A genetically engineered bacterium with high yield of dihydroquercetin, and a preparation method and application thereof

Abstract

The application provides a genetically engineered bacterium with high yield of dihydroquercetin, a preparation method and application thereof, relates to the technical field of genetic engineering, and comprises a malonyl coenzyme A synthesis module, a tyrosine deaminase expression module, a naringenin synthesis module and a dihydroquercetin post-modification module. The enzymatic property of the fusion protein is increased by about 50% of the enzyme activity before fusion, the activity of converting naringenin into dihydroquercetin is greatly improved, and finally the yield of dihydroquercetin expressed in cyanobacteria is greatly improved. Meanwhile, BauA, MCR-C, Sam8, 4CL, CHS, CHI and expression fusion protein N33'H are expressed in cyanobacteria. The yield of dihydroquercetin is greatly increased under seawater culture conditions. The technical problem that the yield of biosynthetic dihydroquercetin is low and cannot meet the requirements of industrial application in the prior art is solved.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a genetically engineered bacterium that produces high levels of dihydroquercetin, its preparation method, and its applications. Background Technology

[0002] Due to the characteristics of cyanobacteria, such as photosynthetic autotrophy, ease of gene manipulation, and rapid growth, there are many examples in the literature and industry of using cyanobacteria as bioreactors to express exogenous genes, such as: mosquito larvae toxin protein gene, fatty acid unsaturated enzyme gene, hydroxybutyrate polymerase gene, dehydrogenase gene catalyzing eicosapentaenoic acid (EPA) synthesis, and cellulose gene.

[0003] Dihydroquercetin (DHQ) is a natural flavonoid compound that reduces oxidative stress-induced cell damage by scavenging free radicals and activating the Nrf2 antioxidant pathway. It can delay oxidative aging of the skin and organs and may improve chronic diseases related to oxidative damage (such as neurodegenerative diseases). Its core functions include antioxidation, anti-inflammation, cardiovascular protection, and immune regulation. Potential benefits include anti-allergy, liver protection, and anti-aging. Dihydroquercetin is mainly found in certain plants (such as Douglas fir), but its content in these plants is low, and the isolation and purification of natural products is complex. Utilizing synthetic biology and fermentation engineering to prepare dihydroquercetin is another promising approach to increasing its yield, but current production levels are still far from industrial application.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide a genetically engineered bacterium that produces high levels of dihydroquercetin, thereby solving the technical problem that the yield of dihydroquercetin in the prior art is low and cannot meet the requirements for industrial application.

[0006] The second objective of this invention is to provide a method for preparing the above-mentioned genetically engineered bacteria.

[0007] A third objective of this invention is to provide the application of the above-mentioned genetically engineered bacteria or the genetically engineered bacteria prepared by the above-mentioned method in the synthesis of dihydroquercetin.

[0008] The fourth objective of this invention is to provide a method for synthesizing dihydroquercetin.

[0009] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a genetically engineered bacterium that produces high levels of dihydroquercetin, comprising a malonyl-CoA synthesis module, a tyrosine ammonia-lyase expression module, a naringenin synthesis module, and a dihydroquercetin post-modification module. The malonyl-CoA synthesis module includes a first promoter, a gene encoding β-alanine-pyruvate transaminase, and a gene encoding malonyl-CoA reductase, wherein the gene encoding β-alanine-pyruvate transaminase; The tyrosine ammonia-lyase expression module includes a second promoter and a gene encoding tyrosine ammonia-lyase; The naringenin synthesis module includes a third promoter, a gene encoding 4-coumaroyl-CoA ligase, a gene encoding chalcone synthase, and a gene encoding chalcone isomerase. The dihydroquercetin post-modification module includes a fourth promoter and a gene encoding the fusion protein N33'H, which is obtained by fusing the C-terminal α-helix domain of α-ketoglutarate-3-oxygenase with the N-terminal α-helix domain of flavonoid 3' hydroxylase. The nucleotide sequence of the fusion protein N33'H is shown in SEQ ID No. 11.

