Recombinant microorganism for high-level synthesis of polyphenol compound and application thereof

Abstract

According to the invention, three typical polyphenol compounds of resveratrol, naringenin and curcumin are taken as example products, yarrowia lipolytica is taken as a main display bacterium, and 20 new genetic modification targets are disclosed for modifying microorganisms so as to improve the level of synthesizing polyphenol compounds taking p-coumaroyl coenzyme A and malonyl coenzyme A as direct metabolism precursors. Therefore, the invention discloses a microorganism for improving synthesis of a polyphenol compound and an application of the microorganism. The microorganism is used for synthesizing the polyphenol compound with p-coumaroyl coenzyme A and malonyl coenzyme A as direct metabolism precursors. The efficient microbial synthesis of the polyphenol compound is an important production mode, the production performance of the strain is a key core, the key problem is solved, and the method has great application value.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and bioengineering, and specifically relates to a microorganism with improved ability to synthesize polyphenolic compounds and application thereof. The microorganism synthesizes polyphenolic compounds with p-coumaroyl-CoA and malonyl-CoA as direct metabolic precursors. Background Art

[0002] Polyphenol compounds with p-coumaroyl-CoA and malonyl-CoA as direct metabolic precursors are widely used in medicine, health care, food and other fields. Such compounds include active ingredients of traditional Chinese medicine such as flavonoids such as naringenin and baicalein, important functional compounds of diphenylethylene such as resveratrol and polydatin, as well as curcumin and anthocyanins. At present, the synthesis of polyphenol compounds is extracted from plants, and the stability of supply is restricted by many problems such as arable land, climate and complex plant extraction process.

[0003] Microbial fermentation can solve many problems faced by plant extraction methods and has the advantage of being green and environmentally friendly. However, low synthesis efficiency is the main reason that limits the efficient synthesis of polyphenolic compounds by microorganisms. Combination modification of key genes is an effective solution to improve the performance of microorganisms in synthesizing polyphenolic compounds. Under the action of different catalytic effector enzymes, p-coumaroyl-CoA and malonyl-CoA are converted in one step to form polyphenolic compounds with p-coumaroyl-CoA and malonyl-CoA as direct metabolic precursors. The biosynthesis process of these polyphenolic compounds uses the common p-coumaroyl-CoA and malonyl-CoA biosynthetic pathways. Therefore, a genetic modification combination that improves the microbial synthesis of one of the polyphenolic compounds can also have a general promoting effect on the microbial synthesis of other polyphenolic compounds.

[0004] In the process of improving the synthesis of polyphenol compounds by microorganisms through gene combination modification, identifying key genes is one of the technical difficulties. In addition, the microbial metabolic pathways and regulatory networks are complex, and the modification of a single gene will produce specific disturbances to the entire complex network, which may cause conflicts in the effects of multi-gene combination modification and reduce the synthesis level. Therefore, identifying effective gene combination modification is the second technical difficulty in improving the performance of microorganisms in synthesizing polyphenol compounds. Summary of the invention

[0005] The technical problem to be solved by the present invention is: how to improve the synthesis of polyphenol compounds with p-coumaroyl-CoA and malonyl-CoA as direct metabolic precursors by microorganisms.

[0006] In order to solve the above technical problems, the present invention uses three typical polyphenol compounds, resveratrol, naringenin and curcumin, as example products (stilbene, flavonoids, and pigment platform compounds), and Yarrowia lipolytica as the main display bacteria, and discloses 20 new genetic modification targets for improving microbial synthesis of polyphenol compounds with p-coumaroyl-CoA and malonyl-CoA as direct metabolic precursors.

[0007] First, the present invention first obtained 20 genetic modification targets for improving microbial synthesis of polyphenol compounds with p-coumaroyl-CoA and malonyl-CoA as direct metabolic precursors. The related modified genes are involved in the synthesis enhancement of polyphenol metabolic precursors, transcriptional regulation of the metabolic synthesis process, and protein regulation.

[0008] The synthesis enhancement of polyphenol metabolic precursors involves amino acids such as tyrosine and phenylalanine, which are precursors of p-coumaroyl-CoA, and acetyl-CoA, which is the synthetic precursor of malonyl-CoA. Genetic targets that have an enhancing effect on the synthesis of polyphenol compounds include: 4-hydroxyphenylpyruvate dioxygenase (4-HPPD, GI: 50547455), aromatic amino acid transaminase II (ARO9, GI: 50547993), 3-phosphoglycerate dehydrogenase (SER3, GI: 50555640), acylglycerol lipase 3 (TGL, GI: 50550941), serine hydroxymethyltransferase 2 (SHMT2, GI: 50547455), and acylglycerol lipase 3 (TGL, GI: 50550942). :50553214), serine hydroxymethyltransferase 1 (SHMT1, GI:50551359), aspartate aminotransferase (AKX1, GI:50553242), anabolic serine and threonine dehydratase precursor (ILV1, GI:50549715), lipoic acid ligase (LipB, GI:50557070), citrate transporter (CTP1, GI:50556988), etc. A total of 10. The polyphenol synthesis enhancement effect produced by the modification of the above targets is mainly achieved by strengthening the metabolic flow of the main synthesis pathway and reducing the diversion of key metabolites. The effective target genes play a role in the upstream synthesis pathway (such as SHMT2) and downstream conversion pathway (such as 4-HPPD) of the key compound shikimic acid. Serine hydroxymethyltransferase 2 (SHMT2) alone or in combination with any other target disclosed in the present invention can achieve optimization of metabolic flux allocation in the upstream synthesis pathway of shikimic acid; 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) alone or in combination with any other target disclosed in the present invention can achieve optimization of metabolic flux allocation in the downstream synthesis pathway of shikimic acid.

