Screening method for amino acid degradation products with inducing activity and application thereof

Abstract

The application discloses a screening method of amino acid degradation products with induction activity and application thereof. The method comprises the following steps: S1, using a molecular docking technology to screen amino acid degradation products with high affinity to auxin receptors; S2, selecting amino acid degradation products with the lowest binding energy, and jointly culturing microalgae under high light nitrogen stress conditions to screen amino acid degradation products capable of synergistically promoting the accumulation of pigments and oils of microalgae. The application further uses a dose gradient biological test to finally determine optimal amino acid degradation products and optimal induction concentrations thereof, investigates the promotion effects of different dose concentrations on the accumulation of pigments and oils of microalgae, and thus screens optimal doses of optimal amino acid degradation products. The method is simple and effective, can quickly screen amino acid degradation products capable of promoting the synergistic high production of pigments and oils of microalgae, and has important economic value and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of industrial biotechnology, and specifically relates to a screening method for amino acid degradation products with inducible activity and its application. Background Technology

[0002] Microalgae are among the most diverse and important biological resources in nature. Under stress conditions such as high light and nitrogen deficiency, they can accumulate large amounts of bioactive substances such as lipids and carotenoids within their cells. Utilizing microalgae fermentation technology to produce high-value products such as polyunsaturated fatty acids and carotenoids is of significant practical importance in addressing current global shortages of food and medicinal resources. Among numerous economically valuable microalgae, *Chromochloris zofingiensis* is a spherical green alga with unique physiological characteristics. It can utilize multiple carbon sources such as CO2 and glucose for autotrophic, heterotrophic, and polytrophic cultivation, exhibiting rapid growth and easily regulated physiological metabolism. Its most notable feature is its ability to utilize organic carbon sources such as glucose for high-density heterotrophic fermentation. Furthermore, under induced conditions, it can accumulate large amounts of high-value products such as lipids and astaxanthin within its cells, which are easily broken down and extracted. It is considered one of the microalgae resources with the greatest potential for commercial production of astaxanthin and lipids, possessing significant economic development value and commercial prospects.

[0003] Current technological developments targeting the accumulation of astaxanthin and lipids in *Zovochrysis galbana* primarily focus on the cellular metabolic responses and mechanisms under different culture methods and conventional induced stress conditions such as high light and low nitrogen. The resulting astaxanthin content is less than 1% of the dry weight, approximately one-third to one-quarter of that in *Haematococcus pluvialis*, and roughly equivalent to that of *Rhodotorula rubrum*. The very low accumulation rate during the co-production of astaxanthin and lipids is the main technological bottleneck, and it has not yet met the requirements for industrialization. Therefore, breaking through efficient induction techniques to significantly improve the synergistic production efficiency of astaxanthin and lipids is the key to the commercial development of astaxanthin from *Zovochrysis galbana*.

[0004] Amino acid degradation products, especially tryptophan catabolites (TCs), are a class of aromatic heterocyclic compounds with subtle structural differences, including indole, indolepropionic acid, indoleacrylic acid, methylindole, tryptophan, tryptophan alcohol, etc. They are derived from amino acid degradation by various Clostridium or Bacillus microorganisms and secreted extracellularly, exhibiting a variety of important biological activities in the physiological metabolism of organisms. Although there are currently no systematic reports on the inducing effects of amino acid degradation products on the synthesis of astaxanthin and lipids in *Green Algae Zoffychinensis*, considering that tryptophan degradation products are also a large class of signaling molecules in algal-microbe symbiotic interactions, it is speculated that they also play an important metabolic regulatory role in the physiological metabolism of microalgal cells.

[0005] Tryptophan degradation products (including structural derivatives) are diverse and abundant, and their standards are expensive. Traditional high-throughput batch screening biological methods, including high-throughput cultures based on culture plates, micro-embedding, diffusion boxes, hollow fibers, and separation chips, are time-consuming and labor-intensive. Even with high-throughput batch screening methods, it is impossible to evaluate the activity of dozens or even hundreds of tryptophan degradation products, making it difficult to guarantee the acquisition of target compounds that have a significant inducing effect on the accumulation of astaxanthin and lipids in chlorophyll. Therefore, new rapid screening technologies are urgently needed.

[0006] Virtual screening is an advanced technique in computer-aided drug design and development, used to rapidly screen small molecule compounds with significant affinity activities for specific target biomolecules. It is characterized by high speed, efficiency, and accuracy, significantly reducing the number of blind screenings in biological experiments, shortening experimental cycles, and saving research and development costs. Molecular docking technology is an important technique in virtual screening. It requires knowledge of the target protein and its three-dimensional crystal structure. Based on this structural information, computers are used to perform conformational and positional calculations between the molecular structure of the test compound and the active site, obtaining the optimal molecular connection conformation and affinity parameters. The best affinity molecule can be selected through scoring. This technique is highly suitable for examining the conformational docking between proteins with known three-dimensional structures and their small molecule ligands, greatly improving the efficiency and accuracy of small molecule drug activity screening, and rapidly screening dozens or even hundreds of compound molecules. Currently, there are no reports on using this technique to screen amino acid degradation products that synergistically promote the accumulation of astaxanthin and lipids in microalgae. Summary of the Invention

[0007] The primary objective of this invention is to overcome the shortcomings and deficiencies of existing technologies for evaluating and screening the inductive activity of amino acid degradation products, and to provide a screening method for amino acid degradation products with inductive activity, thereby improving the synergistic accumulation capacity of microalgae for high-yield pigments and lipids.

[0008] Another object of the present invention is to provide an application of the screening method for the amino acid degradation products with inducing activity.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A method for screening amino acid degradation products with inducible activity includes the following steps:

[0011] S1. Virtual screening of amino acid degradation products using molecular docking technology based on auxin receptors:

[0012] Using auxin receptor protein as the target protein and amino acid degradation products from the KEGG metabolic pathway database as small molecule ligands, the Autodock Vina structural analysis software was used for rapid batch molecular docking of the target protein and small molecule ligands. By calculating the conformational affinity parameters between the target protein and the small molecule ligands of different amino acid degradation products, the binding energy of each small molecule ligand was obtained. Then, based on the binding energy, amino acid degradation products with binding energies between -8.2 kcal / mol and -6.9 kcal / mol (i.e., -8.2 kcal / mol ≤ binding energy ≤ -6.9 kcal / mol) were selected.

[0013] S2. Biological validation of the effect of virtual-screened amino acid degradation products on intracellular pigment accumulation in microalgae:

[0014] ① Microalgae are activated and cultured to prepare microalgae seed culture;

[0015] ② Dissolve the amino acid degradation products obtained in step S1 with a co-solvent to prepare amino acid degradation product mother liquor;

[0016] ③ The biological verification experiment was divided into an experimental group and a control group: In the experimental group, microalgae seed culture was inoculated into a sugar-containing nitrogen-free fermentation medium, and then different doses of amino acid degradation product mother liquor were added to form at least three concentration gradients for each amino acid degradation product in the fermentation culture system. The fermentation culture system was then used to induce algal cell culture. After the culture was completed, the algal cells were collected. The control group did not add amino acid degradation products, and other conditions were the same. Finally, the biomass, pigment content and / or yield of the cells in the experimental group and the control group were measured respectively.

