A process for the continuous production of phenylalanine using carbon dioxide as the main raw material

Abstract

This invention provides a continuous process for producing phenylalanine using carbon dioxide as the main raw material, relating to the field of amino acid production. The process includes: Step 1: Adding styrene, a photocatalyst, a solvent, an electron donor, and an alkaline additive to a photoreactor; introducing CO2 into the photoreactor until the pressure reaches a set level; turning on the light source of the photoreactor; and conducting a photocatalytic reaction under stirring conditions. After the reaction, filtering to recover the catalyst, distilling to recover the solvent, and purifying cinnamic acid by crystallization or chromatography. Step 2: Adding the cinnamic acid obtained in Step 1 to a fermentation medium; inoculating the fermentation medium with genetically engineered bacterial strains and controlling the fermentation conditions; during fermentation, adding carbon source, nitrogen source, and cinnamic acid according to the carbon and nitrogen concentration of the fermentation broth and the growth of the bacterial cells; after fermentation, separating the bacterial cells and purifying L-phenylalanine. This two-stage integrated system of photocatalytic carboxylation and bio-fermentation achieves efficient conversion from inexpensive raw materials to high-value-added amino acids.

Description

Technical Field

[0001] This invention relates to the field of amino acid production technology, specifically a process for the continuous production of phenylalanine using carbon dioxide as the main raw material, belonging to the interdisciplinary field of biochemical engineering and photocatalysis technology. Background Technology

[0002] Phenylalanine is an important essential amino acid widely used in medicine, food, and animal feed. L-phenylalanine, as the main raw material for the artificial sweetener aspartame, has a huge market demand. In addition, phenylalanine also has important applications in the pharmaceutical fields of anti-tumor drugs, anti-inflammatory drugs, and cardiovascular drugs.

[0003] Currently, the industrial production of phenylalanine mainly employs the following methods: 1. Chemical Synthesis: This method uses benzaldehyde and acetaldehyde as raw materials, and involves multiple steps such as condensation, reduction, and ammonolysis to produce the product. This method has many reaction steps, a complex process, and causes serious environmental pollution. Furthermore, the product is a DL-racemic mixture, requiring further resolution, resulting in poor atom economy.

[0004] 2. Fermentation method: This method uses sugars such as glucose as raw materials and ferments them with E. coli or yeast. This method has high raw material costs, a long fermentation cycle (48-72 hours), low yield (40-60%), difficulties in downstream separation and purification, and high equipment investment.

[0005] 3. Enzymatic method: Using cinnamic acid or its derivatives as raw materials, phenylalanine ammonia lyase (PAL) is used for catalytic conversion. Although this method has high selectivity, the substrate cost is high, the enzyme stability is poor, and industrial production is limited.

[0006] 4. Extraction method: Extraction from natural protein hydrolysates. This method has limited raw material sources, high costs, and low purity, and is only suitable for small-scale production.

[0007] In recent years, the utilization of CO2 resources has become a research hotspot. CO2 is a cheap, abundant, and renewable carbon resource, and its use in synthesizing high-value-added chemicals has significant economic and environmental implications. Photocatalysis technology provides a new pathway for the activation and conversion of CO2, but existing CO2 photocatalytic carboxylation technologies mainly focus on small molecule synthesis, and there are no reports of coupling CO2 photocatalytic carboxylation with bio-fermentation for phenylalanine production. Summary of the Invention

[0008] This invention provides a process for the continuous production of phenylalanine using carbon dioxide as the main raw material, in order to solve the technical problem mentioned in the background that "existing CO2 photocatalytic carboxylation technology mainly focuses on small molecule synthesis, and there is no evidence of coupling CO2 photocatalytic carboxylation with bio-fermentation for the production of phenylalanine".

[0009] To address the aforementioned technical problems, this invention discloses a continuous process for producing phenylalanine using carbon dioxide as the main raw material, comprising: Step 1: Photocatalytic carboxylation stage: Styrene, photocatalyst, solvent, electron donor, and alkaline additive are added to the photoreactor. CO2 is introduced into the photoreactor until the pressure inside the photoreactor reaches the set pressure. The light source of the photoreactor is turned on, and the photocatalytic reaction is carried out under stirring conditions. After the reaction is completed, the catalyst is recovered by filtration, the solvent is recovered by distillation, and cinnamic acid is purified by crystallization or chromatographic separation. Step 2: Bio-fermentation stage: Add the cinnamic acid obtained in Step 1 to the fermentation medium, inoculate the genetically engineered strain into the fermentation medium, and control the fermentation conditions; during the fermentation process, add carbon source, nitrogen source and cinnamic acid according to the carbon and nitrogen concentration of the fermentation broth and the growth of the cells; after the fermentation is completed, separate the cells and purify L-phenylalanine.

[0010] Preferably, the photocatalyst is selected from one or more of the following types: Metal oxide photocatalysts, composite oxide photocatalysts, sulfide photocatalysts, nitride photocatalysts, noble metal supported photocatalysts, quantum dot photocatalysts, organic framework material photocatalysts, organic semiconductor photocatalysts, and dye-sensitized photocatalysts.

[0011] Preferably, the solvent is selected from one or a mixture of solvents, wherein the mixture of solvents is a mixture of any two or more of the following: organic solvents, ionic liquids, and aqueous systems.

[0012] Preferably, the electron donor is selected from one or more of the following types: Organic amines, organosulfur compounds, organophosphorus compounds, heterocyclic compounds, inorganic compounds, I-, Br-, S 2- S2O3 2- N2H4, H3PO2, NaBH4.

[0013] Preferably, the specific process parameters for the photocatalytic carboxylation reaction are as follows: Raw material molar ratio: Styrene:CO2 = 1:(1~50); Styrene:catalyst = 1:(0.001~0.5); Styrene:electron donor = 1:(0.1~10); Styrene:alkaline additive = 1:(0.1~5); Reaction temperature: 0~150℃; Reaction pressure: 0.1~10 MPa; Light intensity: 10~2000mW / cm² 2 ; Reaction time: 0.5–48 hours; stirring speed: 100–2000 rpm; Light source types: ultraviolet light, visible light, sunlight, LED light source, xenon lamp, mercury lamp, sodium lamp; among which, the wavelength of ultraviolet light is 200-400nm; the wavelength of visible light is 400-800nm.

[0014] Preferably, in step 2, the strain types include: engineered Escherichia coli, engineered yeast, engineered Pseudomonas, engineered Bacillus, engineered Corynebacterium, Rhodotorula spp. yeast, Aspergillus spp. fungi, and Streptomyces spp. actinomycetes.

[0015] Preferably, in step 2, the culture medium composition includes: carbon source, nitrogen source, inorganic salt, growth factor, precursor substance, pH adjuster, and defoamer.

[0016] Preferably, in step 2, the process parameters include: Seed culture process parameters, including: seed culture medium, including LB medium or M9 basal medium; Culture temperature: 25–40℃; culture time: 6–24 hours; shaker speed: 100–300 rpm; inoculum size: 0.1%–10% (v / v). Fermentation process parameters include: fermentation temperature: 20–45℃; fermentation pH: 5.0–9.0; fermentation time: 12–72 hours; stirring speed: 100–1000 rpm; aeration rate: 0.1–5 vvm; tank pressure: 0.01–0.5 MPa.

[0017] Preferably, the feeding strategy in step 2 is as follows: carbon source feeding: when the residual sugar concentration is below 5 g / L, glucose is added to 10-20 g / L; nitrogen source feeding: when the amino nitrogen concentration is below 0.5 g / L, nitrogen source is added to 1-2 g / L; precursor feeding: when the cinnamic acid concentration is below 1 g / L, cinnamic acid is added to 5-10 g / L; inducer feeding: PAL gene expression is induced by IPTG or lactose, with a final concentration of 0.1-1 mM. The dissolved oxygen control strategy is as follows: the dissolved oxygen level is 10%-100%; the control method is to adjust the stirring speed, aeration rate, tank pressure, or add pure oxygen.

[0018] The pH control strategy involves automatic addition of acid or alkali with a control accuracy of ±0.1 pH units; the temperature control strategy involves automatic adjustment of the cooling water or heating system with a temperature control accuracy of ±0.5℃.

[0019] Preferably, the raw material ratio and reaction conditions in step 2 include: Cinnamic acid dry weight: cell dry weight = 1:(0.1~10); initial cinnamic acid concentration: 1~50 g / L; cell density: OD600 = 0.1~50; transformation time: 1~48 hours; transformation temperature: 20~45℃; transformation pH: 6.0~9.0; coenzyme: pyridoxal phosphate concentration: 0.01~1 mM; metal ion: Mg2+ Mn 2+ Zn 2+ Fe 2+ The concentration of metal ions is 0.1–10 mM.

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. Raw material innovation: Using CO2 as the main raw material, it realizes the resource utilization of CO2, reduces raw material costs, reduces carbon emissions, and is in line with the development direction of green chemistry and carbon neutrality.

[0022] 2. Process innovation: The coupling of photocatalytic carboxylation with bio-fermentation enables the conversion of simple raw materials into high-value-added amino acids, with a short process route and high atom economy.