[0009] The microbial synthesis of polyphenolic compounds is achieved by the catalysis of metabolic enzymes. The expression pattern and functional effect of microbial metabolic reaction enzymes are regulated at the transcription and protein levels. Among them, the regulation at the transcription and post-transcription levels involves the coordination of functional elements such as transcription factors, RNA polymerases, and mRNA splicing factors, which ultimately achieves the effect of changing the synthesis level of polyphenolic compounds. Changing the intracellular content and activity of metabolic reaction enzymes through protein regulation can affect the synthesis of polyphenolic substances. The regulation process is mainly achieved by modifying or degrading proteins. Protein modifications include phosphorylation, acetylation, methylation, etc. These modifications can affect the function and interaction of proteins. In addition, protein degradation is also an important means of regulating metabolic pathways, which adjusts the flow of metabolic pathways and the synthesis of products by degrading proteins. The present invention discloses 10 genetic targets for improving the synthesis of polyphenolic compounds by regulating transcription and protein levels: CYC8 transcription repressor (CYC8, GI: 50546166), gluconeogenic transcription factor (CAT8, GI: 50549061), RNA polymerase II transcription regulator (HDB, GI: 50556186), pre-mRNA splicing factor 1 (LSM1, GI: 50545209), pre-mRNA splicing factor 5 (LSM5, GI: 50546793), serine / threonine protein phosphatase (CNA1, GI: 50543112), RING-type E3 ubiquitin transferase (TUL1, GI: 50556876), GTP binding protein (GTR1, GI: 50543528), WD-repeat protein (SWD1, GI: 50549369), UBC13 E2 ubiquitin conjugating enzyme (UBC13, GI: 50547641). Among them, mRNA pre-splicing factor 1 (LSM1) alone or in combination with any other target disclosed in the present invention can achieve regulation metabolism at the transcriptional level; ring-type E3 ubiquitin transferase (TUL1) alone or in combination with any other target disclosed in the present invention can achieve regulation metabolism at the protein level.

[0010] The amino acid molecules further defined above have more than 80% identity and encode protein molecules having the above enzyme function.

[0011] In the above method, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using the blastp tool, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences to calculate, the value of identity (%) can then be obtained.

[0012] In the above method, the above 80% identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0013] The present invention particularly provides 20 genetic modification targets for improving the microbial synthesis of polyphenolic compounds with p-coumaroyl-CoA and malonyl-CoA as direct metabolic precursors.

[0014] Further, the microorganism has the ability to synthesize stilbene compounds, preferably, it includes resveratrol synthase (EC:2.3.1.95); or, the microorganism has the ability to synthesize flavonoids and anthocyanidins, preferably, it includes chalcone synthase (EC:2.3.1.74) and chalcone isomerase (EC:5.5.1.6); or, the microorganism has the ability to synthesize curcuminoids, preferably, it includes curcumin synthase (EC:2.3.1.217).

[0015] Furthermore, the resveratrol synthase includes but is not limited to Vitis vinifera and Arachis hypogaea; the chalcone synthase includes but is not limited to Arabidopsis thaliana, Perilla frutescens, and Hypericum androsaemum; the chalcone isomerase includes but is not limited to Petunia hybrid, Saussurea medusa, and Allium cepa; and the curcumin synthase includes but is not limited to Curcumalonga and Oryza sativa.

[0016] Furthermore, the resveratrol synthase amino acid sequence is as shown in SEQ ID NO: 1 or its analogue, the chalcone synthase amino acid sequence is as shown in SEQ ID NO: 2 or its analogue, the chalcone isomerase amino acid sequence is as shown in SEQ ID NO: 3 or its analogue, and the curcumin synthase amino acid sequence is as shown in SEQ ID NO: 4 or its analogue.

[0017] The above analogs refer to those having one or a combination of the following characteristics:

[0018] (i) a mutant of the corresponding protein, the amino acid sequence of the mutant having at least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of the corresponding protein;

[0019] (ii) A fusion protein obtained by connecting a protein tag to the N-terminus or / and the C-terminus.

[0020] The 20 genetic modification targets are used individually or in combination not only for the synthesis of resveratrol, naringenin, and curcumin, but also for resveratrol and its derivatives, naringenin and its derived metabolites, curcumin and its derived metabolites. Furthermore, they are used for polyphenol compounds such as stilbene functional compounds, flavonoids, pigments, etc.; and further, they are used for improving microbial synthesis of polyphenol compounds with p-coumaroyl-CoA and malonyl-CoA as direct metabolic precursors.

[0021] Specifically, the polyphenol compound is a polyphenol compound with p-coumaroyl-CoA and malonyl-CoA as direct metabolic precursors;

[0022] Preferably, the polyphenolic compounds are flavonoids, phenolic acids, lignans, and stilbenes;

[0023] Furthermore, the polyphenol compound is a stilbene, flavonoid, or pigment platform compound;

[0024] Further, the stilbene compound is selected from resveratrol, scutellariae, picetol, polydatin, pterostilbene, mulberry glycoside and other resveratrol derivatives; the flavonoid compound is selected from naringenin and its derivatives, preferably, the naringenin and its derivatives are selected from naringenin, baicalein, quercetin, tangerine, luteolin, myricetin, catechin, epicatechin, isoliquiritigenin, xanthohumol, icariin, 7-O-(2-naphthylmethyl) naringenin, The pigment compound is selected from curcumin, anthocyanin and its derivatives. Preferably, the curcumin, anthocyanin and its derivatives are selected from demethoxycurcumin, bisdemethoxycurcumin, cyanidin, pelargonidin, peony pigment, delphinidin, morning glory pigment and malva pigment.

[0025] Preferably, the organism used for the genetic target is a microorganism, and the microorganism is a fungus or a bacterium;

[0026] Preferably, the fungus is selected from the genus Yarrowia, Kluyveromyces, Saccharomyces, Candida, Pichia, Aspergillus or Fusarium;

[0027] The bacterial cell is selected from the genus Lactobacillus or Bacillus;

[0028] Preferably, the Yarrowia is any one of Yarrowia lipolytica, Yarrowia.alimentaria, Yarrowia.Bubula, Yarrowia.hollandica, Yarrowia.deformans or Yarrowia.divulgata;

[0029] Preferably, the Kluyveromyces genus is any one of Kluyveromyces marxianus, Kluyveromyces marxianus variant, Kluyveromyces lactis, Kluyveromyces hubeiensis, Kluyveromyces wickheimii or Kluyveromyces thermotolerans;

[0030] Preferably, the Bacillus is any one of Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis;

[0031] Preferably, the Lactobacillus is any one of Lactobacillus delbrueckii bulgaricus, Lactobacillus acidophilus, Lactobacillus casei subsp. casei, Lactobacillus reuteri, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus plantarum or Lactobacillus salivarius;

[0032] More preferably, the microorganism is Yarrowia lipolytica, Candida lipolytica, Saccharomyces cerevisiae, Pichia pastoris, Rhodosporidium toruloides, Pichia fermentans, Kluyveromyces marxianus, Hansenula, Debaryomyces hansenii, Zygosaccharomyces bailii, Wickhamia anomala or Pichia kudria, Trichoderma reesei or Aspergillus niger.