[0017] ④ Compare the biomass, pigment content and / or yield of cells in the experimental group and the control group, and select the amino acid degradation products corresponding to cells in the experimental group with higher biomass, pigment content and / or yield than those in the control group as amino acid degradation products for synergistically promoting the accumulation of pigments and lipids in microalgae (i.e., the amino acid degradation products with inducing activity).

[0018] The virtual screening method described in step S1 uses molecular docking technology for screening. By calculating the conformational affinity parameters between the target protein and the small molecule ligands of different amino acid degradation products, the amino acid degradation products with the lowest binding energy can be screened quickly.

[0019] The auxin receptor mentioned in step S1 is the auxin receptor SCF. TIR1 / AFB At least one of ABP1, IBR5, and SKP2A; preferably ubiquitin ligase complex SCF. TIR1 / AFB The auxin receptor protein TIR1 in the body.

[0020] The amino acid degradation products mentioned in step S1 are various small molecule compounds produced during amino acid metabolism; preferably, they are tryptophan degradation products, including all precursors and degradation products produced during tryptophan metabolism, further including all tryptophan metabolism degradation products in the KEGG metabolic pathway database, and also including structural derivatives and analogs of tryptophan degradation products; more preferably, they are at least one of 3-indolepyruvate (IP), indole-3-acetamide (IAM), indole-3-lactic acid (ILA), indigo (ID), indole-3-acetonitrile (IA), melatonin (MT), 3,5-dimethyl-2-aminobenzoic acid (AD), indole-3-carboxaldehyde (ICA), 5-hydroxyindoleacetic acid (HIA), pyridine-2,3-dicarboxylic acid (PCC), and tryptophan (TP); more preferably, they are 3-indolepyruvate (IP).

[0021] The microalgae mentioned in step ① are microalgal cells capable of accumulating high-value products such as pigments and lipids; preferably, *C. zofingiensis*.

[0022] The culture medium used for activation culture in step ① is either Basal medium or Bristol medium; preferably, it is a modified Bristol medium with the following formula: 10000 mg / L glucose, 75 mg / L K2HPO4, 5 mg / L FeCl3, 25 mg / L NaCl, 0.061 mg / L H3BO3, 750 mg / L NaNO3, 25 mg / L MgSO4·7H2O, 25 mg / L CaCl2·2H2O, 0.287 mg / L ZnSO4·7H2O, 175 mg / L KH2PO4, 0.0025 mg / L CuSO4·5H2O, 0.169 mg / L MnSO4·H2O, 0.00124 mg / L (NH4)6Mo7O 24 ·7H2O, pH 6.0~7.0.

[0023] The activation culture described in step ① involves transferring Zoff's green algae cells to a liquid culture medium for cyclic shaking culture. The culture conditions are: temperature 20–30℃, light intensity 10–300 μmol m -2 s -1 Rotation speed 50–500 rpm, light cycle 0h:24h (light:dark)–24h:0h (light:dark); preferably: culture temperature 26℃, light intensity 10 μmol / m³. -2 s -1 The light-dark cycle is 24h:0h, and the culture period is 1-2 weeks.

[0024] The co-solvent mentioned in step ② is at least one of water, ethanol and dimethyl sulfoxide; a suitable co-solvent can be selected according to different amino acid degradation products.

[0025] The inoculation amount of the microalgae seed solution mentioned in step ③ is 2.0-3.0 g / L at an initial cell density.

[0026] The fermentation medium mentioned in steps ③ and S3 is either Basal medium or Bristol medium; preferably, it is a sugar-containing, nitrogen-free fermentation medium with the following formula: 10000 mg / L glucose, 75 mg / L K₂HPO₄, 5 mg / L FeCl₃, 25 mg / L NaCl, 0.061 mg / L H₃BO₃, 25 mg / L MgSO₄·7H₂O, 25 mg / L CaCl₂·2H₂O, 0.287 mg / L ZnSO₄·7H₂O, 175 mg / L KH₂PO₄, 0.0025 mg / L CuSO₄·5H₂O, 0.169 mg / L MnSO₄·H₂O, 0.00124 mg / L (NH₄)₆Mo₇O 24 ·7H2O, pH 6.0~7.0.

[0027] The amount of amino acid degradation product added in step ③ is calculated based on its final concentration in the fermentation culture system from 0 μmol / L to 150 μmol / L. During the experiment, the concentration gradients can be set according to the actual situation (more than 3 concentration gradients can be set, preferably more than 5 concentration gradients, and even more than 7 concentration gradients are preferred). For example, low, medium and high concentration levels can be set for induction culture to observe its induction effect on algal cell growth and metabolite accumulation, thereby providing a decision-making basis for the subsequent screening of the best tryptophan degradation product. Preferably, it is calculated based on its final concentration in the fermentation culture system of 25 μmol / L, 50 μmol / L and 100 μmol / L.

[0028] The induction culture described in step ③ involves transferring well-grown microalgae cells obtained after activation culture into a nitrogen-free, sugar-containing fermentation medium, while simultaneously adding one or more tryptophan degradation products at appropriate concentrations. Finally, the cells are placed in a constant-temperature shaker or a microplate shaker with an external temperature-controlled lighting system for induction stress culture under high-intensity white fluorescent light irradiation. The microalgae induction culture device used is a transparent microplate or a similar thin-layer culture device, and the culture is performed by reciprocating shaking on a shaker. The microalgae induction culture device is placed in a light incubator with a built-in light source, and the light source used for light-induced culture is continuous white light irradiation. The preferred induction culture conditions are: temperature 20–30℃, continuous irradiation with a white fluorescent lamp, and a light intensity of 200 ± 30 μmol m². -2 s -1The rotation speed was 250–500 rpm, and the culture was terminated when the intracellular astaxanthin accumulation in the green algae reached its maximum. The preferred conditions were: a temperature of 26℃ and a light intensity of 200 ± 30 μmol / m². -2 s -1 The rotation speed was 350 rpm, and the induction culture time was 2 weeks.

[0029] The pigment mentioned in step ③ is at least one of astaxanthin, lutein, ketolutein, canthaxanthin and chlorophyll; preferably astaxanthin.

[0030] The screening method for amino acid degradation products with inducible activity further includes the following steps after step S2:

[0031] S3. Optimal amino acid degradation product dosage screening for accumulated pigments and lipids in microalgae.

[0032] Microalgae seed culture was inoculated into a sugar-containing, nitrogen-free fermentation medium. Then, different doses of the amino acid degradation product mother liquor obtained in step S2 were added to create a series of concentration gradient fermentation culture systems for each amino acid degradation product. The fermentation culture systems were then used to induce algal cell culture. After the culture was completed, the algal cells were collected, and the algal cell biomass, pigment content and / or yield, and lipid content and / or yield were measured. Finally, the concentration at which the algal cell biomass, pigment content and / or yield, and lipid content and / or yield all increased with increasing concentration were selected as the optimal induction concentration of the amino acid degradation product (or the concentration range of the amino acid degradation product could be further decreased or increased, and the optimal concentration could be the concentration at which the cell biomass, pigment content and / or yield, and lipid content and / or yield were all the highest).

[0033] The amino acid degradation product mentioned in step S3 is preferably 3-indolepyruvic acid (IP), which can be added to the culture system once or multiple times.