[0023] 3. High efficiency conversion: The yield of cinnamic acid in the first stage is ≥90%, the conversion rate of L-phenylalanine in the second stage is ≥95%, and the total yield is ≥85%, which is significantly higher than that of existing technologies.

[0024] 4. Green production: Photocatalysis technology is used to drive the reaction with solar energy; the bio-fermentation conditions are mild and energy consumption is low; solvents and catalysts can be recycled and reused, resulting in less waste emissions.

[0025] 5. Continuous production: It realizes two-stage continuous production, with high production efficiency and high equipment utilization, which is suitable for industrial-scale production.

[0026] 6. Cost advantages: Low raw material costs, low energy consumption, simple processes, low production costs, and strong product competitiveness.

[0027] 7. High product quality: L-phenylalanine purity ≥98%, optical purity ≥99%, meeting food-grade and pharmaceutical-grade standards.

[0028] 8. Wide range of applications: The process of this invention can be applied to the production of various aromatic amino acids, such as tyrosine and tryptophan.

[0029] 9. Advanced technology: It integrates a variety of advanced technologies such as photocatalysis, genetic engineering, fermentation engineering, separation and purification, and has a high technical content.

[0030] 10. Good economic benefits: High return on investment, broad market prospects, and can create significant economic and social benefits. Attached Figure Description

[0031] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This is a process flow diagram of the present invention; Figure 2 This is a schematic diagram of the photocatalytic carboxylation reaction mechanism; Figure 3 This is a schematic diagram of the bio-fermentation transformation mechanism; Figure 4 This is a diagram of a photocatalytic carboxylation reaction apparatus; Figure 5 Diagram of a bio-fermentation apparatus; Figure 6 This is a schematic diagram of a two-stage integrated system; Figure 7 The hydrogen spectrum of cinnamic acid according to the present invention; Figure 8 This is the carbon spectrum of cinnamic acid according to the present invention; Figure 9 For product quality stability assessment Figure 1 ; Figure 10 For product quality stability assessment Figure 2 . Detailed Implementation

[0032] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0033] Furthermore, in this invention, the use of terms such as "first" and "second" is for descriptive purposes only and does not specifically refer to any order or sequence, nor is it intended to limit the invention. They are merely used to distinguish components or operations described using the same technical terms and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the technical solutions and features of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. If a combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0034] The present invention provides the following embodiments: Example 1: This embodiment of the invention provides a process for the continuous production of phenylalanine using carbon dioxide as the main raw material, such as... Figures 1-10 As shown, it includes: Step 1: Photocatalytic carboxylation stage: Styrene, photocatalyst, solvent, electron donor, and alkaline additive are added to the photoreactor. CO2 is introduced into the photoreactor until the pressure inside the photoreactor reaches the set pressure. The light source of the photoreactor is turned on, and the photocatalytic reaction is carried out under stirring conditions. After the reaction is completed, the catalyst is recovered by filtration, the solvent is recovered by distillation, and cinnamic acid is purified by crystallization or chromatographic separation. Step 2: Bio-fermentation stage: Add the cinnamic acid obtained in Step 1 to the fermentation medium, inoculate the genetically engineered strain into the fermentation medium, and control the fermentation conditions; during the fermentation process, add carbon source, nitrogen source and cinnamic acid according to the carbon and nitrogen concentration of the fermentation broth and the growth of the cells; after the fermentation is completed, separate the cells and purify L-phenylalanine.

[0035] The photocatalyst is selected from one or more of the following types: Metal oxide photocatalysts include: TiO2 (anatase, rutile, brookite), ZnO, WO3, Fe2O3, Cu2O, Bi2O3, CeO2, ZrO2, Nb2O5, Ta2O5 and their doped and modified forms; Composite oxides: BiVO4, Bi2MoO6, Bi2WO6, BiFeO3, SrTiO3, BaTiO3, CaTiO3, ZnFe2O4, CuFeO2 and their doped and modified compounds; Sulfides: CdS, ZnS, MoS2, WS2, Cu2S and their doped and modified compounds; Nitrides: g-C3N4, Ta3N5, Ge3N4 and their doped and modified forms; Noble metal supported types: Au / TiO2, Ag / TiO2, Pt / TiO2, Pd / TiO2, Au / g-C3N4, Ag / g-C3N4, Pt / g-C3N4, Pd / g-C3N4, Au / BiVO4, Ag / BiVO4; Quantum dot type: CdSe, CdTe, PbS, ZnSe, CuInS2, CuGaS2 and their core-shell structures; Organic framework materials: MOF-5, UiO-66, MIL-101, ZIF-8 and their modifiers; Organic semiconductors: polypyrrole, polyaniline, polythiophene, polyfuran and their derivatives; Dye-sensitized semiconductor catalysts: Ru(bpy)3Cl2, Ru(phen)3Cl2, Ir(ppy)3, Eosin Y, Rose Bengal, fluorescein, copper phthalocyanine, zinc phthalocyanine, and porphyrin-based compounds.

[0036] Preferred photocatalysts are: g-C3N4, BiVO4, Au / TiO2, Pd / g-C3N4, Ru(bpy)3Cl2 / TiO2, and EosinY / TiO2.

[0037] The solvent is selected from one of the following or is a mixed solvent, wherein the mixed solvent is a mixture of any two or more of the following solvents: Organic solvents: Nitriles: Acetonitrile, propionitrile, butyronitrile, benzonitrile, acrylonitrile; Ethers: Diethyl ether, tetrahydrofuran (THF), 1,4-dioxane, ethylene glycol dimethyl ether, diethylene glycol dimethyl ether, methyl tert-butyl ether; Alcohols: Methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol, glycerol; Amides: N,N-dimethylformamide (DMF), N,N-dimethylacetamide, N-methylpyrrolidone (NMP); Sulfides: Dimethyl sulfoxide, sulfolane; Esters: Ethyl acetate, methyl acetate, dimethyl carbonate, diethyl carbonate; Ketones: Acetone, butanone, cyclohexanone; Aromatic hydrocarbons: Benzene, toluene, xylene, chlorobenzene; Halogenated hydrocarbons: Dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane; Alkanes: n- Hexane, cyclohexane, petroleum ether; Ionic liquids: imidazoles: [BMIM][BF4], [BMIM][PF6], [EMIM][BF4], [EMIM][PF6], [BMIM][Tf2N], [EMIM][Tf2N]; Pyridines: [BPy][BF4], [BPy][PF6]; Quaternary ammoniums: [N4444][BF4], [N4444][PF6], [N8888][BF4]; Pyrrolidines: [BMPYRR][BF4], [BMPYRR][PF6]; Supercritical fluids: supercritical CO2, supercritical CHF3, supercritical C2H6; Aqueous systems: pure water, deionized water, ultrapure water.

[0038] Mixed solvents: such as acetonitrile-water (volume ratio 1:1 to 9:1), DMF-water (volume ratio 1:1 to 9:1), DMSO-water (volume ratio 1:1 to 9:1), THF-water (volume ratio 1:1 to 9:1), ionic liquid-water (volume ratio 1:1 to 9:1).

[0039] Preferred solvent systems are: acetonitrile, DMF, DMSO, acetonitrile-water (volume ratio 7:3), DMF-water (volume ratio 8:2), and [BMIM][BF4]-acetonitrile (volume ratio 1:1).

[0040] The electron donor is selected from one or more of the following types: Organic amines: Aliphatic amines: trimethylamine, triethylamine, tripropylamine, tributylamine, N,N-diisopropylethylamine, N,N,N',N'-tetramethylethylenediamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene; Aromatic amines: N,N-dimethylaniline, N,N-diethylaniline, triphenylamine, N-methylcarbazole, phenothiazine, phenotoxazine; Cyclic amines: pyrrolidine, piperidine, morpholine, piperazine, imidazole, benzimidazole; Organosulfur compounds: Thiols: methanethiol, ethanethiol, propanethiol, butanethiol, benzenethiol, mercaptoethanol, mercaptopropionic acid; Thioethers: dimethyl sulfide, diethyl sulfide, diphenyl sulfide, tetrahydrothiophene; Disulfides: dimethyl disulfide, diphenyl disulfide; Organophosphorus compounds: Phosphines: triphenylphosphine, tributylphosphine, trimethylphosphine, triethylphosphine; Phosphites: trimethyl phosphite, triethyl phosphite, triphenyl phosphite; Heterocyclic compounds: Indoles: indole, 3-methylindole, tryptophan, tryptophan; Furans: furan, 2-methylfuran, furfural; Thiophenes: thiophene, 2-methylthiophene; Phenolic compounds: phenol, hydroquinone, resorcinol, catechol, 1,2,3-trihydroxybenzene, 1,3,5-trihydroxybenzene, ascorbic acid; Carboxylic acids: formic acid, acetic acid, propionic acid, oxalic acid, ascorbic acid; Alcohols: methanol, ethanol, isopropanol, ethylene glycol, glycerol, benzyl alcohol; Inorganic compounds: Metal powders: Zn powder, Mg powder, Al powder, Fe powder, Cu powder; Metal ions: Fe 2+ Cu + Sn 2+ Ti 3+ V 2+ Cr 2+ ; Electron donor types also include I- and Br-. - S 2- S2O3 2- N2H4, H3PO2, NaBH4.