[0010] Furthermore, the first promoter, the second promoter, the third promoter, and the fourth promoter are each independently selected from any one of promoter PcpcBA, promoter Prbcl, promoter PpsbA, or promoter PpsaA; The gene sequence of the promoter PcpcBA is shown in SEQ ID No. 1, the gene sequence of the promoter Prbcl is shown in SEQ ID No. 4, the gene sequence of the promoter PpsbA is shown in SEQ ID No. 6, and the gene sequence of the promoter PpsaA is shown in SEQ ID No. 10. Preferably, the first promoter is promoter PcpcBA; Preferably, the second promoter is the promoter Prbcl; Preferably, the third promoter is promoter PpsbA; Preferably, the fourth promoter is promoter PpsaA.

[0011] Furthermore, the gene sequence encoding β-alanine-pyruvate transaminase is shown in SEQ ID No. 2, and the gene sequence encoding malonyl-CoA reductase is shown in SEQ ID No. 3.

[0012] Furthermore, the gene sequence encoding tyrosine ammonia-lyase is shown in SEQ ID No. 5.

[0013] Furthermore, the gene sequence encoding 4-coumaroyl-CoA ligase is shown in SEQ ID No. 7, the gene sequence encoding chalcone synthase is shown in SEQ ID No. 8, and the gene sequence encoding chalcone isomerase is shown in SEQ ID No. 9.

[0014] Secondly, the present invention provides a method for preparing the above-mentioned genetically engineered bacteria, comprising introducing a vector containing a malonyl-CoA synthesis module, a tyrosine ammonia-lyase expression module, a naringenin synthesis module and a dihydroquercetin post-modification module sequentially into a host cell to obtain genetically engineered bacteria.

[0015] Furthermore, the gene encoding the N33'H fusion protein in the dihydroquercetin post-modification module is obtained by fusion PCR of the gene encoding α-ketoglutarate-3-oxygenase and the gene encoding flavonoid 3' hydroxylase. Preferably, the gene sequence encoding α-ketoglutarate-3-oxygenase is shown in SEQ ID No. 12, and the gene sequence encoding brass 3' hydroxylase is shown in SEQ ID No. 13.

[0016] Furthermore, the host cell includes at least one of cyanobacteria, green algae, or true eye algae; Preferably, the cyanobacteria are selected from at least one of the genera Synechococcus, Synechococcus, Cryptococcus, Anabaena, Nostoc, Oscillatoria, Chlorella, Aggregatibium, Bifidobacteria, or Diplocoidea. Preferably, the green algae are selected from the genera *Chlamydomonas* and / or *Chlorella*. Preferably, the true eye-point algae is from the genus *Micrococcus*. Preferably, the carrier comprises a shuttle plasmid; Preferably, the shuttle plasmid includes pAQE19.

[0017] Thirdly, the present invention provides the application of the above-mentioned genetically engineered bacteria or the genetically engineered bacteria prepared by the above-mentioned method in the synthesis of dihydroquercetin.

[0018] Fourthly, the present invention provides a method for synthesizing dihydroquercetin, comprising culturing the above-mentioned genetically engineered bacteria or the genetically engineered bacteria prepared by the above-mentioned preparation method.

[0019] This invention provides a genetically engineered bacterium that produces high levels of dihydroquercetin, by combining the C-terminal α-helix domain of naringenin, 2-oxoglutarate 3-dioxygenase (N3D), with flavonoid 3'-hydroxylase. The N-terminal α-helical domain of 3'-hydroxylase (F3'H) is fused together to form a new fusion protein, N33'H. This results in an approximately 50% increase in enzyme activity compared to the original fusion protein, significantly enhancing the conversion of naringenin to dihydroquercetin and ultimately leading to a substantial increase in dihydroquercetin production in cyanobacteria. Simultaneously, the fusion protein N33'H is expressed in cyanobacteria using BauA (β-alanine-pyruvate transaminase), MCR-C (malonyl-CoA reductase), Sam8 (tyrosine ammonia-lyase), 4CL (4-coumaroyl-CoA ligase), CHS (chalcone synthase), and CHI (chalcone isomerase). This achieves a significant increase in dihydroquercetin production to 300 mg / L under seawater cultivation conditions. This solves the technical problem of low dihydroquercetin yield in existing biosynthetic technologies, which cannot meet the requirements for industrial application. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram illustrating the preparation of a new fusion protein N33'H by fusing the C-terminal α-helix domain of α-ketoglutarate-3-oxygenase with the N-terminal α-helix domain of brass 3' hydroxylase, as provided in Example 1 of this invention. Figure 2 This is a schematic diagram of the expression vector pAQE19-DHQ of the dihydroquercetin-producing cyanobacterial engineered bacteria provided in Example 1 of the present invention; Figure 3 This is an electrophoresis diagram of the PCR verification results of the engineered positive bacteria provided in Example 1 of the present invention; Figure 4 The results show the detection of dihydroquercetin content in the culture medium of the positive engineered bacteria at different culture stages provided in Example 2 of the present invention. Detailed Implementation