[0033] More preferably, the culture medium comprises a carbon source and a nitrogen source, which may be a combination of one or more of the following:

[0034] (i) the carbon source is selected from a fermentable carbon source or a non-fermentable carbon source;

[0035] (ii) the nitrogen source is selected from an organic nitrogen source or an inorganic nitrogen source;

[0036] (iii) Biomass containing carbon and nitrogen sources.

[0037] More specifically, the fermentable carbon source is selected from glucose, fructose, galactose, xylose, and sucrose, and the non-fermentable carbon source is selected from ethanol, acetic acid and acetate, glycerol, lactate, methanol, and formic acid;

[0038] The organic nitrogen source is selected from peptone, yeast powder, and corn steep liquor; the inorganic nitrogen source is selected from ammonium sulfate, ammonium chloride, nitrate, and ammonia water;

[0039] The biomass containing carbon source and nitrogen source is agricultural, industrial and forestry biomass residues or biomass waste; more preferably, the carbon source in the culture medium is acetic acid or acetate, and the nitrogen source is ammonium sulfate or ammonium chloride as substrates.

[0040] The 20 genetic modification targets provided by the invention are used to improve the synthesis of polyphenol compounds with p-coumaroyl-CoA and malonyl-CoA as direct metabolic precursors by microorganisms, and have wide applications and economic value. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with specific embodiments, and the examples given are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0042] In the content analysis of the following examples, resveratrol (Tanmo Quality Inspection Standard Material Center, product catalog number 501-36-0) and naringenin (Shanghai Jizhi Biochemical Technology Co., Ltd., product catalog number 73692-50-9) were used as standard products, and high performance liquid chromatography was used to draw standard curves to quantitatively analyze the content of resveratrol and naringenin in the test samples.

[0043] Example 1: Prediction of genetic modification targets and construction of elements for improving the synthesis of polyphenolic compounds with p-coumaroyl-CoA and malonyl-CoA as direct metabolic precursors by Yarrowia lipolytica

[0044] First, 20 genetic modification targets were obtained through high-throughput screening to improve the microbial synthesis of polyphenol compounds with p-coumaroyl-CoA and malonyl-CoA as direct metabolic precursors. The relevant modified genes are involved in the synthesis enhancement of polyphenol metabolic precursors, transcriptional regulation of metabolic synthesis, and protein regulation.

[0045] The synthesis enhancement of polyphenol metabolic precursors involves amino acids such as tyrosine and phenylalanine, which are precursors of coumaroyl-CoA, and acetyl-CoA, which is the synthetic precursor of malonyl-CoA. There are 10 genetic targets that have an improving effect on the synthesis of polyphenol compounds, of which 8 inactivated genetic modification targets are 4-hydroxyphenylpyruvate dioxygenase (4-HPPD, GI: 50547455), aromatic amino acid transaminase II (ARO9, GI: 50547993), 3-phosphoglycerate dehydrogenase (SER3, GI: 50555640), acylglycerol lipase 3 (TGL, GI: 50550941), serine hydroxymethyltransferase 2 (SHMT 2, GI:50553214), serine hydroxymethyltransferase 1 (SHMT1, GI:50551359), aspartate aminotransferase (AKX1, GI:50553242), anabolic serine and threonine dehydratase precursor (ILV1, GI:50549715); 2 overexpression genetic modification targets, respectively, lipoate ligase (LipB, GI:50557070) and citrate transporter (CTP1, GI:50556988).

[0046] The microbial synthesis of polyphenolic compounds is achieved by the catalysis of metabolic enzymes. The expression pattern and functional effect of microbial metabolic reaction enzymes are regulated at the transcription and protein levels. Among them, the regulation at the transcription and post-transcription levels involves the coordination of functional elements such as transcription factors, RNA polymerases, and mRNA splicing factors, which ultimately achieves the effect of changing the synthesis level of polyphenolic compounds. Changing the intracellular content and activity of metabolic reaction enzymes through protein regulation can affect the synthesis of polyphenolic substances. The regulation process is mainly achieved by modifying or degrading proteins. Protein modifications include phosphorylation, acetylation, methylation, etc. These modifications can affect the function and interaction of proteins. In addition, protein degradation is also an important means of regulating metabolic pathways, which adjusts the flow of metabolic pathways and the synthesis of products by degrading proteins. The present invention discloses five inactivation targets for improving the synthesis of polyphenol compounds through transcription and post-transcriptional regulation: CYC8 transcription repressor (CYC8, GI: 50546166), gluconeogenesis transcription factor (CAT8, GI: 50549061), RNA polymerase II transcription regulator (HDB, GI: 50556186), mRNA pre-splicing factor 1 (LSM1, GI: 50545209), and mRNA pre-splicing factor 5 (LSM5, GI: 50546793). There are five inactivation targets for improving the synthesis of polyphenolic compounds through protein regulation: serine / threonine protein phosphatase (CNA1, GI: 50543112), RING-type E3 ubiquitin transferase (TUL1, GI: 50556876), GTP-binding protein (GTR1, GI: 50543528), WD-repeat protein (SWD1, GI: 50549369), and UBC13 E2 ubiquitin conjugating enzyme (UBC13, GI: 50547641).

[0047] 2) Construction of 18 knockout target elements: The upstream and downstream homology arms of the knockout gene were amplified by PCR, and the upstream and downstream homology arms were further assembled by fusion PCR to obtain the donor fragments of the 18 genes to be knocked out.

[0048] 3) Construction of two up-regulated gene elements: Using the Gibson assembly method, the two gene sequences were assembled with the promoter PrTefintron and the terminator Tlip2 modules, respectively, to construct two expression plasmids targeting the same genomic integration site.

[0049] 4) The two plasmids were double-digested to obtain two integrated DNA fragments.