[0041] Preferred electron donors are: triethylamine, DIPEA, TMEDA, triphenylamine, phenothiazine, triphenylphosphine, indole, ascorbic acid, Zn powder, and NaBH4.

[0042] In the photocatalytic carboxylation reaction, an alkaline additive is added, and the alkaline additive is selected from one or more of the following types: Inorganic bases: Alkali metal hydroxides: LiOH, NaOH, KOH, RbOH, CsOH; Alkali earth metal hydroxides: Be(OH)2, Mg(OH)2, Ca(OH)2, Sr(OH)2, Ba(OH)2; Alkali metal carbonates: Li2CO3, Na2CO3, K2CO3, Rb2CO3, Cs2CO3; Alkali metal bicarbonates: LiHCO3, NaHCO3, KHCO3, RbHCO3, CsHCO3; Alkali metal phosphates: Na3PO4, K3PO4, Na2HPO4, K2HPO4; Alkali metal acetates: NaOAc, KOAc, LiOAc; Organic bases: Aliphatic amines: trimethylamine, triethylamine, tributylamine, DBU, DBN, TMEDA; Aromatic amines: pyridine, 4-dimethylaminopyridine, N-methylimidazolium, 1,8-bis(dimethylaminonaphthalene); Alkoxides: sodium methoxide, sodium ethoxide, sodium tert-butoxide, potassium tert-butoxide; Organometallic bases: n-butyllithium, sec-butyllithium, tert-butyllithium, diisopropylaminolithium, hexamethyldisilaminolithium, sodium hexamethyldisilamino, potassium hexamethyldisilamino. Buffer systems: phosphate buffer, borate buffer, carbonate buffer, acetate buffer, Tris-HCl buffer; phosphate buffer (PBS): NaH2PO4-Na2HPO4, KH2PO4-K2HPO4, pH range 6.0–8.0; borate buffer: H3BO3-Na2B4O7, pH range 8.0–10.0; carbonate buffer: NaHCO3-Na2CO3, pH range 9.0–11.0; acetate buffer: HOAc-NaOAc, pH range 4.0–6.0; Tris-HCl buffer: pH range 7.0–9.0.

[0043] Molecular sieves and alkaline resins: Molecular sieves: 3A, 4A, 5A, 13X type molecular sieves; Alkaline ion exchange resins: Dowex 1×8, Amberlite IRA-400, Amberlyst A-26.

[0044] Preferred alkaline additives include: K2CO3, Na2CO3, K3PO4, triethylamine, DBU, pyridine, potassium tert-butoxide, and PBS buffer (pH 7.4).

[0045] The specific process parameters for the photocatalytic carboxylation reaction are as follows: Raw material molar ratio: Styrene:CO2 = 1:(1-50); preferably 1:(5-20), more preferably 1:10; Styrene:catalyst = 1:(0.001~0.5); preferably 1:(0.01~0.1), more preferably 1:0.05; Styrene:electron donor = 1:(0.1-10); preferably 1:(0.5-3), more preferably 1:1.5; Styrene:alkaline additive = 1:(0.1-5); preferably 1:(0.5-2), more preferably 1:1; Reaction temperature: 0–150°C; preferably 20–100°C, more preferably 40–80°C; Reaction pressure: 0.1–10 MPa; preferably 0.5–5 MPa, more preferably 1–3 MPa; Light intensity: 10~2000mW / cm 2 Preferred strength: 50–500 mW / cm 2 More preferably 100–300 mW / cm 2 ; Reaction time: 0.5–48 hours; preferably 2–24 hours, more preferably 6–12 hours Stirring speed: 100–2000 rpm; preferably 300–1000 rpm Light source types: ultraviolet light, visible light, sunlight (natural sunlight or simulated sunlight), LED light source (ultraviolet LED, blue LED, green LED, white LED), xenon lamp, mercury lamp, sodium lamp; wherein, the wavelength of ultraviolet light is 200-400nm (preferably 300-380 nm); the wavelength of visible light is 400-800nm ​​(preferably 420-700 nm).

[0046] Reactor types: Batch reactors: quartz reactors, glass reactors, stainless steel reactors; Continuous flow reactors: tubular reactors, microchannel reactors, fixed-bed reactors; photocatalytic membrane reactors: photocatalytic membrane coupled with reaction separation.

[0047] To reduce the production cost of the first-stage synthesis process, this invention proposes the following strategy: Catalyst cost optimization: Replace precious metal catalysts with non-precious metal catalysts (such as g-C3N4, BiVO4); develop recyclable supported catalysts; improve the photostability and recycling rate of catalysts (≥10 times); use solar energy as a light source to reduce energy consumption costs.

[0048] Raw material cost optimization: Utilizing CO2 from industrial waste gas as a raw material to achieve resource recycling; Use inexpensive and readily available electron donors (such as ascorbic acid and Zn powder). Optimize the solvent system and select low-toxicity, recyclable green solvents.

[0049] Process cost optimization: Adopting continuous flow process to improve production efficiency; optimizing reaction conditions to shorten reaction time; increasing product yield (≥90%) and reducing raw material consumption; simplifying post-processing to reduce separation costs.

[0050] Equipment cost optimization: Using LED light sources reduces equipment investment and operating costs; designing efficient photoreactors improves light energy utilization; and achieving automated control reduces labor costs.

[0051] Phase Two: Bio-fermentation Transformation This invention utilizes genetically engineered strains to convert cinnamic acid into L-phenylalanine. It systematically investigated key factors such as fermentation strains, culture medium composition, and fermentation process conditions, establishing a highly efficient fermentation conversion system.

[0052] Fermentation strain system: The fermentation strains applicable to this invention include, but are not limited to, the following types: Engineered Escherichia coli strains: Escherichia coli overexpressing phenylalanine aminolyase (PAL); Escherichia coli with knockout genes related to the phenylalanine degradation pathway; Escherichia coli overexpressing key enzyme genes of the shikimic acid pathway; and Escherichia coli integrating exogenous PAL genes and amino acid transporter genes.

[0053] Engineered yeast strains (Saccharomyces cerevisiae): yeast strains expressing plant PAL genes; yeast strains optimizing the shikimic acid pathway; yeast strains enhancing amino acid synthesis capabilities.

[0054] Engineered Pseudomonas bacteria: Pseudomonas bacteria with highly efficient cinnamic acid metabolism capabilities; Pseudomonas aeruginosa expressing the exogenous PAL gene.

[0055] Engineered Bacillus bacteria: Bacillus bacteria expressing the heat-resistant PAL gene; Bacillus bacteria with highly efficient amino acid secretion capabilities.

[0056] Corynebacterium engineered bacteria: Corynebacterium with highly efficient amino acid synthesis capabilities; Corynebacterium with optimized metabolic pathways.

[0057] Other strains: Rhodotorula yeast; Aspergillus fungus; Streptomyces actinomycetes.

[0058] The preferred fermentation strains are: Escherichia coli BL21(DE3) / pET-PAL, Escherichia coli W3110 / ΔpheA / pACYC-PAL, and yeast BY4741 / pYES-PAL.

[0059] Culture medium composition: The culture medium composition applicable to this invention is as follows: Carbon sources: Sugars: glucose, fructose, sucrose, lactose, maltose, xylose, arabinose; Sugar alcohols: glycerol, sorbitol, mannitol; Organic acids: acetic acid, propionic acid, citric acid, succinic acid; Carbon source dosage: 10-100g / L, preferably 20-50g / L.

[0060] Nitrogen sources: Inorganic nitrogen sources: ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium phosphate, urea; Organic nitrogen sources: peptone, yeast powder, beef extract, corn steep liquor, soybean meal powder; Nitrogen source dosage: 1-50 g / L, preferably 5-20 g / L.

[0061] Inorganic salts: MgSO4·7H2O: 0.1–5 g / L, preferably 0.5–2 g / L; K2HPO4: 0.5–10 g / L, preferably 1–5 g / L; KH2PO4: 0.1–5 g / L, preferably 0.5–2 g / L; NaCl: 0.1–10 g / L, preferably 1–5 g / L; CaCl2·2H2O: 0.01–1 g / L, preferably 0.05–0.5 g / L; FeSO4 4·7H2O: 0.001~0.1g / L, preferably 0.01~0.05g / L; MnSO4·H2O: 0.001~0.1g / L, preferably 0.01~0.05g / L; ZnSO4·7H2O: 0.001~0.1g / L, preferably 0.01~0.05g / L; CuSO4·5H2O: 0.00~0.05g / L, preferably 0.01~0.02g / L.

[0062] Growth factors: Vitamins: Vitamin B1, Vitamin B6, Vitamin B12, Biotin, Calcium Pantothenate, Niacin; Amino acids: L-Tyrosine, L-Tryptophan, L-Histidine, L-Proline; Nucleotides: Adenine, Guanine, Cytosine, Uracil; Growth factor dosage: 0.001-0.1 g / L, preferably 0.01-0.05 g / L.