[0034] The concentration of the amino acid degradation products mentioned in step S3 can be set according to the different amino acid degradation products obtained by screening. Preferably, it is calculated based on the final concentration of 0 to 150 μmol / L in the fermentation culture system; more preferably, it is calculated based on the final concentration of 0, 25, 50, 75, 100, 125 and 150 μmol / L in the fermentation culture system.

[0035] The screening method for amino acid degradation products with inducible activity is applied in promoting the production of pigments and / or algal oils by microalgae.

[0036] The microalgae mentioned are microalgal cells capable of accumulating high-value products such as pigments and lipids; preferably, *C. zofingiensis*.

[0037] The pigment is at least one of astaxanthin, lutein, ketolutein, canthaxanthin and chlorophyll; preferably astaxanthin.

[0038] A method for synergistically promoting the accumulation of pigments and lipids in microalgae includes the following steps:

[0039] (1) Microalgae are activated and cultured to prepare microalgae seed culture;

[0040] (2) The amino acid degradation products are dissolved in a co-solvent to prepare an amino acid degradation product stock solution; wherein the amino acid degradation product is at least one of 3-indolepyruvic acid (IP), indole-3-acetamide (IAM), indole-3-lactic acid (ILA), indigo (ID), indole-3-acetonitrile (IA), melatonin (MT), 3,5-dimethyl-2-aminobenzoic acid (AD), indole-3-carboxaldehyde (ICA), 5-hydroxyindoleacetic acid (HIA), pyridine-2,3-dicarboxylic acid (PCC) and tryptamine (TP);

[0041] (3) Inoculate the microalgae seed liquid into a sugar-containing nitrogen-free fermentation medium, and then add the amino acid degradation product mother liquid to induce the culture of algal cells in order to promote the accumulation of pigments and lipids in microalgae cells.

[0042] The microalgae mentioned in step (1) are microalgae cells that can accumulate high-value products such as pigments and oils; preferably, Zoffingiensis.

[0043] The culture medium used for activation culture in step (1) is either Basal medium or Bristol medium; preferably, it is modified Bristol medium with the following formula: 10000 mg / L glucose, 75 mg / L K2HPO4, 5 mg / L FeCl3, 25 mg / L NaCl, 0.061 mg / L H3BO3, 750 mg / L NaNO3, 25 mg / L MgSO4·7H2O, 25 mg / L CaCl2·2H2O, 0.287 mg / L ZnSO4·7H2O, 175 mg / L KH2PO4, 0.0025 mg / L CuSO4·5H2O, 0.169 mg / L MnSO4·H2O, 0.00124 mg / L (NH4)6Mo7O 24 ·7H2O, pH 6.0~7.0.

[0044] The activation culture described in step (1) involves transferring Zoff's green algae cells to a liquid culture medium for cyclic shaking culture. The culture conditions are: temperature 20–30℃, light intensity 10–300 μmol m -2 s -1Rotation speed 50–500 rpm, light cycle 0h:24h (light:dark)–24h:0h (light:dark); preferably: culture temperature 26℃, light intensity 10 μmol / m³. -2 s -1 The light-dark cycle is 24h:0h, and the culture period is 1-2 weeks.

[0045] The co-solvent mentioned in step (2) is at least one of water, ethanol and dimethyl sulfoxide; a suitable co-solvent can be selected according to different amino acid degradation products.

[0046] The amino acid degradation product described in step (2) is preferably 3-indolepyruvic acid (IP).

[0047] The inoculation amount of the microalgae seed solution mentioned in step (3) is 2.0 to 3.0 g / L at an initial cell density.

[0048] The fermentation medium mentioned in step (3) is either Basal medium or Bristol medium; preferably, it is a sugar-containing, nitrogen-free fermentation medium with the following formula: 10000 mg / L glucose, 75 mg / L K2HPO4, 5 mg / L FeCl3, 25 mg / L NaCl, 0.061 mg / L H3BO3, 25 mg / L MgSO4·7H2O, 25 mg / L CaCl2·2H2O, 0.287 mg / L ZnSO4·7H2O, 175 mg / L KH2PO4, 0.0025 mg / L CuSO4·5H2O, 0.169 mg / L MnSO4·H2O, 0.00124 mg / L (NH4)6Mo7O 24 ·7H2O, pH 6.0~7.0.

[0049] The concentration of the amino acid degradation product mentioned in step (3) can be set according to different amino acid degradation products, preferably calculated based on the final concentration of 0 to 150 μmol / L (excluding 0) in the fermentation culture system.

[0050] The preferred conditions for induction culture in step (3) are: temperature 20–30℃, continuous irradiation with a white fluorescent lamp, and light intensity of 200 ± 30 μmol m. -2 s -1 The rotation speed was 250–500 rpm, and the culture was terminated when the intracellular astaxanthin accumulation in the green algae reached its maximum. The preferred conditions were: a temperature of 26℃ and a light intensity of 200 ± 30 μmol / m². -2 s -1 The rotation speed was 350 rpm, and the induction culture time was 2 weeks.

[0051] The pigment mentioned in step (3) is at least one of astaxanthin, lutein, ketolutein, canthaxanthin and chlorophyll; preferably astaxanthin.

[0052] The present invention has the following advantages and effects compared with the prior art:

[0053] 1. To synergistically promote the high production of pigments and lipids (algal oil) in microalgae and thus enhance their market value for commercial application, this invention provides a method for screening amino acid degradants that synergistically promote the accumulation of pigments and lipids in microalgae, comprising the following steps: S1, screening amino acid degradants with high affinity for auxin receptors using molecular docking technology; S2, selecting amino acid degradants with the lowest binding energy and inducing microalgae cultivation under high light and nitrogen deficiency stress conditions to verify the effect of these compounds on astaxanthin accumulation; S3, using dose gradient biological experiments to finally determine the optimal amino acid degradants and their optimal induction concentration, examining the promoting effect of different dose concentrations on the accumulation of pigments and lipids in microalgae, thereby realizing the biological efficacy of amino acid degradants in inducing the synergistic accumulation of intracellular pigments and lipids in *Zov's Green Algae*, solving the huge practical difficulties and technical challenges brought about by the structural diversity of amino acid degradants in their screening and evaluation technology, and thus effectively screening and obtaining amino acid degradants that help synergistically promote the high production of pigments and lipids in microalgae.

[0054] 2. This invention studies various amino acid degradation products with similar structures, using the synergistic promotion of pigment and lipid accumulation in microalgal cells as the screening criterion. This allows for the rapid screening of highly efficient, fast, and low-cost amino acid degradation products, promoting the synergistic high production of pigments and lipids in Zovella.

[0055] 3. This invention provides a novel induction process technology, opening up new fields for the practical application of amino acid degradation products. It provides important technical support for using amino acid degradation products to regulate microalgal metabolism and accumulate high-value products, directly promoting the commercial development and utilization of Zov's green algae. It has the advantages of being simple, effective, and rapid, significantly reducing the number of blind screenings and research costs in biological experiments, and has important application prospects.

[0056] 4. The virtual screening strategy of this invention transforms the conventional blind biological screening of amino acid degradation products into a rationally designed batch screening evaluation. This not only ensures the stability, reliability, and accuracy of batch screening but also significantly reduces screening time and labor costs. Furthermore, it is simpler and more efficient in terms of operation and technology, representing a significant technical improvement and enhancement over existing biological batch screening evaluation technologies.