[0022] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0023] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0024] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] The present invention provides a genetically engineered bacterium that produces high levels of dihydroquercetin, comprising a malonyl-CoA synthesis module, a tyrosine ammonia-lyase expression module, a naringenin synthesis module, and a dihydroquercetin post-modification module. The malonyl-CoA synthesis module includes a first promoter, a gene encoding β-alanine-pyruvate transaminase, and a gene encoding malonyl-CoA reductase, wherein the gene encoding β-alanine-pyruvate transaminase; The tyrosine ammonia-lyase expression module includes a second promoter and a gene encoding tyrosine ammonia-lyase; The naringenin synthesis module includes a third promoter, a gene encoding 4-coumaroyl-CoA ligase, a gene encoding chalcone synthase, and a gene encoding chalcone isomerase. The dihydroquercetin post-modification module includes a fourth promoter and a gene encoding the fusion protein N33'H, which is obtained by fusing the C-terminal α-helix domain of α-ketoglutarate-3-oxygenase with the N-terminal α-helix domain of flavonoid 3' hydroxylase. The nucleotide sequence of the fusion protein N33'H is shown in SEQ ID No. 11.

[0026] Independent promoters were set for each of the four functional modules to ensure that the expression sequence of each module matches the metabolic requirements and eliminate endogenous competition in the pathway. A non-natural two-enzyme cascade pathway composed of β-alanine pyruvate transaminase (BauA) and malonyl-CoA reductase (McrC) was used to achieve efficient synthesis of malonyl-CoA. The C-terminal α-helix domain of α-ketoglutarate-3-oxygenase (N3D) was combined with that of brass 3' hydroxylase (Flavonoid)... The N-terminal α-helical domain of 3'-hydroxylase (F3'H) is fused together to form a new fusion protein, N33'H. This new fusion protein exhibits approximately 50% increased enzyme activity compared to the original protein, significantly enhancing the activity of converting naringenin to dihydroquercetin and ultimately leading to a substantial increase in dihydroquercetin production in cyanobacteria. Simultaneously, the fusion protein N33'H is expressed in cyanobacteria using BauA (β-alanine-pyruvate transaminase), MCR-C (malonyl-CoA reductase), Sam8 (tyrosine ammonia-lyase), 4CL (4-coumaroyl-CoA ligase), CHS (chalcone synthase), and CHI (chalcone isomerase). This results in a significant increase in dihydroquercetin production to 225 mg / L under seawater cultivation conditions. This solves the technical problem of low dihydroquercetin production in existing biosynthetic technologies, which cannot meet the requirements for industrial application.

[0027] The β-alanine-pyruvate transaminase (bauA) gene and malonyl-CoA reductase (mcr-C) gene are expressed using the strong promoter PcpcBA; the sam8 gene is expressed using the strong promoter PrbcL; the 4cl, chs, and chi genes are expressed using the strong promoter PpsbA; and the N33'H gene of the fusion protein is expressed using the strong promoter PpsaA. In some specific embodiments, the first, second, third, and fourth promoters are independently selected from any one of the promoters PcpcBA, PrbcL, PpsbA, or PpsaA; the gene sequence of promoter PcpcBA is shown in SEQ ID No. 1, the gene sequence of promoter PrbcL is shown in SEQ ID No. 4, the gene sequence of promoter PpsbA is shown in SEQ ID No. 6, and the gene sequence of promoter PpsaA is shown in SEQ ID No. 10.

[0028] In some specific embodiments, the first promoter is promoter PcpcBA; in some specific embodiments, the second promoter is promoter Prbcl; in some specific embodiments, the third promoter is promoter PpsbA; and in some specific embodiments, the fourth promoter is promoter PpsaA.