[0063] Precursor substances: Cinnamic acid: 0.1-50 g / L, preferably 1-20 g / L; Shikimic acid: 0.1-10 g / L, preferably 0.5-5 g / L; Phosphoenolpyruvic acid (PEP): 0.1-5 g / L, preferably 0.5-2 g / L.

[0064] pH adjusters: Acids: hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, citric acid; Bases: sodium hydroxide, potassium hydroxide, ammonia, sodium carbonate; Buffer systems: phosphate buffer, Tris-HCl buffer, citrate buffer.

[0065] Defoamers: silicone oils, polyethers, and higher alcohols; Defoamer dosage: 0.01–1 g / L, preferably 0.1–0.5 g / L.

[0066] Fermentation process conditions: This invention studies in detail the effects of fermentation process conditions on conversion rate and establishes a complete process parameter window.

[0067] Seed culture: Seed culture medium: LB medium or M9 basal medium; Culture temperature: 25-40℃, preferably 30-37℃; Culture time: 6-24 hours, preferably 8-16 hours; Shaking speed: 100-300 rpm, preferably 150-250 rpm; Inoculation amount: 0.1-10% (v / v), preferably 1-5% (v / v).

[0068] Fermentation culture: Fermentation temperature: 20-45℃, preferably 28-37℃; Fermentation pH: 5.0-9.0, preferably 6.5-7.5; Fermentation time: 12-72 hours, preferably 24-48 hours; Stirring speed: 100-1000 rpm, preferably 200-600 rpm; Aeration rate: 0.1-5 vvm, preferably 0.5-2 vvm; Tank pressure: 0.01-0.5 MPa, preferably 0.05-0.2 MPa.

[0069] Feeding strategy: Carbon source supplementation: When the residual sugar concentration is below 5 g / L, supplement glucose to 10-20 g / L; Nitrogen source supplementation: When the amino nitrogen concentration is below 0.5 g / L, supplement nitrogen source to 1-2 g / L; Precursor supplementation: When the cinnamic acid concentration is below 1 g / L, supplement cinnamic acid to 5-10 g / L; Inducer supplementation: Induce PAL gene expression with IPTG or lactose at a final concentration of 0.1-1 mM.

[0070] Dissolved oxygen control: Dissolved oxygen level: 10-100%, preferably 20-60%; Control method: Adjust stirring speed, aeration rate, tank pressure or add pure oxygen.

[0071] pH control: Automatic addition of acid (HCl or H2SO4) or alkali (NaOH or NH4OH); control accuracy: ±0.1 pH units.

[0072] Temperature control: Automatically adjusts cooling water or heating system; control accuracy: ±0.5℃.

[0073] Raw material ratio and reaction conditions: Cinnamic acid: bacterial cells (dry weight) = 1:(0.1-10), preferably 1:(0.5-3), more preferably 1:1.

[0074] Substrate concentration: initial concentration of cinnamic acid 1-50 g / L, preferably 5-20 g / L.

[0075] Cell density: OD600 = 0.1-50, preferably 1-20, more preferably 5-10.

[0076] Conversion time: 1 to 48 hours, preferably 6 to 24 hours, more preferably 12 to 18 hours.

[0077] Conversion temperature: 20-45℃, preferably 25-37℃, more preferably 30℃.

[0078] Conversion pH: 6.0–9.0, preferably 6.5–7.5, more preferably 7.0.

[0079] Coenzyme: PLP (pyridoxal phosphate) 0.01-1 mM, preferably 0.1-0.5 mM.

[0080] Metal ion: Mg 2+ Mn 2+ Zn 2+ Fe 2+ The concentration is 0.1–10 mM, preferably 1–5 mM.

[0081] Specific strategies and methods for reducing costs: To reduce the production cost of the second-stage fermentation synthesis process, this invention proposes the following strategies: Strain cost optimization: Constructing engineered strains with high-efficiency expression to increase enzyme activity per unit; improving strain stability and reusability; developing immobilized cell technology to achieve cell recycling; and using inexpensive carbon sources (such as crude glycerol and lignocellulose hydrolysate).

[0082] Culture medium cost optimization: Use industrial by-products (such as corn steep liquor and molasses) to replace some culture medium components; optimize culture medium formulations to reduce the amount of expensive components; develop low-cost serum-free culture media.

[0083] Fermentation cost optimization: High-density fermentation technology is adopted to increase the yield per unit volume; fermentation conditions are optimized to shorten the fermentation cycle; substrate conversion rate is increased (≥95%) to reduce raw material consumption; continuous or semi-continuous fermentation is achieved to improve equipment utilization.

[0084] Optimize separation and purification costs: Develop efficient product separation technologies (such as membrane separation and crystallization); simplify purification processes and reduce solvent consumption; realize the resource utilization of by-products.

[0085] Energy cost optimization: adopt energy-saving fermentation equipment; optimize the cooling system to reduce energy consumption; and utilize fermentation heat for energy recovery.

[0086] The invention enables continuous production in two stages: Continuous photocatalytic carboxylation: using a fixed-bed photoreactor or a microchannel photoreactor; continuous feeding (styrene, CO2, solvent, etc.); continuous discharge (cinnamic acid solution); online monitoring and control of reaction parameters; To achieve 24-hour continuous production.

[0087] Continuous bio-fermentation: Employs a multi-stage fermentation system; Stage 1: Inoculum activation; Stage 2: Seed culture expansion; Stage 3: Main fermentation; Continuous feeding (carbon source, nitrogen source, precursors); Continuous or intermittent discharge; Online monitoring and control of fermentation parameters; Achieves continuous production for 7-30 days.

[0088] Continuous product separation and purification: Membrane separation technology (ultrafiltration, nanofiltration, reverse osmosis); continuous ion exchange technology; continuous crystallization technology; online quality monitoring and automatic control.

[0089] The L-phenylalanine produced by the process has a purity of ≥98% and an optical purity of ≥99%, meeting food-grade and pharmaceutical-grade standards.

[0090] This invention establishes a complete quality control system: Raw material quality control: Styrene purity ≥ 99%; CO2 purity ≥ 99.9%; other reagents are analytical grade or industrial grade.

[0091] Process quality control: Online monitoring of reaction temperature, pressure, pH, dissolved oxygen and other parameters; periodic sampling and analysis of substrate and product concentrations; analysis using methods such as HPLC, GC, LC-MS and other methods.

[0092] Product quality control: Cinnamic acid: appearance, melting point, infrared spectrum, nuclear magnetic resonance spectrum, HPLC purity (≥95%); L-phenylalanine: appearance, specific rotation, infrared spectrum, nuclear magnetic resonance spectrum, HPLC purity (≥98%), heavy metal content, microbial limits.

[0093] Example 1: Preparation of cinnamic acid by photocatalytic carboxylation reaction (preferred conditions) (1) Reaction raw materials and reagents: Styrene: purity ≥99%, industrial grade, purchased from Sinopharm Chemical Reagent Co., Ltd.; CO2: purity ≥99.9%, food grade, purchased from Beijing Helium North Branch Gas Industry Co., Ltd.; g-C3N4 photocatalyst: laboratory-made, specific surface area 120m² / g; Acetonitrile: chromatographic grade, purchased from Merck; Triethylamine: analytical grade, purchased from Sinopharm Chemical Reagent Co., Ltd.; K2CO3: analytical grade, purchased from Sinopharm Chemical Reagent Co., Ltd. (2) Preparation of g-C3N4 photocatalyst; 10g of melamine was placed in a crucible and heated to 550℃ at a heating rate of 5℃ / min. The temperature was maintained for 2 hours, and after natural cooling, yellow g-C3N4 powder was obtained. The obtained g-C3N4 powder was ground and sieved (200 mesh), washed three times each with deionized water and ethanol, and dried under vacuum at 80℃ for 12 hours to obtain the final product.

[0094] Catalyst characterization: XRD: Characteristic peaks appear at 2θ = 13.1° and 27.4°, corresponding to the (100) and (002) crystal planes, respectively. FTIR: CN and C=N stretching vibration peaks appear in the range of 1200-1650 cm⁻¹; UV-Vis: Absorption edge approximately 470 nm, band gap approximately 2.7 eV; BET specific surface area: 120 m² / g Reaction steps: Add the following components to a 250 mL quartz photoreactor: Styrene: 5.2 g (0.05 mol); g-C3N4 catalyst: 0.5 g (9.6% of the mass of styrene); Acetonitrile: 150 mL; Triethylamine: 7.6 g (0.075 mol); K2CO3: 6.9 g (0.05 mol); Deionized water: 50 mL; Seal the reactor and replace the air inside with CO2 three times. Then, introduce CO2 until the pressure reaches 2.0 MPa. Turn on the magnetic stirrer and set the speed to 600 rpm. Turn on the xenon lamp source (wavelength range 420-700 nm, light intensity 200 mW / cm²) and react at 60°C for 8 hours.