[0057] 5. This invention establishes biological validation of amino acid degradation products and comparative experiments on optimal concentration doses. First, it screens numerous high-affinity amino acid degradation products to obtain those that promote efficient astaxanthin accumulation. Then, it uses dose-response curve experiments to determine the optimal inducing dose. This technical solution effectively supplements and improves the results of virtual screening, greatly ensuring the accuracy and reliability of the research results of this invention.

[0058] 6. The screening scheme established in this invention has the advantages of simple operation, accuracy and stability, and suitability for batch processing. It can significantly reduce the number of blind screenings in biological experiments, greatly improve the efficiency and accuracy of screening, significantly improve or enhance the existing production level of astaxanthin, shorten the experimental cycle, and save research and development costs. It provides technical support for the process development and utilization of Zovella pulcherrima to synergistically produce high-yield pigments and oils. It has very important economic value and application prospects in the research and application of inducing and promoting high-yield pigments and oils in microalgae. Attached Figure Description

[0059] Figure 1 This is a structural diagram of the active site and auxin-binding domain of the auxin receptor SCFTIR1 / AFB.

[0060] Figure 2 This is a search space information diagram for molecular docking of the active amino acid regions of auxin receptor target proteins.

[0061] Figure 3 This is a graph showing the effects of different tryptophan degradation products on the cell growth of Zovella green algae.

[0062] Figure 4 This is a graph showing the effect of different tryptophan degradation products on the astaxanthin content accumulated in Zoff's green algae cells.

[0063] Figure 5 This is a graph showing the effect of different tryptophan degradation products on the intracellular astaxanthin production of Zoff's green algae.

[0064] Figure 6 This is a graph showing the effect of different doses and concentrations of indolepyruvic acid on the growth of Zoff's green algae cells.

[0065] Figure 7 This is a graph showing the effect of different doses and concentrations of indolepyruvic acid on astaxanthin accumulation in Zoff's green algae cells.

[0066] Figure 8 This is a graph showing the effect of different doses and concentrations of indolepyruvic acid on lipid accumulation in Zoff's green algae cells. Detailed Implementation

[0067] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0068] This invention develops a method for screening amino acid degradation products that synergistically promote the accumulation of pigments and lipids in microalgae. First, a virtual screening strategy based on molecular docking technology is used to screen for small molecules of tryptophan degradation products with high affinity for auxin receptor target proteins. Then, biological experiments are used to further determine the optimal tryptophan degradation product and its optimal dosage concentration. Finally, this method is applied to the fermentation culture of *Chlorella zovichii* to significantly improve the synergistic accumulation of intracellular pigments and lipids. This invention evaluates the promoting effect of amino acid degradation products on the accumulation of pigments and lipids in *Chlorella zovichii*, confirming the close correlation between the molecular structure and biological activity of amino acid degradation products. This provides a significantly efficient induction technology solution for synergistically improving the accumulation of intracellular pigments and lipids in *Chlorella zovichii*. This invention utilizes computer-aided virtual screening technology, which can significantly improve the screening efficiency and accuracy of active amino acid degradation products. It also eliminates the need for expensive amino acid degradation product standards, reduces the number of experiments in biological batch screening, and significantly lowers research and development costs. It has obvious advantages such as high speed, high efficiency, and high accuracy.

[0069] Example 1: Virtual Screening Based on Auxin Receptor Protein Molecular Docking Technology

[0070] Tryptophan degradation products are a large class of aromatic heterocyclic compounds produced by the degradation of tryptophan. These compounds exhibit subtle structural differences in their side chains, resulting in a variety of physiological and metabolic regulatory activities. Due to their similar molecular structure to the auxin indoleacetic acid (IAA), it is hypothesized that tryptophan degradation products can also bind to auxin receptors, thereby utilizing auxin receptor-mediated signaling pathways to regulate the physiological and metabolic activities of microalgal cells. This example utilizes the ubiquitin ligase complex SCF, whose active site is conserved. TIR1 / AFB Using the auxin receptor protein TIR1 as the target protein, a series of tryptophan degradation products were used as ligands for molecular docking, thereby virtually screening out tryptophan degradation products with high affinity for the target protein. These products were then used as small molecule inducers with potential physiological metabolic regulatory activities for subsequent induction culture of Zov's green algae.

[0071] 1.1 Preparation of molecular structure information files for auxin protein receptor and tryptophan degradation products

[0072] The target protein used in this embodiment is the ubiquitin ligase complex SCF derived from Arabidopsis thaliana. TIR1 / AFB The auxin receptor protein TIR1 (TAN X, CALDERON-VILLALOBOS LIA, SHARON M, et al. Mechanism of auxin perception by the TIR1 ubiquitin ligase[J]. Nature, 2007, 446(7136):640–645.) has structural information that can be retrieved from the PDB protein database (Protein DataBank, www.rcsb.org), with the number 2P1Q. The structure and active site of this receptor protein are conserved among different species. Figure 1 The dashed box area is the auxin receptor SCF. TIR1 / AFB The structural diagram of the active site and auxin binding domain.

[0073] Normally, auxin primarily binds to the LRRs (Leucine-rich-repeats) domain at the top of the TIR1 receptor protein, through molecular binding via the carboxyl groups of its amino acid residues, thereby influencing the signaling pathways involved by the receptor. The three-dimensional structure of the auxin receptor protein described above was processed using the specialized molecular docking software AutoDock, including dehydration ("Edit" → "Delete Water"), hydrogenation ("Edit" → "Hydrogens" → "Add"), and charge addition ("Edit" → "Charges" → "Compute Gasteiger"). Finally, it was saved as a PDBQT file for later use.

[0074] In this embodiment, the tryptophan degradation products were selected from the tryptophan metabolic pathway in the KEGG metabolic pathway database (KEGG: Kyoto Encyclopedia of Genes and Genomes), with the number map00380. The name information of all tryptophan degradation products was obtained from this database. Then, the three-dimensional structure diagrams of all these molecules were obtained from the PubChem small molecule database (https: / / pubchem.ncbi.nlm.nih.gov / ). Finally, the three-dimensional structure information files of these tryptophan degradation small molecules were converted into PDBQT format files using the professional structure conversion software OpenBabel for later use.

[0075] To screen for active tryptophan degradation products, this embodiment uses PyMol, a professional protein structure analysis software, to analyze the specific binding domain between the auxin receptor and auxin, obtaining the search space near the active amino acid of the target protein, thereby determining the coordinate parameters (or docking box) of the binding space region of the tryptophan degradation product in the target protein during molecular docking. The specific operation steps and parameter conditions are as follows: First, ensure that the "GetBoxPlugin" plugin is installed in the "Plugin" menu of PyMol software. Open the 3D structure diagram of the 2P1Q protein receptor, click "Molecules" in the "Selecting" section of the parameter settings area, and then select the auxin molecule; then, click "GetBox Plugin" → "Autodetect box" in the "Plugin" menu. The software will automatically analyze and calculate the coordinate parameters and size of the search space, as shown below. Figure 2 As shown, its parameters are: center_x = 6.6, center_y = -112.4, center_z = -25.7, size_x = 14.7, size_y = 13.7, size_z = 15.2. This study uses this search space as a grid box for molecular docking of small tryptophan degradation molecules with auxin receptor target proteins, for subsequent batch virtual screening of tryptophan degradation products.