[0050] Example 2: Application Test Example 1 - Resveratrol to Improve the Synthesis of Polyphenolic Compounds Using p-Coumaryl-CoA and Malonyl-CoA as Direct Metabolism Precursors by Yarrowia lipolytica

[0051] 1. Construction of the Yarrowia lipolytica strain Res1 for synthesizing resveratrol:

[0052] Construction of a resveratrol-synthesizing Yarrowia lipolytica strain: First, a tyrosine ammonia lyase encoding gene (sequence shown in SEQ ID NO: 5), a 4-coumarinoyl-CoA ligase encoding gene (sequence shown in SEQ ID NO: 6) and a resveratrol synthase encoding gene (sequence shown in SEQ ID NO: 7) were integrated into a neutral site of the Yarrowia lipolytica chromosome to obtain the resveratrol-synthesizing Yarrowia lipolytica strain Res1.

[0053] 2. Construction of genetic target recombinant Yarrowia lipolytica for the synthesis enhancement module of phenolic metabolic precursors and the level of resveratrol synthesis

[0054] 1) The eight knockout genes and two integrated DNA fragments of the polyphenol metabolic precursor synthesis enhancement module obtained in Example 1 were further transformed into strain Res1, and confirmed by genomic PCR, thereby obtaining 10 recombinant Yarrowia lipolytica strains.

[0055] 2) The above 10 recombinant Yarrowia lipolytica strains were inoculated into the basic mineral salt culture medium and pre-cultured for 30-36 hours to obtain the 600 The basal mineral salt culture medium was inoculated with 0.05, cultured for 39 hours, 100 μL of the culture was taken, 100 μL of anhydrous ethanol was added, vortexed and filtered, and 100 μL was taken into a liquid phase injection bottle for HPLC detection of resveratrol. The results are shown in Table 1, indicating that regulating the 10 genetic modification targets can increase the level of resveratrol synthesis by Yarrowia lipolytica to varying degrees (7.47%-32.60%).

[0056] Table 1. Genetic targets of the synthesis enhancement module of polyphenol metabolic precursors and the level of resveratrol synthesis by recombinant Yarrowia lipolytica

[0057] Target Resveratrol yield (mg / L) Improvement rate (%) None (Res1) 121.25±0.37 / 4HPPD 138.07±0.54 13.87 ARO9 135.81±0.23 12.01 SER3 130.31±0.23 7.47 TGL 130.45±0.58 7.59 SHMT1 153.41±1.03 26.52 SHMT2 160.78±0.89 32.60 AKX1 151.21±0.85 24.71 ILV1 143.58±0.56 18.42 LipB 152.36±0.91 25.66 CTP1 133.75±0.96 10.31

[0058] 3. Transcriptional regulation of metabolic synthesis and genetic targets for protein regulation Construction of recombinant Yarrowia lipolytica and the level of resveratrol synthesis

[0059] 1) The fragments of the 10 knockout genes of transcriptional regulation and protein regulation of the metabolic synthesis process obtained in Example 1 were further transformed into strain Res1, and confirmed by genomic PCR, thereby obtaining 10 recombinant Yarrowia lipolytica strains.

[0060] 2) The above 10 recombinant Yarrowia lipolytica strains were inoculated into the basic mineral salt culture medium and pre-cultured for 30-36 hours to obtain the 600The basal mineral salt culture medium was inoculated with 0.05, cultured for 39 hours, 100 μL of the culture was taken, 100 μL of anhydrous ethanol was added, vortexed and filtered, and 100 μL was taken into a liquid phase injection bottle for HPLC detection of resveratrol. The results are shown in Table 2, indicating that regulating the expression of the above 10 genetic modification targets can increase the level of resveratrol synthesis by Yarrowia lipolytica to varying degrees (11.23%-48.91%).

[0061] Table 2. Genetic targets for transcriptional regulation and protein regulation of metabolic synthesis process Levels of resveratrol synthesis by recombinant Yarrowia lipolytica fermentation

[0062] Target Resveratrol yield (mg / L) Improvement rate (%) None (Res1) 121.25±0.37 / CYC8 150.17±0.63 23.85 CAT8 150.25±0.45 23.92 HDB 164.32±0.57 35.52 LSM1 178.90±0.56 47.55 LSM5 168.32±0.69 38.82 CNA1 134.87±0.28 11.23 TUL1 160.78±0.58 32.60 GTR1 180.55±0.54 48.91 SWD1 160.35±0.58 32.25 UBC13 154.21±0.34 27.18

[0063] Example 3: Target stacking application test example 2 for improving the synthesis of polyphenolic compounds with p-coumaroyl-CoA and malonyl-CoA as direct metabolic precursors by Yarrowia lipolytica - resveratrol

[0064] 1) The genetic targets of the synthesis enhancement module of the polyphenol metabolic precursor obtained in Example 1 were randomly selected and superimposed and combined. The combination pattern is shown in Table 3. The corresponding knockout or integration fragments were transformed into the strain Res1 and confirmed by genomic PCR, thereby obtaining 6 recombinant Yarrowia lipolytica strains.

[0065] 2) The above 6 recombinant Yarrowia lipolytica strains were inoculated into the basic mineral salt culture medium and pre-cultured for 30-36 hours to obtain the 600 The basal mineral salt culture medium was inoculated with 0.05, cultured for 39 hours, 100 μL of the culture was taken, 100 μL of anhydrous ethanol was added, vortexed and mixed, and then filtered, and 100 μL was taken into a liquid injection bottle for HPLC detection of resveratrol. The results are shown in Table 3, indicating that the combination of targets has a superimposed enhancement effect on the synthesis of resveratrol (better than one target).

[0066] Table 3. The level of resveratrol synthesis by recombinant Yarrowia lipolytica fermentation using the genetic targets of the synthesis enhancement module of polyphenol metabolic precursors

[0067] Target Resveratrol yield (mg / L) Improvement rate (%) None (Res1) 121.25±0.37 / 4HPPD+ARO9 140.07±0.38 15.52 4HPPD+AKX1 160.23±0.42 32.14 SHMT1+SHMT2 170.35±0.81 40.49 SHMT1+AKX1 160.31±0.58 32.21 4HPPD+CTP1 140.42±0.48 15.81 AKX1+CTP1 155.89±0.56 28.57 4HPPD+SHMT1+SHMT2 175.28±0.45 44.56

[0068] 3) The genetic targets of the transcriptional regulation and protein regulation modules of the metabolic synthesis process obtained in Example 1 were randomly selected and superimposed and combined. The combination pattern is shown in Table 4. The corresponding knockout or integration fragments were transformed into the strain Res1 and confirmed by genomic PCR, thereby obtaining 9 recombinant Yarrowia lipolytica strains.