[0095] During the reaction, samples were taken for analysis every 2 hours. After the reaction was completed, the light source was turned off, and the reactor was allowed to cool to room temperature before the pressure was slowly released and the reaction solution was removed.

[0096] Product separation and purification: The reaction solution was filtered through a 0.22 μm filter membrane to recover the catalyst. The filtrate was evaporated using a rotary evaporator (50 °C, -0.08 MPa) to remove the solvent acetonitrile. The residue was acidified with 1 mol / L HCl to pH 2, and then extracted three times with ethyl acetate (50 mL each time). The combined organic phases were washed with saturated NaCl solution until neutral and dried over anhydrous Na₂SO₄. After filtration, the ethyl acetate was evaporated under reduced pressure to obtain the crude product.

[0097] The crude product was recrystallized from ethanol-water (volume ratio 1:3) to obtain white crystals. These crystals were then dried under vacuum at 60°C for 12 hours to obtain the final product, cinnamic acid.

[0098] Product characterization: Yield: 92.5%; Melting point: 132-134℃ (literature value: 133-134℃); ¹H NMR (400 MHz, DMSO-d6) δ (ppm): 12.20 (s, 1H, COOH), 7.68-7.52 (m, 3H, Ar-H), 7.42-7.28 (m, 3H, Ar-Hand =CH), 6.55 (d, J=16.0 Hz, 1H, =CH); ¹³C NMR (100 MHz, DMSO-d6) δ (ppm): 168.2 (COOH), 143.8 (=CH), 134.5 (Ar-C), 129.8 (Ar-CH), 128.9 (Ar-CH), 127.6 (Ar-CH), 120.5 (=CH); FTIR (KBr, cm⁻¹): 3430 (OH), 3020 (=CH), 2800-2900 (CH), 1680 (C=O), 1625 (C=C),1575, 1490 (Ar C=C), 1280 (CO), 970 (trans CH); HPLC purity: 96.8%; Chromatographic conditions: C18 column (250 mm × 4.6 mm, 5 μm), mobile phase: methanol-water (containing 0.1% phosphoric acid) (70:30), flow rate: 1.0 mL / min, detection wavelength: 280 nm, column temperature: 30℃; Example 2: Preparation of cinnamic acid by photocatalytic carboxylation (continuous flow process): (1) Microchannel photoreactor A microchannel photoreactor was used for continuous flow reaction. Microchannel dimensions: 500 μm wide, 200 μm deep, and 50 cm long. Reactor material: quartz glass. Light source: LED array (wavelength 450 nm, light intensity 300 mW / cm²).

[0099] (2) Preparation of reaction solutions: Solution A: 52 g / L styrene (acetonitrile solution); Solution B: 5 g / L g-C3N4 catalyst (acetonitrile suspension); Solution C: 76 g / L triethylamine (acetonitrile solution); Solution D: 69 g / L K2CO3 (aqueous solution) (3) Continuous flow reaction conditions: Total flow rate: 1.0 mL / min; Solution A flow rate: 0.4 mL / min; Solution B flow rate: 0.2 mL / min; Solution C flow rate: 0.3 mL / min; Solution D flow rate: 0.1 mL / min; CO2 flow rate: 20 mL / min; reaction temperature: 60℃; reaction pressure: 2 MPa; residence time: 30 minutes (4) Reaction results: Cinnamic acid yield: 94.5%; Selectivity: 96.2%; Space time yield: 12.5 g / (L·h) After 24 hours of continuous operation, the yield remained stable at 93-95%. Example 3: Construction of genetically engineered strains: (1) Cloning of the PAL gene and construction of the expression vector: The PAL gene (AtPAL1, GenBank accession number: AT2G37040) was amplified from Arabidopsis thaliana cDNA.

[0100] Primer sequences: Forward primer: 5'-CATATGGTGAGAGAGTCTTGGTTC-3' (NdeI site); Reverse primer: 5'-CTCGAGCTAAGACGAGGCAGAGC-3' (XhoI site); PCR amplification conditions: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 56℃ annealing for 30 seconds, 72℃ extension for 2 minutes, for a total of 30 cycles; 72℃ final extension for 10 minutes; the PCR product and pET-28a(+) vector were digested with NdeI and XhoI respectively, ligated, transformed into E. coli DH5α, positive clones were screened, and sequenced for verification.

[0101] (2) Construction of expression strain: The recombinant plasmid pET-28a-PAL was transformed into Escherichia coli BL21(DE3) to obtain the expression strain E. coli BL21(DE3) / pET-28a-PAL.

[0102] (3) Construction of gene knockout strains: The pheA gene (encoding branch acid mutase / prephenyl acid dehydratase) of Escherichia coli W3110 was knocked out using CRISPR-Cas9 technology.

[0103] sgRNA sequence: 5'-GCTGGTGCGTATGCGCTAGA-3' The sgRNA expression vector and donor DNA (a linear DNA fragment containing homologous arms) were co-transformed into E. coli W3110, and knockout strains were screened and verified by PCR and sequencing.

[0104] (4) Optimization of the shikimic acid pathway: Overexpression of key enzyme genes in the shikimic acid pathway: aroG: encoding DAHP synthase; aroB: encoding 3-dehydroquinic acid synthase; aroD: encoding 3-dehydroquinic acid dehydratase; aroE: Encodes shikimate dehydrogenase; the above gene was cloned into the pACYC-Duet vector to construct the multi-gene expression vector pACYC-aroGBDE.

[0105] (5) Construction of integrated strain: pET-28a-PAL and pACYC-aroGBDE were co-transformed into Escherichia coli W3110ΔpheA to obtain integrated strain E. coli W3110ΔpheA / pET-PAL / pACYC-aro.

[0106] Example 4: Bio-fermentation conversion to prepare L-phenylalanine (preferred conditions): (1) Culture medium composition: Seed medium (LB medium): peptone: 10 g / L; yeast extract: 5 g / L NaCl: 10 g / L; pH: 7.0; Fermentation medium: Glucose: 30 g / L; (NH4)2SO4: 10 g / L; K2HPO4: 3 g / L; KH2PO4: 1 g / L; MgSO4·7H2O: 0.5 g / L; NaCl: 1 g / L; CaCl2·2H2O: 0.05 g / L; FeSO4·7H2O: 0.01 g / L; MnSO4·H2O: 0.01 g / L; ZnSO4·7H2O: 0.01 g / L; CuSO4·5H2O: 0.005 g / L; Vitamin B1: 0.01 g / L; Biotin: 0.001 g / L; Cinnamic acid: 10 g / L; pH: 7.0.

[0107] (2) Seed culture: Take the strain W3110ΔpheA / pET-PAL / pACYC-aro from the -80℃ glycerol tube, inoculate it into 5 mL LB medium, and culture at 37℃ and 200 rpm for 12 hours. Then, transfer it to 100 mL LB medium at a 1% inoculation rate and culture at 37℃ and 200 rpm for 8 hours until OD600 = 2.0-3.0.

[0108] (3) Fermentation culture: The seed liquid was inoculated into 2 L of fermentation medium (5 L fermenter) at an inoculation rate of 5%. The fermentation conditions were as follows: temperature: 30℃; pH: 7.0 (adjusted by automatic addition of 2 mol / L NaOH and 1 mol / L HCl); stirring speed: 400 rpm; aeration rate: 1.0 vvm (air); tank pressure: 0.1 MPa; fermentation time: 36 hours.

[0109] (4) Induction of expression: When the OD600 reaches 0.6-0.8, add IPTG to a final concentration of 0.5 mM to induce PAL gene expression. At the same time, cool down to 25℃ and continue culturing.

[0110] (5) Substrate supplementation: During fermentation, 5 g / L of cinnamic acid was added every 6 hours for a total of 4 times (total supplementation amount 20 g / L).

[0111] (6) Carbon source replenishment: When the residual sugar concentration is below 5 g / L, add glucose to 15 g / L.

[0112] (7) Product separation and purification: After fermentation, the fermentation broth was centrifuged at 8000 rpm for 10 minutes and the supernatant was collected.

[0113] ① Ion exchange chromatography A strong acidic cation exchange resin (type 001×7) was used. Resin pretreatment: alternating treatment with 2 mol / L HCl and 2 mol / L NaOH, followed by washing with deionized water until neutral.

[0114] The pH of the fermentation supernatant was adjusted to 3.0, and the solution was passed through an ion exchange column at a flow rate of 2 BV / h. Impurities were washed away with deionized water, followed by elution with 2 mol / L ammonia solution, and the L-phenylalanine elution peak was collected.

[0115] ② Decolorization: Add activated carbon (1%, w / v), stir at 60℃ for 30 minutes, and filter.

[0116] ③ Concentration and crystallization: Concentrate the filtrate under reduced pressure to 1 / 3 of its original volume, cool to 4°C, and allow to crystallize for 12 hours. Filter and collect the crystals, then wash with a small amount of cold water.

[0117] ④ Recrystallization: The crude crystals were recrystallized with a 70% aqueous ethanol solution to obtain white crystals. The crystals were then dried under vacuum at 60°C for 12 hours.