[0076] 1.2 Bulk molecular docking of tryptophan degradation products with auxin protein receptors and comparison of their affinity parameters

[0077] This embodiment utilizes the professional structural analysis software Autodock Vina for rapid batch molecular docking between auxin receptors and small molecule ligands of tryptophan degradation products. By creating docking parameter files and automatically running the docking program, the three-dimensional conformation and optimal binding energy information of each small molecule ligand of tryptophan degradation product binding to the auxin receptor target protein can be obtained, ultimately yielding docking conformations and binding energies for hundreds of molecules. The specific docking parameter file settings are as follows: receptor = TIR.pdbqt, center_x = 6.6, center_y = -112.4, center_z = -25.7, size_x = 14.7, size_y = 13.7, size_z = 15.2. The docking program uses Autodock Vina software: The specific method is to place the target protein structure file, ligand structure file, parameter setting file, etc. in the same folder, and then, keeping the above parameters unchanged, use all small molecule ligands as variables, click to run Vina.exe software, and through multiple loop operations, obtain 10 three-dimensional conformations (parameter set to "num_modes" = 10) of each tryptophan degradation small molecule ligand binding to the auxin receptor target protein, as well as the binding energy information file of the corresponding conformations.

[0078] By integrating and comparing the binding energy and other data obtained above, it is possible to quickly screen for auxin receptor SCFs. TIR1 / AFB By combining high-affinity ligand molecules with the KEGG metabolic pathway diagram and extensive literature review to infer their potential regulatory mechanisms, the potential active (low binding energy) tryptophan degradation products were finally identified. The experimental results are shown in Table 1.

[0079] Table 1. Tryptophan degradation products and their molecular structures obtained by virtual screening based on auxin receptor as the target protein.

[0080]

[0081]

[0082] The tryptophan degradation products shown in Table 1 share the common feature of possessing a benzene ring or aromatic heterocycle structure, while their main differences lie in their side chain groups. Most of these tryptophan degradation products obtained through virtual screening are precursors or degradation products of indoleacetic acid-based auxins, suggesting they can replace auxin and directly bind to auxin receptors, thereby affecting the signal transduction pathways mediated by auxin receptors and exerting the same or better physiological regulatory activities as auxin. In target protein-ligand molecular docking, these tryptophan degradation products can indeed bind to auxin receptor proteins, forming various binding conformations. When the binding energy is high, the likelihood of molecular binding between the two is very small, and the feasibility of conformational existence is low. Only when the binding energy is lowest will the target protein and small molecule ligand be in the optimal binding conformation and then exist stably. Table 1 shows the minimum binding energy of each tryptophan degradation product to the auxin receptor protein. Due to differences in their molecular structures, the optimal binding conformation with the target protein also varies, resulting in different minimum binding energies for each tryptophan degradation product. The data ranges from -8.2 kcal / mol to -6.9 kcal / mol.

[0083] Currently, there are relatively few studies comparing the effects of tryptophan degradation products on the physiological activities of microalgae, and no reports have been found analyzing the correlation between the differences in binding energy between tryptophan degradation products and auxin receptors and their physiological activities. This study hypothesizes that tryptophan degradation products can bind to different amino acid residues of auxin receptor proteins, thus existing in different optimal conformations. These conformational changes have a significant impact on the inducing activity of tryptophan degradation products. Currently, the relationship between specific conformational changes of tryptophan degradation products and their inducing activity lacks scientific research. This invention will conduct subsequent biological experiments on the actual inducing effect of virtually screened tryptophan degradation products to verify their biological effect in inducing the synergistic accumulation of pigments and lipids in *Zovochrysis galbana*.

[0084] Example 2: Comparison of the effects of different tryptophan degradation products on the growth and metabolism of Zoff's green algae

[0085] This embodiment primarily employs the aforementioned virtual screening technology to conduct in-depth comparative studies of various tryptophan degradation products, comprehensively analyzing their effects on the cell growth and physiological metabolism of *Zovella zovis*, ultimately identifying the active tryptophan degradation molecule that facilitates the synergistic accumulation of pigments and lipids. In plants, small molecules such as auxin and indoleacetic acid possess crucial physiological and metabolic regulatory activities; however, they are highly unstable, easily photodegraded, and readily destroyed by oxidases in vivo, generating inactive degradation products. Therefore, this embodiment aims to verify the biological activity of the aforementioned different tryptophan degradation products through biological experiments. More importantly, it seeks to screen for tryptophan degradation products with high affinity for auxin receptors and resistance to oxidative degradation to replace plant auxins, thereby enhancing the ability and production level of *Zovella zovis* to accumulate pigments and lipids. This is of great significance for the development and utilization of *Zovella zovis* biomass resources.

[0086] Therefore, based on the virtual screening described above, this embodiment sets up high, medium and low concentration levels for each tryptophan degrader to induce the culture of Zoff's green algae, thereby observing its induction effect on algal cell growth and astaxanthin accumulation, thus providing a decision-making basis for the subsequent screening of the best tryptophan degrader.

[0087] 2.1 Algal Activation and Seed Culture Preparation

[0088] The Zofingiensis strain, preserved in the laboratory, was transferred to test tube slants containing modified Bristol medium (agar content of 1.5–2.0% (w / v) to prepare solid slants) and cultured in a light incubator at 26°C and 10 μmol / m² light intensity. -2 s -1 The light-dark cycle was 24h:0h, and the growth of Zov's green algae was observed. After culturing for 1-2 weeks, it was stored in a refrigerator at 4℃. The culture was transferred for preservation every 3-6 months. The components of the modified Bristol medium (pH 6.5) are shown in Table 2.

[0089] Table 2. Liquid culture medium formulations (unit: mg / L)

[0090]

[0091]

[0092] 2.2 Induction culture experiment of different tryptophan degradation products on the growth and metabolism of Zoff's green algae

[0093] 2.2.1 Experimental protocol for inducing growth and metabolism of Zoff's green algae by different tryptophan degradation products

[0094] The Zoff's green algae seed culture prepared above was centrifuged and washed with sterile water, and then resuspended in nitrogen-free Bristol fermentation medium containing sugar (glucose concentration of 10 g / L) (the medium formula is shown in Table 2, where the NaNO3 content is 0 mg / L), ensuring that the initial cell density is about 2.0 to 3.0 g / L, and various types of tryptophan degradation products obtained in the previous virtual screening were added. Among these compounds, pyridine-2,3-dicarboxylic acid and indolepyruvic acid were prepared using water as a cosolvent, while melatonin, 3,5-dimethyl-2-aminobenzoic acid, 5-hydroxyindoleacetic acid, and tryptophan were prepared using ethanol as a cosolvent. All other compounds were prepared using dimethyl sulfoxide as a cosolvent. These stock solutions were then added to the culture medium at a concentration not exceeding 0.1% (v / v). The final concentrations of tryptophan degradation products in the culture medium were controlled at three different concentration gradients (25 μmol / L, 50 μmol / L, and 100 μmol / L) (named according to concentration, e.g., for melatonin, 25 μmol / L, 50 μmol / L, and 100 μmol / L were named "MT-L", "MT-M", and "MT-H", respectively, and so on). Algal cells were subjected to induced stress culture, with a culture without tryptophan degradation products serving as a control (Ctl). Both were cultured under high light irradiation and nitrogen-free stress conditions. Finally, the microalgal cell culture medium was dispensed into microplates using a pipette and placed in a microplate shaker at 350 rpm. The plates were then subjected to high-light nitrogen-deficiency induction culture for 2 weeks in a light incubator at 26°C with a light intensity of 200 ± 30 μmol / m². -2 s -1 Algal cells were induced and cultured for two weeks to promote the efficient accumulation of high-value metabolites such as astaxanthin and lipids. After the culture was completed, the algal cells were collected, and various analytical techniques were used to measure indicators such as cell biomass and astaxanthin accumulation, which were then used to evaluate the induction effect and screen tryptophan degradation products.