[0029] In some specific embodiments, the gene sequence encoding β-alanine-pyruvate transaminase is shown in SEQ ID No. 2, and the gene sequence encoding malonyl-CoA reductase is shown in SEQ ID No. 3. In some specific embodiments, the gene sequence encoding tyrosine ammonia-lyase is shown in SEQ ID No. 5. In some specific embodiments, the gene sequence encoding 4-coumaroyl-CoA ligase is shown in SEQ ID No. 7, the gene sequence encoding chalcone synthase is shown in SEQ ID No. 8, and the gene sequence encoding chalcone isomerase is shown in SEQ ID No. 9.

[0030] According to another aspect of the present invention, a method for preparing the above-mentioned genetically engineered bacteria is also provided, comprising introducing a vector containing a malonyl-CoA synthesis module, a tyrosine ammonia-lyase expression module, a naringenin synthesis module and a dihydroquercetin post-modification module sequentially into a host cell to obtain genetically engineered bacteria.

[0031] In some specific embodiments, the host cell includes at least one of cyanobacteria, green algae, or true-eye algae; in some specific embodiments, the cyanobacteria are selected from at least one of the genera *Synechococcus*, *Synechococcus*, *Cryptococcus*, *Anabaena*, *Nostoc*, *Oscillatoria*, *Chlorella*, *Agga*, *Bifidobacterium*, or *Dermophyta*; in some specific embodiments, the green algae are selected from the genera *Chlamydomonas* and / or *Chlorella*; in some specific embodiments, the true-eye algae is *Micrococcus*.

[0032] In some specific embodiments, the vector includes a shuttle plasmid; in some specific embodiments, the shuttle plasmid includes pAQE19.

[0033] In some specific embodiments, the gene encoding the N33'H fusion protein in the dihydroquercetin post-modification module is obtained by fusion PCR of the gene encoding α-ketoglutarate-3-oxygenase and the gene encoding flavonoid 3' hydroxylase; in some specific embodiments, the gene sequence encoding α-ketoglutarate-3-oxygenase is shown in SEQ ID No. 12, and the gene sequence encoding flavonoid 3' hydroxylase is shown in SEQ ID No. 13.

[0034] According to another aspect of the present invention, the application of the above-described genetically engineered bacteria or the genetically engineered bacteria prepared by the above-described method in the synthesis of dihydroquercetin is also provided.

[0035] According to another aspect of the present invention, a method for synthesizing dihydroquercetin is also provided, comprising culturing the above-described genetically engineered bacteria or the genetically engineered bacteria prepared by the above-described preparation method.

[0036] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0037] Material: Synechococcus sp. PCC7002 and its derived mutants, grown in A + Liquid culture medium or A + In solid culture medium. A + Solid culture medium: in A + Agar was added to the liquid culture medium to a final concentration of 1.2% (mass percentage). During liquid culture, a fluorescent lamp with a light intensity of 100 uE / m² was used for illumination. 2 •s, temperature 30℃, and air containing 1% CO2 (v / v) is introduced.

[0038] The *E. coli* strain DH5α used for plasmid construction was grown on standard LB medium. The antibiotic concentrations used for growth were 100 µg / ml Amp. + 50 µg / ml Kan + .

[0039] The shuttle cyanobacterial plasmid pAQE19 used in the experiment was purchased from a biotechnology company.

[0040] A + Culture medium: per liter of culture medium components: MgSO4·7H2O 5.0g, NaNO3 1.0g, NaCl 18g, KCl 0.6g, CaCl2 0.27g, KH2PO4 0.05g, Na2CO3 0.02g, FeCl3·6H2O 3.89mg, EDTA·Na2 30mg, Tris·Cl (pH 8.2) 1.0g and Trace metal mix 1.0mL, plus vitamin VB12 (4µg / mL).

[0041] The above-mentioned trace metal mix contains 2.860 mg / L H3BO3, 1.810 mg / L MnCl2·4H2O, 0.222 mg / L ZnSO4·7H2O, 0.390 mg / L Na2MoO4·2H2O, 0.079 mg / L CuSO4·5H2O, and 0.0494 mg / L Co(NO3)2·6H2O.