[0118] (8) Product characterization: Yield: 96.8% (based on total cinnamic acid); Purity: 99.2% (HPLC); Specific rotation: [α]D² 0 = -34.5° (c=2, H2O) (Literature value: -34.5°); ¹H NMR (400 MHz, D2O) δ (ppm): 7.25-7.15 (m, 5H, Ar-H), 3.85 (dd, J=7.6, 5.2 Hz, 1H, α-CH), 3.15 (dd, J=14.0, 5.2 Hz, 1H, β-CH2), 2.95 (dd, J=14.0, 7.6 Hz, 1H, β-CH2); ¹³C NMR (100 MHz, D2O) δ (ppm): 174.5 (COOH), 137.2 (Ar-C), 129.8 (Ar-CH), 129.2 (Ar-CH), 127.5 (Ar-CH), 56.8 (α-CH), 38.5 (β-CH2); FTIR (KBr, cm⁻¹): 3400-3200 (NH, OH), 3030 (Ar CH), 2960 (CH), 1600 (NH2), 1500 (ArC=C), 1400 (COO⁻), 1130 (CN); Elemental analysis: Theoretical values: C 65.45%, H 6.71%, N 8.48%; Measured values: C 65.38%, H 6.75%, N 8.45%; Optical purity: 99.5% ee (chiral HPLC); Chiral HPLC conditions: Chiralpak AD-H column (250 mm × 4.6 mm, 5 μm), mobile phase: n-hexane-isopropanol-trifluoroacetic acid (90:10:0.1), flow rate: 1.0 mL / min, detection wavelength: 254 nm, column temperature: 25 °C; Specific measures to reduce costs: Catalyst cost reduction: Using g-C3N4 non-precious metal catalysts, the cost is only 1 / 40th of that of precious metal catalysts; the catalyst can be recycled more than 10 times, reducing the cost per use by 90%. The catalyst preparation process is simple, and the raw materials are inexpensive and readily available. Solvent costs are reduced because an acetonitrile-water mixed solvent is used, with a solvent recovery rate of >97%. The solvent can be recycled through simple distillation, and the aqueous phase can be reused, reducing wastewater discharge. Reduced energy consumption and costs: The reaction temperature is 60℃, resulting in lower energy consumption; LED light source (wavelength 420-700 nm) is used, resulting in high photoelectric conversion efficiency; the reaction time is 8 hours, leading to high production efficiency. Reduced raw material costs: CO2 is an industrial waste gas with extremely low costs; styrene is a bulk chemical product with stable prices; triethylamine and K2CO3 are commonly used chemical raw materials with low costs. The beneficial effects of the above technical solution are as follows: 1. Raw material innovation: Using CO2 as the main raw material, it realizes the resource utilization of CO2, reduces raw material costs, reduces carbon emissions, and is in line with the development direction of green chemistry and carbon neutrality.

[0119] 2. Process innovation: The coupling of photocatalytic carboxylation with bio-fermentation enables the conversion of simple raw materials into high-value-added amino acids, with a short process route and high atom economy.

[0120] 3. High efficiency conversion: The yield of cinnamic acid in the first stage is ≥90%, the conversion rate of L-phenylalanine in the second stage is ≥95%, and the total yield is ≥85%, which is significantly higher than that of existing technologies.

[0121] 4. Green production: Photocatalysis technology is used to drive the reaction with solar energy; the bio-fermentation conditions are mild and energy consumption is low; solvents and catalysts can be recycled and reused, resulting in less waste emissions.

[0122] 5. Continuous production: It realizes two-stage continuous production, with high production efficiency and high equipment utilization, which is suitable for industrial-scale production.

[0123] 6. Cost advantages: Low raw material costs, low energy consumption, simple processes, low production costs, and strong product competitiveness.

[0124] 7. High product quality: L-phenylalanine purity ≥98%, optical purity ≥99%, meeting food-grade and pharmaceutical-grade standards.

[0125] 8. Wide range of applications: The process of this invention can be applied to the production of various aromatic amino acids, such as tyrosine and tryptophan.

[0126] 9. Advanced technology: It integrates a variety of advanced technologies such as photocatalysis, genetic engineering, fermentation engineering, separation and purification, and has a high technical content.

[0127] 10. Good economic benefits: High return on investment, broad market prospects, and can create significant economic and social benefits.

[0128] In one embodiment, the process for determining the process combination (for photocatalytic reaction) of the current batch and current step 1 raw material combination (when the current batch and current step 1 raw material combination is mass-produced, step 1 is executed based on the corresponding process combination) includes: Step 11: Obtain the pre-determined pre-selected process combination corresponding to the current raw material combination in Step 1. The pre-selected process combination includes: pre-selected power, pre-selected stirring speed, theoretical average gas pressure drop rate, and set pressure; and obtain the stirring speed-CO2 gas pressure drop rate change trajectory within the preset stirring speed range under the pre-determined power corresponding to the current raw material combination in Step 1. Step 12: Add the raw material from Step 1 to the photoreactor, and introduce CO2 into the photoreactor until the pressure inside the photoreactor reaches the set pressure (reaction pressure range 0.1–10 MPa); turn on the light source of the photoreactor with the power of the light source being the pre-selected power corresponding to Step 1, and carry out the photocatalytic reaction for the preset time at the pre-selected stirring speed determined in Step 1; and repeatedly monitor the gas pressure (gas pressure inside the photoreactor), light source power, and stirring speed during the reaction; and construct the first trajectory, which is the reaction time-gas pressure change trajectory inside the photoreactor. Step 14: Determine the actual average pressure drop rate for the preset duration, and combine it with the theoretical average pressure drop rate determined in Step 11 to determine the actual power rate factor and the drop rate deviation. When the actual power rate factor is greater than or equal to the first threshold (within the range of 0.85 to 0.95), determine the pre-selected process combination as the set process combination. Step 15: When the power rate factor is less than the first threshold, analyze the slope fluctuation of the first trajectory. When the slope fluctuation of the first trajectory is greater than the preset fluctuation threshold (e.g., 10%), select the corresponding matching stirring speed based on the falling rate deviation in the stirring speed-CO2 gas pressure falling rate change trajectory, and only adjust the actual stirring speed to different matching stirring speeds, continue the photocatalytic reaction, and obtain the actual average gas pressure falling rate within the preset time corresponding to each matching stirring speed, and determine the matching stirring speed with the largest actual average gas pressure falling rate as the target matching stirring speed. And determine the power rate factor corresponding to the target matching stirring speed. When the actual power rate factor is greater than or equal to the first threshold, replace the pre-selected stirring speed in the pre-selected process combination with the target matching stirring speed to obtain the set process combination. Step 16: When the actual power rate factor is less than the first threshold, the corrected power is determined based on the power rate factor determined in step 15. Then, in the pre-selected process combination, the pre-selected stirring speed is replaced with the target matching stirring speed, and the pre-selected power is replaced with the corrected power to obtain the set process combination.

[0129] In step 11: Multiple raw material combinations: This refers to the existence of multiple different initial material configuration schemes in step 1. Each scheme is an independent raw material combination, and the differences between the different raw material combinations are reflected in the types, proportions, or states of materials.

[0130] Step 1 Raw Material Combination: This refers to the characteristic combination of all raw materials added to the photocatalytic reaction in Step 1, including material type, ratio (completely the same or within the same small range), concentration (liquid phase; completely the same or within the same small range), initial temperature, and pretreatment state.

[0131] For each combination of raw materials in step 1, experiments are conducted before mass production to determine the pre-selected process combination; The pre-selected power is determined based on a power pre-screening process (all rate data are from the same experimental calibration batch). The power pre-screening process is as follows: for the current raw material combination, within a preset power range (UV LED: electric power 10~50W), through experimental calibration, the stirring motor input power that can make the reaction yield / efficiency greater than the preset value (such as yield 90%) and has the lowest energy consumption is determined.

[0132] The power with a reaction yield / efficiency greater than a preset value was determined as the first power, and the actual pressure drop rate corresponding to each first power was determined during the experimental calibration process, and a first power correlation matrix was constructed. The row dimension of the first power correlation matrix represents the first power selected during the experimental calibration process.

[0133] The first power correlation matrix columns represent the actual pressure drop rate measured experimentally at each first power level, with each column corresponding to the rate data at that power level. This matrix is ​​a collection of results from multiple parallel experiments and is used for subsequent rate ratio calculations. Preselected stirring speed refers to the stirring speed of the stirrer that is experimentally calibrated within a preset stirring speed range (e.g., 100-2000 rpm) under the premise of fixed preselected power, which can enable the reaction system to achieve the target yield / efficiency (e.g., yield ≥90%) and minimize stirring energy consumption.

[0134] Preset stirring speed range: refers to the adjustable stirring speed range formed by floating up or down a certain percentage / value with the pre-selected stirring speed corresponding to the current raw material combination as the center value; this range is determined by experimental calibration before formal production, which can ensure that the reaction yield / efficiency is stable within the preset range (e.g., ≥85%), and the stirring energy consumption of all speeds within the range is not higher than 120% of the energy consumption corresponding to the pre-selected stirring speed.