[0069] 5) The above 9 recombinant Yarrowia lipolytica strains were inoculated into the basic mineral salt culture medium and pre-cultured for 30-36 hours to obtain the 600The basal mineral salt culture medium was inoculated with 0.05, cultured for 39 hours, 100 μL of the culture was taken, 100 μL of anhydrous ethanol was added, vortexed and mixed, and then filtered, and 100 μL was taken into a liquid injection bottle for HPLC detection of resveratrol. The results are shown in Table 4, indicating that the combination of targets has a superimposed enhancement effect on the synthesis of resveratrol (better than one target).

[0070] Table 4. The level of resveratrol synthesis by recombinant Yarrowia lipolytica fermentation with the combination of transcriptional regulation and protein regulation genetic targets in the metabolic synthesis process

[0071] Target Resveratrol yield (mg / L) Improvement rate (%) None (Res1) 121.25±0.37 / CAT8+LSM1 185.27±0.42 52.80 CYC8+HDB 195.23±0.54 61.01 LSM1+LSM5 182.25±0.37 50.13 LSM1+TUL1 186.32±0.75 53.67 LSM5+TUL1 175.90±0.53 44.40 TUL1+GTR1 185.32±0.79 52.84 TUL1+SWD1 170.47±0.38 40.59 GTR1+SWD1 185.89±0.48 53.31 LSM1+LSM5+TUL1 190.55±0.34 57.15

[0072] 5) The genetic targets of the synthesis enhancement module of the polyphenol metabolic precursor obtained in Example 1 and the transcriptional regulation and protein regulation modules of the metabolic synthesis process were randomly selected for superposition and combination between the modules. The combination pattern is shown in Table 5. The corresponding knockout or integration fragments were transformed into the strain Res1, and confirmed by genomic PCR, thereby obtaining 6 recombinant Yarrowia lipolytica strains.

[0073] 6) The above 6 recombinant Yarrowia lipolytica strains were inoculated into the basic mineral salt culture medium and pre-cultured for 30-36 hours to obtain the 600 The basal mineral salt culture medium was inoculated with 0.05, cultured for 39 hours, 100 μL of the culture was taken, 100 μL of anhydrous ethanol was added, vortexed and filtered, and 100 μL was taken into a liquid phase injection bottle for HPLC detection of resveratrol. The results are shown in Table 5, indicating that the combination of targets between modules has a superimposed enhancement effect on the synthesis of resveratrol (better than one target).

[0074] Table 5. Levels of resveratrol synthesis by fermentation of recombinant Yarrowia lipolytica using superposition of two modular genetic target sites

[0075] Target Resveratrol yield (mg / L) Improvement rate (%) None (Res1) 121.25±0.37 / 4HPPD+ARO9+LSM1 185.26±0.45 52.79 AKX1+CTP1+TUL1 165.89±0.55 36.82 4HPPD+AKX1+SWD1 170.35±0.85 40.49 4HPPD+SHMT1+SHMT2+TUL1 185.35±0.77 52.87 AKX1+CTP1+TUL1+SWD1 190.32±0.75 56.96 4HPPD+ARO9+CYC8+HDB 198.90±0.66 64.04

[0076] Example 4: Application of key targets for improving the synthesis of polyphenolic compounds with p-coumaroyl-CoA and malonyl-CoA as direct metabolic precursors in Yarrowia lipolytica Test Example 1 - Naringenin

[0077] 1) Construction of a Yarrowia lipolytica strain that synthesizes naringenin: A tyrosine ammonia lyase encoding gene (sequence shown in SEQ ID NO: 5), a 4-coumarinoyl-CoA ligase encoding gene (sequence shown in SEQ ID NO: 6), a chalcone synthase encoding gene (sequence shown in SEQ ID NO: 8) and a chalcone isomerase encoding gene (sequence shown in SEQ ID NO: 9) were integrated into a neutral site of the Yarrowia lipolytica chromosome to obtain a Yarrowia lipolytica strain Nar1 that synthesizes naringenin.

[0078] 2) The eight knockout genes and two integrated DNA fragments of the polyphenol metabolic precursor synthesis enhancement module obtained in Example 1 were further transformed into strain Nar1 and confirmed by genomic PCR, thereby obtaining 10 recombinant Yarrowia lipolytica strains.

[0079] 3) The above 10 recombinant Yarrowia lipolytica strains were inoculated into the basic mineral salt culture medium and pre-cultured for 30-36 hours to obtain the OD 600 The basal mineral salt culture medium was inoculated with 0.05, cultured for 39 hours, 100 μL of the culture was taken, 100 μL of anhydrous ethanol was added, vortexed and filtered, and 100 μL was taken into a liquid phase injection bottle for HPLC detection of naringenin. The results are shown in Table 6, indicating that regulating the 10 genetic modification targets can increase the level of naringenin synthesis by Yarrowia lipolytica to varying degrees (10.80%-36.86%).

[0080] Table 6. Genetic targets of the synthesis enhancement module for polyphenol metabolic precursors. Levels of naringenin synthesized by recombinant Yarrowia lipolytica fermentation

[0081] Inactivation target Naringenin production (mg / L) Improvement rate (%) None (Nar1) 81.89±0.66 / 4HPPD 94.98±0.75 10.80 ARO9 98.25±0.23 13.49 SER3 95.66±0.78 11.36 TGL 98.85±0.34 13.99 SHMT1 110.25±0.68 23.39 SHMT2 120.95±0.89 32.21 AKX1 115.36±0.48 27.60 ILV1 126.58±0.75 36.86 LipB 99.25±0.98 14.32 CTP1 123.58±0.56 34.38

[0082] 6) The fragments of the 10 knockout genes of transcriptional regulation and protein regulation of the metabolic synthesis process obtained in Example 1 were further transformed into the strain Nar1 and confirmed by genomic PCR, thereby obtaining 10 recombinant Yarrowia lipolytica strains.

