to adjust the pH value to 2.0-2.5, let it stand at 7℃-10℃ for 18-24 hours to crystallize, and then dry it after separation and washing to obtain dried apigenin.

10. The method according to claim 8, characterized in that, After collecting the supernatant, anhydrous ethanol with a volume equal to that of the supernatant was added and the mixture was heated to 60°C and stirred for 1 hour. Then, it was cooled to 4°C and allowed to stand for crystallization for 12 hours. After separation and washing, it was dried to obtain apigenin dried product.

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