[0095] 2.2.2 Methods for determining growth and metabolic indicators of Zoff's green algae

[0096] 2.2.2.1 Methods for determining biomass

[0097] The biomass of *Zovella zovis* during its culture was determined using the dry weight method. A specific volume (V) of the sampled algal solution was placed in a pre-weighed centrifuge tube (m1). The tube was centrifuged at 6000 rpm for 1 min to collect the lower layer of algal cells. The cells were then resuspended in pure water by shaking, and the process was repeated twice by centrifugation and washing. The supernatant was removed, and the centrifuge tube containing the algal residue was dried in a 60℃ oven until constant weight. The total weight (m2) of the algal residue and the centrifuge tube was measured. The biomass of the *Zovella zovis* culture medium was calculated by converting the weight of the dried algal residue to the volume of the algal solution. The formulas are: Biomass (g / L) = (Total weight of centrifuge tube and dried algal residue m2 (g) – Weight of centrifuge tube m1 (g)) / Algal solution volume V (L); Biomass yield (g / L / d) = Biomass (g / L) / Culture time (d).

[0098] 2.2.2.2 Determination method of astaxanthin

[0099] The astaxanthin content was determined using a YMC column equipped with a carotene analytical column. TM The determination was performed using a reversed-phase liquid chromatograph (P680, DIONEX, USA) with a Carotenoid C30 column (4.6 × 150 mm, 3 μm, Waters, USA) and a PDA detector (PDA-100, Thermo Scientific, USA). The mobile phase consisted of methanol (A), methyl tert-butyl ether (MTBE) (B), and deionized water (C), with a flow rate of 0.8 mL / min. The linear gradient program was as follows: 0–6 min, 95% A / 5% B to 80% A / 20% B; 6–12 min, 60% A / 38% B / 2% C; 12–28 min, 50% A / 48% B / 2% C and hold for 5 min. The injection volume was 20 μL, the column temperature was 30 °C, and the detection wavelength was 480 nm. Astaxanthin was quantitatively analyzed using a standard curve established with standards.

[0100] 2.2.2.3 Method for determining total lipids

[0101] Total lipid content was determined using an organic solvent extraction method, briefly described as follows: Approximately 100 mg of freeze-dried algal powder was accurately weighed and placed in a cryovial. An appropriate amount of ceramic beads was added, and extraction was performed using a methanol / dichloromethane mixture (1:3, v / v). The mixture was first rapidly shaken in a high-speed bead mill, then frozen and crushed with liquid nitrogen to extract lipids. The supernatant was collected by centrifugation multiple times until complete extraction. All combined supernatant extracts were dried under nitrogen and weighed using a balance. The total lipid content (%), DW, was obtained from the difference in weight before extraction. Based on the measured biomass (g / L) and culture time (d), the total lipid yield (mg / L) and yield (mg / L / d) could be calculated.

[0102] 2.3 Results of the induction culture experiment on the growth and metabolism of Zoff's green algae by different tryptophan degradation products

[0103] The induction culture experiment described in Example 2.2 of this document can be used to obtain the effects of different tryptophan degradation products at different dosage levels on the growth and physiological metabolism of Zoff's green algae cells, such as... Figures 3-5 As shown.

[0104] Depend on Figure 3 The results showed that different tryptophan degraders had a significant impact on the biomass of *Zov's Chlorella* after cultivation (p<0.05). Except for tryptophan TP, indoleacetonitrile IA, brilliant blue ID, and indolepyruvate IP, which promoted algal cell growth at their optimal concentrations, other tryptophan degraders all had a certain degree of inhibitory effect on algal cell biomass. Among them, benzoic acid derivative AD had the strongest inhibitory effect on algal cell growth at high concentrations, with the biomass obtained at the end of cultivation being only about 2.8 g / L, a decrease of 55.3% compared to the control group. This indicates that excessively high concentrations of exogenous indole additives have an inhibitory effect on algal cell growth. Therefore, it is speculated that different tryptophan degraders have different dose-response effects on the cell growth of *Zov's Chlorella*.

[0105] Figure 4The figure shows the effect of different tryptophan degradation products on the astaxanthin content in *Chlorophytum zoversum* cells. As can be seen from the figure, the tryptophan degradation products obtained in the previous virtual screening of this invention all have a certain degree of inducing and promoting effect on astaxanthin accumulation at their optimal dosage concentrations. Among them, indoleformin (ICA), melatonin (MT), indoleglyoxylate (ILA), and IA all have good inducing effects on intracellular astaxanthin in *Chlorophytum zoversum*, especially when ICA is used at a medium dosage concentration, the astaxanthin content in the algal cells is significantly increased compared with the control group (p<0.05), reaching a maximum of 3.5 mg / g. However, this example found that as the concentration of ICA continued to increase, the accumulation of astaxanthin was inhibited. This physiological activity of ICA may be due to the presence of a special carbonyl group in indoleformin (ICA), which can react with C-C and CN bonds to generate various active compounds of indole alkaloids, which have important physiological regulatory activities in organisms under abiotic stress. Similarly, MT, TP, ID, PCC, ILA, and IAM at appropriate concentrations significantly promoted astaxanthin accumulation in *Chlorophytum zoversum*, while the addition of exogenous IAM did not significantly promote cell biomass or astaxanthin accumulation. This study also found that these inducers, at high concentrations, inhibited intracellular astaxanthin content in *Chlorophytum zoversum*. However, for IA (indoleacetonitrile) and IP (indolepyruvate) induction, intracellular astaxanthin content in *Chlorophytum zoversum* increased significantly with increasing concentration (p<0.05).

[0106] The effects of different tryptophan degradation products on the astaxanthin synthesis yield of the final Zoff's Chlorella cells obtained from the culture are as follows: Figure 5 As shown in the figure, different tryptophan degradation products and their concentrations have different inducing effects on the growth of *Chlorophytum zovichense* cells and the accumulation of astaxanthin, resulting in varying astaxanthin yields. Indoleformin (ICA) and melatonin (MT) significantly increased astaxanthin accumulation without significantly inhibiting cell growth, thus achieving higher astaxanthin yields, reaching up to 20.5 mg / L, a significant increase compared to the control group (p<0.05). However, the study also found that further increases in the concentration of these two compounds led to a significant decrease in astaxanthin yield. Conversely, under the induction of compounds IP and IA, although the astaxanthin yield was slightly lower than the former two, it showed a significant increasing trend with increasing concentration. This indicates that by controlling the concentration of these two compounds, it is possible to further significantly increase the intracellular astaxanthin accumulation in *Chlorophytum zovichense*.