[0083] 5) The above 10 recombinant Yarrowia lipolytica strains were inoculated into the basic mineral salt culture medium and pre-cultured for 30-36 hours to obtain the 600 The basal mineral salt culture medium was inoculated with 0.05, cultured for 39 hours, 100 μL of the culture was taken, 100 μL of anhydrous ethanol was added, vortexed and filtered, and 100 μL was taken into a liquid phase injection bottle for HPLC detection of naringenin. The results are shown in Table 7, indicating that regulating the expression of the above 10 genetic modification targets can increase the level of naringenin synthesis by Yarrowia lipolytica to varying degrees (11.02%-54.27%).

[0084] Table 7. Genetic targets for transcriptional regulation and protein regulation of metabolic synthesis. Levels of naringenin synthesis by recombinant Yarrowia lipolytica fermentation

[0085]

[0086]

[0087] Example 5: Target stacking application test example 2 for improving the synthesis of polyphenolic compounds with p-coumaroyl-CoA and malonyl-CoA as direct metabolic precursors by Yarrowia lipolytica - Naringenin

[0088] 1) The genetic targets of the synthesis enhancement module of the polyphenol metabolic precursor obtained in Example 1 were randomly selected and superimposed and combined. The combination pattern is shown in Table 3. The corresponding knockout or integration fragments were transformed into the strain Nar1 and confirmed by genomic PCR, thereby obtaining 6 recombinant Yarrowia lipolytica strains.

[0089] 2) The above 6 recombinant Yarrowia lipolytica strains were inoculated into the basic mineral salt culture medium and pre-cultured for 30-36 hours to obtain the 600 The basal mineral salt culture medium was inoculated with 0.05, cultured for 39 hours, 100 μL of the culture was taken, 100 μL of anhydrous ethanol was added, vortexed and filtered, and 100 μL was taken into a liquid phase injection bottle for HPLC detection of naringenin. The results are shown in Table 8, indicating that the combination of targets has a superimposed enhancement effect on the synthesis of naringenin (better than one target).

[0090] Table 8 Levels of naringenin synthesized by superposition of genetic targets of the synthesis enhancement module of polyphenol metabolic precursors by recombinant Yarrowia lipolytica fermentation

[0091] Target Naringenin production (mg / L) Improvement rate (%) None (Res1) 81.89±0.66 / 4HPPD+ARO9 105.89±0.48 29.31 4HPPD+AKX1 125.33±0.23 53.05 SHMT1+SHMT2 130.39±0.25 59.23 SHMT1+AKX1 130.41±0.77 59.25 4HPPD+CTP1 130.22±0.58 130.22 AKX1+CTP1 145.82±0.64 78.07 4HPPD+SHMT1+SHMT2 135.38±0.59 65.32

[0092] 3) The genetic targets of the transcriptional regulation and protein regulation modules of the metabolic synthesis process obtained in Example 1 were randomly selected and superimposed and combined. The combination pattern is shown in Table 4. The corresponding knockout or integration fragments were transformed into the strain Nar1 and confirmed by genomic PCR, thereby obtaining 10 recombinant Yarrowia lipolytica strains.

[0093] 4) The above 10 recombinant Yarrowia lipolytica strains were inoculated into the basic mineral salt culture medium and pre-cultured for 30-36 hours to obtain the 600 The basal mineral salt culture medium was inoculated with 0.05, cultured for 39 hours, 100 μL of the culture was taken, 100 μL of anhydrous ethanol was added, vortexed and filtered, and 100 μL was taken into a liquid phase injection bottle for HPLC detection of naringenin. The results are shown in Table 9, indicating that the combination of targets has a superimposed enhancement effect on the synthesis of naringenin (better than one target).

[0094] Table 9. Levels of naringenin synthesized by recombinant Yarrowia lipolytica fermentation in combination with transcriptional and protein-regulated genetic targets in metabolic synthesis

[0095] Target Naringenin production (mg / L) Improvement rate (%) None (Res1) 81.89±0.66 / CAT8+LSM1 155.23±0.45 89.49 CYC8+HDB 151.32±0.42 84.78 LSM1+LSM5 148.22±0.66 81.00 LSM1+TUL1 150.55±0.46 83.84 LSM5+TUL1 146.22±0.77 78.56 TUL1+GTR1 135.44±0.63 65.39 TUL1+SWD1 150.25±0.88 83.48 GTR1+SWD1 155.85±0.36 90.32 LSM1+LSM5+TUL1 156.33±0.78 90.90

[0096] 5) The genetic targets of the synthesis enhancement module of the polyphenol metabolic precursor obtained in Example 1 and the transcriptional regulation and protein regulation module of the metabolic synthesis process were randomly selected for superposition and combination between the modules. The combination pattern is shown in Table 5. The corresponding knockout or integration fragments were transformed into the strain Nar1 and confirmed by genomic PCR, thereby obtaining 6 recombinant Yarrowia lipolytica strains.

[0097] 7) The above 6 recombinant Yarrowia lipolytica strains were inoculated into the basic mineral salt culture medium and pre-cultured for 30-36 hours. 600 The basal mineral salt culture medium was inoculated with 0.05, cultured for 39 hours, 100 μL of the culture was taken, 100 μL of anhydrous ethanol was added, vortexed and filtered, and 100 μL was taken into a liquid injection bottle for HPLC detection of naringenin. The results are shown in Table 10, indicating that the combination of targets between modules has a superimposed enhancement effect on the synthesis of naringenin (better than one target).

[0098] Table 10. Levels of naringenin synthesis by recombinant Yarrowia lipolytica fermentation with superposition of two modular genetic targets

[0099] Target Naringenin production (mg / L) Improvement rate (%) None (Res1) 81.89±0.66 / 4HPPD+ARO9+LSM1 155.35±0.20 89.71 AKX1+CTP1+TUL1 154.99±0.46 89.27 4HPPD+AKX1+SWD1 146.45±0.72 78.84 4HPPD+SHMT1+SHMT2+TUL1 151.23±0.62 84.67 AKX1+CTP1+TUL1+SWD1 158.22±0.62 93.21 4HPPD+ARO9+CYC8+HDB 162.58±0.67 98.53

[0100] In summary, it is shown that the 20 genetic modification targets can increase the levels of resveratrol and naringenin, representative polyphenol compounds, which use p-coumaroyl-CoA and malonyl-CoA as direct metabolic precursors, in Yarrowia lipolytica, indicating that these modification targets can reshape the biological metabolic network and enhance the ability to synthesize products. Furthermore, these targets can increase the production of curcumin, a polyphenol compound that also uses p-coumaroyl-CoA and malonyl-CoA as direct metabolic precursors. Furthermore, the above 20 genetic modification targets can be applied to compounds such as stilbenes, naringenin and its derivative metabolites, curcuminoids and anthocyanidin pigments to improve the ability to synthesize products.