[0135] The aforementioned stirring speed-CO2 pressure drop rate curve refers to a single-valued function curve formed by fitting data points to different stirring speeds within a preset stirring speed range, under the premise of a fixed pre-selected power and the current raw material combination. The peak point of this curve corresponds to the pre-selected stirring speed and is the core basis for determining the optimal mass transfer efficiency.

[0136] In step 14: Power rate factor = Actual average air pressure drop rate over a preset duration ÷ Theoretical average air pressure drop rate; In step 15: the reaction time-gas pressure change trajectory in the photoreactor is divided into continuous, non-overlapping segments of equal duration according to a preset time (e.g., a range of 2 to 10 minutes). The slope of each segment (i.e., the instantaneous average gas pressure drop rate within that segment) is calculated. The slope fluctuation is obtained by quantifying the slope fluctuation by the relative standard deviation of the slopes of all segments. Slope fluctuation = standard deviation of the slopes of all segments ÷ average slope of all segments. The specific matching stirring speed is selected based on the power rate factor and the trajectory of the change in stirring speed versus CO2 pressure decrease rate: First, determine the descent rate deviation = theoretical average air pressure descent rate - actual average air pressure descent rate determined in step 11; Then, in the trajectory of the change in stirring speed and CO2 pressure decrease rate, the matching stirring speed is obtained by deviating from the decrease rate deviation to both sides of the pre-selected stirring speed position; Step 16 specifically involves obtaining the first power correlation matrix corresponding to the current raw material combination in Step 1; Determine the rate ratio corresponding to each first power in the first power correlation matrix; The rate ratio corresponding to the current first power = the CO2 pressure drop rate obtained by the current first power in the corresponding power pre-screening process ÷ the CO2 pressure drop rate obtained by the pre-selected power in the corresponding power pre-screening process; The result of the rate ratio × the power rate factor determined in step 15 is between 1.05 and 1.15 (the setting of this target range (1.05 to 1.15) is based on the core principle that "the power correction range must accurately match the efficiency gap and avoid over-correction"). The median value of the target first power is determined to be the corrected power.

[0137] If the target first power is multiple discrete values, take their arithmetic mean as the corrected power; if the average power is not within the preset power range, select the closest power level.

[0138] The beneficial effects of the above technical solution are as follows: 1. The solution employs a pre-experimental calibrated power screening process to precisely identify the stirring motor input power that results in a reaction yield / efficiency higher than a preset value (e.g., 90%) while minimizing energy consumption. Simultaneously, it determines the stirring speed that achieves the target yield / efficiency (e.g., ≥92.5%) while minimizing stirring energy consumption. This ensures reaction efficiency from the outset, avoiding yield fluctuations and material waste caused by improper parameters.

[0139] When the power rate factor fails to reach the threshold, the system first analyzes the slope fluctuation of the gas pressure change trajectory to accurately and quickly match the optimal speed within the stirring speed-CO2 pressure decrease rate change trajectory. If the speed adjustment still fails to meet the standard, the system will also quickly and accurately correct the light source power based on the first power correlation matrix. This dynamic closed-loop correction mechanism ensures that the reaction always operates within the optimal efficiency range, maintaining a stable high reaction yield and significantly improving production continuity and stability.

[0140] This solution calibrates and establishes a quantitative data model, including a first power correlation matrix, a pre-process group, and a trajectory of the change in the stirring speed versus the rate of CO2 pressure drop, through a single pre-experiment. This forms a dynamically adaptable parameter system, completely eliminating the drawback of existing technologies that use "fixed process parameters" for all batches of raw materials. Unlike existing technologies, it avoids the significant drop in reaction efficiency due to raw material differences, and also avoids the high cost and long cycle of conducting numerous pre-experiments to redetermine parameters before each batch of production. In actual production, only quantitative indicators such as the power rate factor and slope fluctuation need to be calculated based on real-time monitored pressure data. Through a progressive dynamic closed-loop correction of "first matching the optimal stirring speed, then accurately correcting the light source power," the optimal process parameters for the current raw material combination can be quickly locked, achieving rapid and accurate parameter determination. This ensures that the reaction always operates within the optimal efficiency range, significantly improving the continuity, stability, and overall capacity of multi-batch production.

[0141] 2. The preset stirring speed range is an adjustable interval centered on a pre-selected speed, fluctuating up and down by a certain percentage. This interval, calibrated experimentally, ensures stable yield. When raw material fluctuations or equipment status changes occur during production, the system can quickly adjust the speed within this safe range without needing to repeat the full-range experiment, thus shortening the response time for parameter adjustments and reducing the risk of production stoppage.

[0142] 3. From power selection to speed calibration, the solution consistently prioritizes "lowest energy consumption" as one of its core objectives. For example, power selection directly targets the input power that achieves the desired yield while minimizing energy consumption, and speed calibration focuses on minimizing stirring energy consumption. In the dynamic correction phase, both speed adjustments and power corrections are based on efficiency gaps to avoid over-correction. This reduces ineffective energy consumption from multiple dimensions, including light source and stirring, resulting in a significant reduction in electricity costs over the long term.

[0143] For each independent raw material combination (with differences in material types, ratios, concentrations, etc.), the solution can complete the calibration of exclusive process parameters and the formulation of dynamic correction rules through pre-experimentation. This standardized adaptability of "one set of parameters for one set of raw materials" enables the process to be quickly adapted to the production of multiple raw materials, and is especially suitable for flexible production scenarios with multiple batches and small quantities.

[0144] In one embodiment, the purification of cinnamic acid by crystallization or chromatography includes: Step S11: Obtain the melting process temperature-reference cinnamic acid solubility mapping table within the melting process temperature range of crude cinnamic acid; determine the target melting process temperature corresponding to the median value of the reference cinnamic acid solubility. The median value of the reference cinnamic acid solubility is the median value of the sequence formed by arranging the reference cinnamic acid solubility in the dissolution process temperature-reference cinnamic acid solubility mapping table in ascending order. When there are two median values, the average of the two median values ​​is taken as the median value of the reference cinnamic acid solubility. In this cinnamic acid purification process, the temperature range (65-75℃) for dissolving crude cinnamic acid is a temperature constraint that ensures dissolution efficiency and product stability. The target volume ratio (1:3) is the optimal feeding ratio verified by experiments within this temperature range, with the lowest solubility as a safety benchmark. The two are matched to ensure that the crude cinnamic acid is completely dissolved in the entire range of 65-75℃, while avoiding the increase in crystallization energy consumption caused by excessive solvent. Together, they provide a stable solution concentration basis for subsequent crystallization processes.

[0145] For one or more batches of crude cinnamic acid produced by photocatalytic reaction under step 1 based on experimental qualified production, crude cinnamic acid is mixed with ethanol aqueous solution at a target volume ratio (1:3), and dissolution test is carried out under the temperature range of crude cinnamic acid dissolution process. Based on the experimental results, a dissolution process temperature-reference cinnamic acid solubility mapping table under the temperature range of crude cinnamic acid dissolution process is constructed. The target dissolution process temperature is the dissolution process temperature corresponding to the median value of the reference cinnamic acid solubility in the dissolution process temperature-reference cinnamic acid solubility mapping table. Step S12: Obtain the predetermined process parameters of the cinnamic acid solution (the above filtrate), including: process crystallization feed rate and process temperature of each sub-crystallization chamber; Step S13: Crude cinnamic acid and aqueous ethanol solution are continuously mixed at the target volume ratio (1:3), and stirred at the target dissolution process temperature until completely dissolved. Insoluble impurities are removed by precision filtration, and the actual solubility of cinnamic acid is tested to determine the solubility coefficient of cinnamic acid. Cinnamic acid solubility coefficient = actual cinnamic acid solubility determined in step S13 ÷ median value of the reference cinnamic acid solubility mentioned in step S11; Step S14: The cinnamic acid solution enters the segmented crystallizer at the process crystallization feed rate determined in step S11. The inlet solution temperature of the segmented crystallizer is detected to determine the solution temperature deviation. Solution temperature deviation = inlet solution temperature of segmented crystallizer - target dissolution process temperature; In step S11, the dissolution process temperature-reference cinnamic acid solubility mapping table is used to deviate the target dissolution process temperature from the filtrate temperature to obtain the first cinnamic acid solubility. First, the filtrate temperature deviation is calculated. Then, the "temperature point corresponding to the deviation of the target dissolution process temperature from this temperature deviation" is found in the mapping table, and the solubility data corresponding to this temperature point is read and defined as the first cinnamic acid solubility (first cinnamic acid solubility in the mapping table - target dissolution process temperature = solution temperature deviation). This corrected solubility parameter will replace the original baseline solubility and be used for core operations such as supersaturation calculation and crystallization rate control in subsequent crystallization processes. This solves the problem of the theoretical and actual solubility values ​​not matching due to temperature fluctuations, ensuring the stability of the crystallization process and the consistency of product yield and purity.