[0107] The above results indicate that the tryptophan degradation products virtually screened in this embodiment have varying degrees of promoting effects on the cell growth and physiological metabolism of *Chlorella zovichii*, especially IP, a tryptophan degradation product, which exhibits significant inducing activity in promoting astaxanthin accumulation in *Chlorella zovichii*. Therefore, it is speculated that further optimization of the concentration and dosage of substances such as IP could potentially further enhance the ability of *Chlorella zovichii* to accumulate astaxanthin.

[0108] Example 3: Effects of indolepyruvate (IP) and its concentration / dosage on the growth and metabolism of Zoff's green algae.

[0109] Microalgae have developed a dose-response effect in response to external compounds or stimuli during natural evolution, which is prevalent in nature. Therefore, this embodiment focuses on indolepyruvate (IP), the optimal tryptophan degradation product screened in the aforementioned experiments, as the research object. A series of different concentration dose gradients (0–150 μmol / L) were used to induce the culture of *Chlorella zovichii* cells to investigate the effects of this tryptophan degradation product on the physiological and biochemical indicators of *Chlorella zovichii* cells. The aim is to further significantly improve the synergistic accumulation level of intracellular astaxanthin and lipids, thereby providing a basis and reference for the cultivation and production of high-value metabolic products from *Chlorella zovichii*.

[0110] 3.1 Algal Activation and Seed Culture Preparation

[0111] The Zoffingiensis algal strain preserved on laboratory slant was used to prepare microalgal seed solution for subsequent experiments, following the activation and culture method described in 2.1.

[0112] 3.2 Experimental methods for investigating the effects of indolepyruvate concentration gradients on the physiological and biochemical properties of Zoff's green algae.

[0113] 3.2.1 Induction culture protocol based on indolepyruvate concentration gradient

[0114] The prepared *Zovella pulcherrima* seed culture was centrifuged and resuspended according to the culture method described in 2.2. Indolepyruvate stock solution was added to the culture medium at the start of fermentation, and the final concentration of indolepyruvate in the culture medium was controlled at 0, 25, 50, 75, 100, 125, and 150 μmol / L for algal cell induction stress culture. High-light, nitrogen-free culture without tryptophan degradation products was used as a control. Subsequent algal cell culture methods and times were as described in 2.2 of the aforementioned examples. After culture, algal cells were collected, and various analytical techniques were used to measure cell biomass, astaxanthin, and lipid accumulation, among other indicators, to evaluate the induction effect of indolepyruvate.

[0115] 3.2.2 Methods for determining the growth and metabolic indicators of Zoff's green algae

[0116] After the algal cells obtained at the end of the culture were freeze-dried, the biomass, astaxanthin and total lipid accumulation of the algal cells were measured using the measurement method described in Example 2.2 above, so as to determine the optimal induction concentration of indolepyruvic acid in the concentration gradient experiment.

[0117] 3.3 Results of the induction culture experiment on the effects of indolepyruvate concentration gradient on the physiological and biochemical properties of Zoff's green algae.

[0118] The induction culture experiment described in Example 3.2 of this paper can be used to obtain the effects of indolepyruvic acid (IP) at concentration gradients of 0–150 μmol / L on the growth and physiological metabolism of Zoff's green algae cells. Figures 6-8 As shown.

[0119] Depend on Figure 6 It is evident that indolepyruvate (IP) in this embodiment did not significantly inhibit algal cell growth, although algal cell growth was somewhat affected with increasing IP concentration during cultivation. However, under the induction of high-concentration IP, algal cell growth was slightly reduced compared to the control, but not significantly different. This indicates that IP has less physiological toxicity to algal cells than IA, which may be due to the unique molecular structures of these two compounds. The aforementioned analysis has shown that indolepyruvate (IP) has the lowest binding energy to auxin receptors among many tryptophan degradation products, suggesting that it can bind to auxin receptors and participate in plant hormone signaling pathways. Furthermore, its unique structure exhibits keto-enol tautomerization, thus demonstrating good stability in solvents and consequently better biological activity.

[0120] Figure 7 The results show that different concentrations of IP significantly affected astaxanthin accumulation in *Chlorophytum zoversum* cells (p<0.05). With increasing IP concentration, the astaxanthin content in the algal cells also showed a significant increasing trend. Furthermore, since high concentrations of IP did not inhibit algal cell growth, the astaxanthin yield and productivity also increased with increasing IP concentration. When the IP concentration was at its maximum, the astaxanthin yield and productivity also reached their maximum values, at 18.5 mg / L and 1.3 mg / L / d, respectively.

[0121] The induction effect of IP on total lipids is basically similar to its induction effect on astaxanthin; both show a continuously increasing trend. Figure 8As shown, the total lipid content reached a maximum of 55.3% at the highest IP concentration, indicating that high concentrations of IP do not inhibit the accumulation of total lipids in Zoff Green algae. Simultaneously, Zoff Green algae can synthesize both astaxanthin and total lipids under IP induction, exhibiting a certain degree of synergistic effect in their metabolic synthesis. This result suggests that IP, as a tryptophan degradation product, has a higher affinity for auxin receptors and, due to its unique structure, exhibits keto-enol tautomerism, making it more stable in solvents. Therefore, using IP for the induction culture of Zoff Green algae demonstrates a better induction effect on the synergistic accumulation of astaxanthin and lipids in algal cells.

[0122] In summary, the innovative virtual screening technology employed in this embodiment can efficiently, rapidly, and in large quantities obtain potentially active tryptophan degradation products. These tryptophan degradation products exhibit different induction effects on cell growth and metabolite accumulation in *Chlorella zovichii* due to their varying molecular structures, and different concentrations and dosages significantly influence their induction effects. Most importantly, by studying the dose-response biological effect of indolepyruvate on *Chlorella zovichii*, using indolepyruvate as an inducer, the synergistic and efficient accumulation of astaxanthin and lipids within *Chlorella zovichii* cells can be achieved at the optimal induction concentration. This has significant application value for the commercial development and utilization of *Chlorella zovichii* biomass resources.

[0123] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for screening amino acid degradation products with inducible activity, characterized in that, Includes the following steps: S1. Virtual screening of amino acid degradation products using molecular docking technology based on auxin receptors: Using auxin receptor protein as the target protein and amino acid degradation products from the KEGG metabolic pathway database as small molecule ligands, the Autodock Vina structural analysis software was used for batch rapid molecular docking of the target protein and small molecule ligands. By calculating the conformational affinity parameters between the target protein and the small molecule ligands of different amino acid degradation products, the binding energy of each small molecule ligand was obtained. Then, based on the binding energy, amino acid degradation products with binding energies between -8.2 kcal / mol and -6.9 kcal / mol were selected. S2. Biological validation of the effect of virtual-screened amino acid degradation products on intracellular pigment accumulation in microalgae: ① Microalgae are activated and cultured to prepare microalgae seed culture; ② Dissolve the amino acid degradation products obtained in step S1 with a co-solvent to prepare amino acid degradation product mother liquor; ③ The biological verification experiment was divided into an experimental group and a control group: In the experimental group, microalgae seed culture was inoculated into a sugar-containing nitrogen-free fermentation medium, and then different doses of amino acid degradation product mother liquor were added to form at least three concentration gradients for each amino acid degradation product in the fermentation culture system. The fermentation culture system was then used to induce algal cell culture. After the culture was completed, the algal cells were collected. The control group did not add amino acid degradation products, and other conditions were the same. Finally, the biomass, pigment content and / or yield of the cells in the experimental group and the control group were measured respectively. ④ Compare the biomass, pigment content and / or yield of cells in the experimental group and the control group, and select the amino acid degradation products corresponding to cells in the experimental group whose biomass, pigment content and / or yield are higher than those in the control group as amino acid degradation products for synergistically promoting the accumulation of pigments and lipids in microalgae. The auxin protein receptor mentioned in step S1 is the ubiquitin ligase complex SCF. TIR1 / AFB The auxin receptor protein TIR1; The amino acid degradation product mentioned in step S1 is 3-indolepyruvic acid; The microalgae mentioned in step ① is Zoff's green algae (Zoff's green algae) C. zofingiensis ); The pigment mentioned in step ③ is astaxanthin; The amount of amino acid degradation product added in step ③ is calculated based on its final concentration in the fermentation culture system from 0 μmol / L to 150 μmol / L.