[0101] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A recombinant microorganism that synthesizes polyphenolic compounds at a high level, characterized in that: The microorganism has genetic target modification that has an enhancing effect on the anabolism of polyphenol compounds, and / or genetic target modification that has an optimizing effect on the transcription and protein level regulation of polyphenol compound synthesis, wherein: (i) The genetic targets that have an enhancing effect on the anabolism of polyphenolic compounds include: one or two, three, four, five or more of 4-hydroxyphenylpyruvate dioxygenase 4-HPPD, aromatic amino acid transaminase II ARO9, 3-phosphoglycerate dehydrogenase SER3, acylglycerol lipase 3TGL, serine hydroxymethyltransferase 2 SHMT2, serine hydroxymethyltransferase 1 SHMT1, aspartate aminotransferase AKX1, anabolic serine and threonine dehydratase precursor ILV1, lipoic acid ligase LipB, and citrate transporter CTP1; (ii) The genetic targets that optimize the transcriptional and protein level regulation of polyphenol compound synthesis include one, two, three, four, five or more of CYC8 transcription repressor CYC8, gluconeogenic transcription factor CAT8, RNA polymerase II transcription regulator HDB, mRNA pre-splicing factor 1LSM1, mRNA pre-splicing factor 5LSM5, serine / threonine protein phosphatase CNA1, RING-type E3 ubiquitin transferase TUL1, GTP-binding protein GTR1, WD-repeat protein SWD1, and UBC13 E2 ubiquitin conjugating enzyme UBC13.

2. The microorganism according to claim 1, characterized in that The genetic modification specifically includes one of the following: (i) 4-hydroxyphenylpyruvate dioxygenase 4-HPPD in combination with one, two, three, four, five or more genetic modifications of (i) or (ii) of claim 1; (ii) a combination of serine hydroxymethyltransferase 2 SHMT2 and one, two, three, four, five or more genetic modifications of (i) or (ii) of claim 1; (iii) a combination of pre-mRNA splicing factor 1LSM1 and one, two, three, four, five or more genetic modifications of (i) or (ii) of claim 1; (iv) RING-type E3 ubiquitin transferase TUL1 A combination of one, two, three, four, five or more genetic modifications according to (i) or (ii) of claim 1.

3. The microorganism according to claim 1 or 2, characterized in that The genetic modification is selected from one of the following: 1) 4HPPD, 4HPPD+AKX1, 4HPPD+AKX1+SWD1, 4HPPD+ARO9, 4HPPD+ARO9+CYC8+HDB, 4HPPD+ARO9+LSM1, 4HPPD+CTP1, 4HPPD+SHMT1+SHMT2 or 4HPPD+SHMT1+SHMT2+TUL1, or 2) AKX1, AKX1+CTP1, AKX1+CTP1+TUL1, AKX1+CTP1+TUL1+SWD1, or 3) ARO9, or 4) CAT8, CAT8+LSM1, or 5) CNA1, or 6) CTP1, or 7) CYC8, CYC8+HDB, or 8) GTR1, GTR1+SWD1, or 9) HDB, or 10) ILV1, or 11) LipB, or 12) LSM1, LSM1+LSM5, LSM1+LSM5+TUL1, LSM1+TUL1, or 13) LSM5, LSM5+TUL1, or 14) SER3, or 15) SHMT1, SHMT1+AKX1, SHMT1+SHMT2, or 16) SHMT2, or 17) SWD1, or 18) TGL, or 19) TUL1, TUL1+GTR1, TUL1+SWD1, or 20) UBC13.

4. The microorganism according to any one of claims 1 to 3, characterized in that The genetic modification of the lipoic acid ligase LipB and the citrate transporter CTP1 is a modification of the genetic target points for overexpression thereof by genetic engineering methods, and the genetic modification of the remaining genes is a modification of the genetic target points for downregulating the expression amount.

5. The genetic modification for improving the ability of a microorganism to synthesize polyphenolic compounds according to any one of claims 1 to 4, wherein: The 4-hydroxyphenylpyruvate dioxygenase 4-HPPD, aromatic amino acid transaminase II ARO9, 3-phosphoglycerate dehydrogenase SER3, acylglycerol lipase 3 TGL, serine hydroxymethyltransferase 2 SHMT2, serine hydroxymethyltransferase 1 SHMT1, aspartate transaminase AKX1, anabolic serine and threonine dehydratase precursor ILV1, lipoic acid ligase LipB, citrate transporter CTP1, CYC8 transcription repressor CYC8, gluconeogenic transcription factor CAT8, RNA polymerase II transcription regulator HDB, pre-mRNA splicing factor 1LSM1, pre-mRNA splicing factor 5LSM5, serine / threonine protein phosphatase CNA1, RING-type E3 ubiquitin transferase TUL1, GTP-binding protein GTR1, WD-repeat protein SWD1, and UBC13 E2 ubiquitin conjugating enzyme UBC13 are derived from yeast, preferably from Yarrowia lipolytica ( Yarrowia lipolytica ), Saccharomyces cerevisiae, Pichia pastoris, Rhodosporidium toruloides, Pichia fermentans, Kluyveromyces marxianus, Hansenula, Debaryomyces hansenula, Zygosaccharomyces bailii, Wickham's anomalous yeast, or Pichia kudriasis.