[0146] Step S15: Determine the adjustment temperature of each sub-crystallization chamber based on the solubility of the first cinnamic acid and the solubility coefficient of cinnamic acid; Step S16: The cinnamic acid solution is fed into the segmented crystallizer at the process crystallization feed rate determined in step S11, and each sub-crystallization chamber is controlled to operate at the corresponding adjusted temperature to achieve crystallization of cinnamic acid.

[0147] Solubility deviation factor = (first cinnamic acid solubility - median value of the reference cinnamic acid solubility described in step S11) ÷ first cinnamic acid solubility; The current sub-crystallization chamber adjustment temperature = the current sub-crystallization chamber process temperature × (1 + solubility deviation factor × cinnamic acid solubility coefficient × temperature adjustment coefficient). Temperature adjustment coefficient (value is 0.8 to 1.2): Through offline small-scale tests, for multiple dynamic combinations of target process temperature, solubility deviation factor and solubility coefficient, the temperature correction ratio that can simultaneously achieve the target crystal size distribution and yield is tested, and then the ratio is substituted into the formula to calculate the initial adjustment coefficient. The process parameters for the cinnamic acid solution in step S11 are based on an overall control system determined by coupling the crystallization kinetics of cinnamic acid with industrial production conditions (ensuring qualified production, which guarantees stable crystallization process, compliant production efficiency, and compliant product quality). First, the solubility curves of cinnamic acid at different temperatures are measured to clarify the process crystallization feed concentration range, ensuring that this concentration provides sufficient crystallization driving force while remaining within the metastable region to avoid uncontrolled spontaneous nucleation. Then, based on the crystal growth rate and nucleation rate at this concentration, combined with the effective volume of the sub-crystallization chamber, the process crystallization feed rate is determined by reverse calculation, ensuring dynamic matching between the feed rate and crystal growth rate, and maintaining stable supersaturation in the system. Finally, based on the influence of segmented temperature control on crystal morphology and particle size distribution, and combined with the phase transition point determined by the solubility curve, a corresponding process temperature is set for each sub-crystallization chamber.

[0148] The beneficial effects of the above technical solution are as follows: First, an experimental mapping table of "dissolution process temperature - baseline cinnamic acid solubility" was established, using the median value of the solubility sequence as the baseline value. Simultaneously, the optimal dissolution process conditions within the 65–75℃ range were determined based on the target volume ratio (1:3), providing a stable solution concentration basis for subsequent production.

[0149] By taking the median value of the solubility sequence, extreme data interference is avoided, and a stable anchor point that fits the normal production situation is established. This makes it easy to judge the deviation status. Combined with the "solution coefficient" to quantify the deviation of the current batch of material from the benchmark, and the "solution temperature deviation" to compensate for equipment temperature loss, there is no need to repeatedly test the solubility in the crystallization process. The actual solubility at the inlet of the crystallization equipment can be predicted by the mapping table alone. Furthermore, by adjusting the sub-crystallization chamber, the process parameters can be automatically adapted to material fluctuations and temperature changes, which not only improves the control efficiency, but also reduces the detection cost and the risk of lag.

[0150] This solution requires only one actual solubility test during the dissolution stage; repeated testing is unnecessary during crystallization. Dual correction for solubility coefficient and temperature deviation allows process parameters to automatically adapt to the characteristic fluctuations of each batch of material. Predictive control based on a mapping table can precisely maintain supersaturation stability during the crystallization process.

[0151] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A process for the continuous production of phenylalanine using carbon dioxide as the main raw material, characterized in that: include: Step 1: Photocatalytic carboxylation stage: Styrene, photocatalyst, solvent, electron donor, and alkaline additive are added to the photoreactor. CO2 is introduced into the photoreactor until the pressure inside the photoreactor reaches the set pressure. The light source of the photoreactor is turned on, and the photocatalytic reaction is carried out under stirring conditions. After the reaction is completed, the catalyst is recovered by filtration, the solvent is recovered by distillation, and cinnamic acid is purified by crystallization or chromatographic separation. Step 2: Bio-fermentation stage: Add the cinnamic acid obtained in Step 1 to the fermentation medium, inoculate the genetically engineered strain into the fermentation medium, and control the fermentation conditions; during the fermentation process, add carbon source, nitrogen source and cinnamic acid according to the carbon and nitrogen concentration of the fermentation broth and the growth of the cells; after the fermentation is completed, separate the cells and purify L-phenylalanine.

2. The process for continuous production of phenylalanine using carbon dioxide as the main raw material according to claim 1, characterized in that: The photocatalyst is selected from one or more of the following types: Metal oxide photocatalysts, composite oxide photocatalysts, sulfide photocatalysts, nitride photocatalysts, noble metal supported photocatalysts, quantum dot photocatalysts, organic framework material photocatalysts, organic semiconductor photocatalysts, and dye-sensitized photocatalysts.

3. The process for continuous production of phenylalanine using carbon dioxide as the main raw material according to claim 1, characterized in that: The solvent is selected from one or a mixture of solvents, where the mixture is a mixture of any two or more of the following solvents: organic solvents, ionic liquids, and aqueous systems.

4. The process for continuous production of phenylalanine using carbon dioxide as the main raw material according to claim 1, characterized in that: The electron donor is selected from one or more of the following types: Organic amines, organosulfur compounds, organophosphorus compounds, heterocyclic compounds, inorganic compounds, I-, Br-, S 2- S2O3 2- N2H4, H3PO2, NaBH4.

5. The process for continuous production of phenylalanine using carbon dioxide as the main raw material according to claim 1, characterized in that: The specific process parameters for the photocatalytic carboxylation reaction are as follows: Raw material molar ratio: Styrene:CO2 = 1:(1~50); Styrene:catalyst = 1:(0.001~0.5); Styrene : Electron donor = 1 : (0.1~10); Styrene:alkaline additive = 1:(0.1~5); Reaction temperature: 0~150℃; Reaction pressure: 0.1–10 MPa; Light intensity: 10~2000mW / cm 2 ; Reaction time: 0.5–48 hours; Stirring speed: 100–2000 rpm; Light source types: ultraviolet light, visible light, sunlight, LED light source, xenon lamp, mercury lamp, sodium lamp; among which, the wavelength of ultraviolet light is 200-400nm; the wavelength of visible light is 400-800nm.

6. The process for continuous production of phenylalanine using carbon dioxide as the main raw material according to claim 1, characterized in that: In step 2, the strain types include: engineered Escherichia coli, engineered yeast, engineered Pseudomonas, engineered Bacillus, engineered Corynebacterium, Rhodotorula spp. yeast, Aspergillus spp. fungi, and Streptomyces spp. actinomycetes.

7. The process for continuous production of phenylalanine using carbon dioxide as the main raw material according to claim 1, characterized in that: In step 2, the culture medium consists of: carbon source, nitrogen source, inorganic salt, growth factor, precursor substance, pH adjuster, and defoamer.

8. The process for continuous production of phenylalanine using carbon dioxide as the main raw material according to claim 1, characterized in that: In step 2, the process parameters include: Seed culture process parameters, including: Seed culture media include: LB medium or M9 basal medium; Incubation temperature: 25–40℃; Incubation time: 6–24 hours; Shaking machine speed: 100-300 rpm; Inoculation dosage: 0.1%–10% (v / v); Fermentation culture process parameters, including: Fermentation temperature: 20~45℃; Fermentation pH: 5.0–9.0; Fermentation time: 12–72 hours; Stirring speed: 100–1000 rpm; Ventilation rate: 0.1–5 vvm; Tank pressure: 0.01~0.5 MPa.

9. The process for continuous production of phenylalanine using carbon dioxide as the main raw material according to claim 1, characterized in that: The feeding strategy in step 2 is as follows: Carbon source replenishment: When the residual sugar concentration is below 5 g / L, add glucose to bring the concentration up to 10-20 g / L; Nitrogen source replenishment: When the amino nitrogen concentration is below 0.5 g / L, add nitrogen source to 1-2 g / L; Precursor feeding: When the concentration of cinnamic acid is below 1 g / L, add cinnamic acid to bring the concentration to 5-10 g / L; Inducer feeding: PAL gene expression was induced by IPTG or lactose, with a final concentration of 0.1–1 mM; The dissolved oxygen control strategy is as follows: the dissolved oxygen level is 10-100%; the control method is to adjust the stirring speed, aeration rate, tank pressure or add pure oxygen. The pH control strategy is to automatically add acid or alkali with a control accuracy of ±0.1 pH unit. The temperature control strategy is to automatically adjust the cooling water or heating system; temperature control accuracy: ±0.5℃.

10. The process for continuous production of phenylalanine using carbon dioxide as the main raw material according to claim 1, characterized in that: The raw material ratio and reaction conditions in step 2 include: Cinnamic acid dry weight: bacterial dry weight = 1:(0.1~10); Initial concentration of cinnamic acid: 1–50 g / L; Cell density: OD600 = 0.1–50; Conversion time: 1–48 hours; Conversion temperature: 20~45℃; Conversion pH: 6.0–9.0; Coenzyme: Pyridoxal phosphate concentration is 0.01–1 mM; Metal ion: Mg 2+ Mn 2+ Zn 2+ Fe 2+ The concentration of metal ions is 0.1–10 mM.

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