2. The screening method for amino acid degradation products with inducible activity according to claim 1, characterized in that, The following steps are included after step S2: S3. Optimal amino acid degradation product dosage screening for accumulated pigments and lipids in microalgae. Microalgae seed culture was inoculated into a sugar-containing, nitrogen-free fermentation medium. Then, different doses of the amino acid degradation product mother liquor screened in step S2 were added to form a series of concentration gradient fermentation culture systems for each amino acid degradation product. The fermentation culture systems were then used to induce algal cell culture. After the culture was completed, the algal cells were collected, and the biomass, pigment content and / or yield, and lipid content and / or yield of the algal cells were measured. Finally, the concentration that increased with increasing concentration was selected as the optimal induction concentration of the amino acid degradation product.

3. The screening method for amino acid degradation products with inducible activity according to claim 2, characterized in that: The concentration of the amino acid degradation product mentioned in step S3 is calculated based on its final concentration of 0 to 150 μmol / L in the fermentation culture system.

4. The screening method for amino acid degradation products with inducible activity according to claim 1, characterized in that: The activation culture medium used in step ① is a modified Bristol medium with the following formula: 10000 mg / L glucose, 75 mg / L K₂HPO₄, 5 mg / L FeCl₃, 25 mg / L NaCl, 0.061 mg / L H₃BO₃, 750 mg / L NaNO₃, 25 mg / L MgSO₄·7H₂O, 25 mg / L CaCl₂·2H₂O, 0.287 mg / L ZnSO₄·7H₂O, 175 mg / L KH₂PO₄, 0.0025 mg / L CuSO₄·5H₂O, 0.169 mg / L MnSO₄·H₂O, 0.00124 mg / L (NH₄)₆Mo₇O₇ 24 ·7H2O, pH 6.0 ~ 7.0; The activation culture conditions described in step ① are: temperature 20–30 °C, light intensity 10–300 μmol m². -2 s -1 Rotation speed 50-500 rpm, illumination cycle 0 h: 24 h ~ 24 h: 0 h; The co-solvent mentioned in step ② is at least one of water, ethanol, and dimethyl sulfoxide; The inoculation amount of the microalgae seed solution mentioned in step ③ is 2.0 ~ 3.0 g / L at an initial cell density; The formulation of the sugar-containing nitrogen-free fermentation medium described in steps ③ and S3 is as follows: 10000 mg / L glucose, 75 mg / L K₂HPO₄, 5 mg / L FeCl₃, 25 mg / L NaCl, 0.061 mg / L H₃BO₃, 25 mg / L MgSO₄·7H₂O, 25 mg / L CaCl₂·2H₂O, 0.287 mg / L ZnSO₄·7H₂O, 175 mg / L KH₂PO₄, 0.0025 mg / L CuSO₄·5H₂O, 0.169 mg / L MnSO₄·H₂O, 0.00124 mg / L (NH₄)₆Mo₇O 24 • 7H₂O, pH 6.0 ~ 7.0; The induction culture conditions described in step ③ are: temperature 20–30 ℃, continuous irradiation with a white fluorescent lamp, and a light intensity of 200 ± 30 μmol m. -2 s -1 The rotational speed is 250–500 rpm.

5. The screening method for amino acid degradation products with inducible activity according to any one of claims 1 to 4, used in promoting the production of pigments and / or algal oil by microalgae, characterized in that: The microalgae mentioned is Zoff's green algae (Zoff's green algae) C. zofingiensis ); The pigment mentioned is astaxanthin.

6. A method for synergistically promoting the accumulation of pigments and lipids in microalgae, characterized in that, Includes the following steps: (1) Microalgae are activated and cultured to prepare microalgae seed culture; (2) Dissolve the amino acid degradation products separately with a co-solvent to prepare amino acid degradation product stock solutions; wherein, the amino acid degradation product is 3-indolepyruvic acid; (3) Inoculate the microalgae seed liquid into a sugar-containing nitrogen-free fermentation medium, and then add the amino acid degradation product mother liquor to induce the culture of algal cells in order to promote the accumulation of pigments and lipids in microalgae cells; The concentration of the amino acid degradation product mentioned in step (3) is calculated based on its final concentration in the fermentation culture system being 0 to 150 μmol / L; wherein, the concentration of the amino acid degradation product is not 0; The pigment mentioned in step (3) is astaxanthin; The microalgae mentioned in step (1) is Zoff's green algae (Zoff's green algae) C. zofingiensis ).

7. The method for synergistically promoting the accumulation of pigments and lipids in microalgae according to claim 6, characterized in that: The activation culture medium used in step (1) is a modified Bristol medium with the following formula: 10000 mg / L glucose, 75 mg / L K2HPO4, 5 mg / L FeCl3, 25 mg / L NaCl, 0.061 mg / L H3BO3, 750 mg / L NaNO3, 25 mg / L MgSO4·7H2O, 25 mg / L CaCl2·2H2O, 0.287 mg / L ZnSO4·7H2O, 175 mg / L KH2PO4, 0.0025 mg / L CuSO4·5H2O, 0.169 mg / L MnSO4·H2O, 0.00124 mg / L (NH4)6Mo7O 24 ·7H2O, pH 6.0 ~ 7.0; The activation culture conditions described in step (1) are: temperature 20–30 °C, light intensity 10–300 μmol m -2 s -1 Rotation speed 50-500 rpm, illumination cycle 0 h: 24 h ~ 24 h: 0 h; The co-solvent mentioned in step (2) is at least one of water, ethanol and dimethyl sulfoxide; The inoculation amount of the microalgae seed solution mentioned in step (3) is 2.0 ~ 3.0 g / L at an initial cell density; The formula for the sugar-containing nitrogen-free fermentation medium mentioned in step (3) is as follows: 10000 mg / L glucose, 75 mg / L K2HPO4, 5 mg / L FeCl3, 25 mg / L NaCl, 0.061 mg / L H3BO3, 25 mg / L MgSO4·7H2O, 25 mg / L CaCl2·2H2O, 0.287 mg / L ZnSO4·7H2O, 175 mg / L KH2PO4, 0.0025 mg / L CuSO4·5H2O, 0.169 mg / L MnSO4·H2O, 0.00124 mg / L (NH4)6Mo7O 24 • 7H₂O, pH 6.0 ~ 7.0; The induction culture conditions described in step (3) are: temperature 20–30 °C, continuous irradiation with a white fluorescent lamp, and a light intensity of 200 ± 30 μmol m. -2 s -1 The rotational speed is 250–500 rpm.

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