6. A genetic modification for improving the ability of a microorganism to synthesize polyphenolic compounds according to any one of claims 1 to 5, wherein the amino acid sequence of the 4-hydroxyphenylpyruvate dioxygenase 4-HPPD is GI: 50547455 or an analog thereof, the amino acid sequence of the aromatic amino acid transaminase II ARO9 is GI: 50547993 or an analog thereof, the amino acid sequence of the 3-phosphoglycerate dehydrogenase SER3 is GI: 50555640 or an analog thereof, the amino acid sequence of the acylglycerol lipase 3 TGL is GI: 50550941 or an analog thereof, the amino acid sequence of the serine hydroxymethyltransferase 2 SHMT2 is GI: 50553214 or an analog thereof, the amino acid sequence of the serine hydroxymethyltransferase 1 SHMT1 is GI: 50551359 or an analog thereof, the amino acid sequence of the aspartate aminotransferase AKX1 is GI: 50553242 or its analogs, the amino acid sequence of the anabolic serine and threonine dehydratase precursor ILV1 is GI: 50549715 or its analogs, the amino acid sequence of the lipoic acid ligase LipB is GI: 50557070 or its analogs, the amino acid sequence of the citrate transporter CTP1 is GI: 50556988 or its analogs, the amino acid sequence of the CYC8 transcription repressor CYC8 is GI: 50546166 or its analogs, the amino acid sequence of the gluconeogenic transcription factor CAT8 is GI: 50549061 or its analogs, the amino acid sequence of the RNA polymerase II transcription regulator HDB is GI: 50556186 or its analogs, the amino acid sequence of the mRNA pre-splicing factor 1LSM1 is GI: 50545209 or its analogs, and the amino acid sequence of the mRNA pre-splicing factor 5LSM5 is GI: 50546793 or its analogues, the amino acid sequence of the serine / threonine protein phosphatase CNA1 is GI: 50543112 or its analogues, the amino acid sequence of the RING-type E3 ubiquitin transferase TUL1 is GI: 50556876 or its analogues, the amino acid sequence of the GTP-binding protein GTR1 is GI: 50543528 or its analogues, the amino acid sequence of the WD-repeat protein SWD1 is GI: 50549369 or its analogues, and the amino acid sequence of the UBC13 E2 ubiquitin conjugating enzyme UBC13 is GI: 50547641 or its analogues.

7. The genetic modification for improving the ability of a microorganism to synthesize polyphenolic compounds according to any one of claims 1 to 6, wherein the analog has one or a combination of the following characteristics: (i) a mutant of a corresponding genetic target site, the amino acid sequence of the mutant having at least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of the corresponding genetic target site; (ii) A fusion protein obtained by connecting a protein tag at the N-terminus or / and the C-terminus of the lipoic acid ligase LipB and the citrate transporter CTP1.

8. The microorganism according to any one of claims 1 to 7, characterized in that The microorganism is a fungus or a bacterium; Preferably, the fungus is selected from the genus Yarrowia, Kluyveromyces, Saccharomyces, Candida, Pichia, Aspergillus or Fusarium; The bacterial cell is selected from the genus Lactobacillus or Bacillus; Preferably, the Yarrowia spp. is Yarrowia lipolytica ( Yarrowia lipolytica )、 Yarrowia.alimentaria, Yarrowia. Bubula, Yarrowia. hollandica, Yarrowia. deformans or Yarrowia divulgata Any one of; Preferably, the Kluyveromyces genus is any one of Kluyveromyces marxianus, Kluyveromyces marxianus variant, Kluyveromyces lactis, Kluyveromyces hubeiensis, Kluyveromyces wickheimii or Kluyveromyces thermotolerans; Preferably, the Bacillus is any one of Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis; Preferably, the Lactobacillus is any one of Lactobacillus delbrueckii bulgaricus, Lactobacillus acidophilus, Lactobacillus casei subsp. casei, Lactobacillus reuteri, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus plantarum or Lactobacillus salivarius; More preferably, the microorganism is Yarrowia lipolytica ( Yarrowia lipolytica ), Saccharomyces cerevisiae, Pichia pastoris, Rhodosporidium toruloides, Pichia fermentans, Kluyveromyces marxianus, Hansenula, Debaryomyces hansenii, Zygosaccharomyces bailii, Wickhamella anomala, or Pichia kudria, Trichoderma reesei, or Aspergillus niger.

9. Use of the microorganism according to claims 1 to 8 in synthesizing polyphenolic compounds, by fermenting the microorganism in a medium with a suitable bottom to synthesize the polyphenolic compounds; Preferably, the polyphenol compound is a polyphenol compound with p-coumaroyl-CoA and malonyl-CoA as direct metabolic precursors; More preferably, the polyphenolic compounds are flavonoids, phenolic acids, lignans, and stilbenes; Furthermore, the polyphenol compound is a stilbene, flavonoid, or pigment platform compound; Further, the stilbene compound is selected from resveratrol, scutellariae, picetol, polydatin, pterostilbene, morin and other resveratrol derivatives; the flavonoid compound is selected from naringenin and its derivatives, preferably, the naringenin and its derivatives are selected from naringenin, baicalein, quercetin, tangerine, luteolin, myricetin, catechin, epicatechin, isoliquiritigenin, xanthohumol, icariin, 7-O-(2-naphthylmethyl) naringenin, quercetin, tangerine, luteolin, myricetin, catechin, epicatechin, isoliquiritigenin, xanthohumol, icariin, 7-O-(2-naphthylmethyl) naringenin, quercetin, tangerine, catechin ... The pigment compounds are selected from curcumin, anthocyanin and derivatives thereof. Preferably, the curcumin, anthocyanin and derivatives thereof are selected from demethoxycurcumin, bisdemethoxycurcumin, cyanidin, pelargonidin, peony pigment, delphinidin, petunidin and malva pigment.

10. The use according to claim 9, characterized in that: The culture medium includes a carbon source and a nitrogen source, which may be a combination of one or more of the following: (i) the carbon source is selected from a fermentable carbon source or a non-fermentable carbon source. Preferably, the fermentable carbon source includes but is not limited to glucose, fructose, galactose, xylose, and sucrose, and the non-fermentable carbon source includes but is not limited to ethanol, acetic acid and acetate, glycerol, lactate, methanol, and formic acid; (ii) the nitrogen source is selected from an organic nitrogen source or an inorganic nitrogen source. Preferably, the organic nitrogen source includes but is not limited to peptone, yeast powder, corn steep liquor, and the inorganic nitrogen source includes but is not limited to ammonium sulfate, ammonium chloride, nitrate and ammonia water; (iii) Biomass containing carbon and nitrogen sources, including but not limited to agricultural, industrial and forestry residues and wastes.

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