[0042] Example 1: Preparation of engineered cyanobacteria producing dihydroquercetin 1. Preparation of n33'd gene by fusion PCR The prediction results show that the α-helix regions of the N3D and F3'H proteins perfectly fuse into a single α-helix region. The schematic diagram of the fusion of the n33'd gene is shown below. Figure 1 Optimized N3D and F3'H gene templates from cyanobacteria were obtained through gene synthesis. The gene fragment encoding amino acids 1-342 of the N3D protein (nucleotide sequence shown in SEQ ID No. 12) was amplified by PCR using primers P1 / P2 (primer sequences are shown in Table 1). The F3'H gene fragment (nucleotide sequence shown in SEQ ID No. 13) was amplified by PCR using primers P3 / P4. Then, the n33'd fusion gene was obtained by fusion PCR using primers P1 / P4, and its nucleotide sequence is shown in SEQ ID No. 11.

[0043] 2. Construction of the expression vector pAQE19-DHQ for dihydroquercetin The construction process of the vector pAQE19-DHQ expressing dihydroquercetin is as follows: Figure 2Using total genomic DNA from wild-type Synechococcus sp. PCC7002 as a template, gene fragments with four strong promoters, PcpcBA, PrbcL, PpsbA, and PpsaA, were amplified by PCR using primer pairs P5 / P6, P7 / P8, P9 / P10, and P11 / P12, respectively. The nucleotide sequences are shown in SEQ ID No. 1, SEQ ID No. 4, SEQ ID No. 6, and SEQ ID No. 10, respectively. Gene templates optimized for bauA (β-alanine-pyruvate transaminase), mcr-C (malonyl-CoA reductase), sam8 (tyrosine ammonia-lyase), 4cl (4-coumaroyl-CoA ligase), chs (chalcone synthase), and chi (chalcone isomerase) from cyanobacteria were obtained through gene synthesis. Gene fragments of bauA, mcr-C, sam8, 4cl, chs, and chi were amplified by PCR using primer pairs P13 / P14, P15 / P16, P17 / P18, P19 / P20, P21 / P22, and P23 / P24, respectively. The sequences of the bauA gene fragment are shown in SEQ ID No. 2, the mcr-C gene fragment in SEQ ID No. 3, the sam8 gene fragment in SEQ ID No. 5, the 4cl gene fragment in SEQ ID No. 7, the chs gene fragment in SEQ ID No. 8, and the chi gene fragment in SEQ ID No. 9 are shown in SEQ ID No. 10. As shown in ID No. 9, gene fragments of the cyanobacterial shuttle vector pAQE19 (Dongyi Xu 2005; Plant Physiology, July 2005, Vol. 138, pp. 1586~1595) were amplified using primers P25 / P26. All the amplified gene fragments were then integrated into a single vector using a Gibson Assemble kit with homologous fragments, resulting in the expression vector pAQE19-DHQ.

[0044] (3) Screening of positive cyanobacterial engineered bacteria and PCR verification that they are homozygous strains The expression vector pAQE19-DHQ was transformed into wild-type Synechococcus PCC7002. pAQE19 is a shuttle vector for cyanobacteria and Escherichia coli, and can replicate autonomously in cyanobacteria. The transformation method for Synechococcus PCC 7002 is as follows: Take 600 μl of cultured *Synechococcus polyphylla* PCC7002 (OD730nm value 0.8), centrifuge at 6000 r / min for 2 min, discard 400 μl of supernatant, add 10 μl of the prepared plasmid, mix gently, and incubate in the dark for 4–8 hours. Spread the algal solution evenly on A+ solid medium, incubate for 12–14 hours, and pour Top agar (5 ml Top agar contains 50 μl of kanamycin (100 mg / ml)). Incubate for approximately 15 days.

[0045] From A + Single colonies were selected from solid culture media and then further cultured in kanamycin-resistant A culture medium. + High-copy-ratio stable positive engineered bacteria were obtained by streaking and passaged on solid culture medium. The positive strains were identified by PCR using primer pairs P4 and P5 (identification results are shown in the figure). Figure 3 As shown, the wild-type PCR showed no band, while the mutant PCR band was 11.5 kb in size. The identification results indicate that the dihydroquercetin-positive cyanobacterial engineered bacterium 19DHQ has been obtained.

[0046] Table 1

[0047] Comparative Example 1 The difference from Example 1 is that the expression vector pAQE19-DHQ for expressing dihydroquercetin does not contain a malonyl-CoA synthesis module and is free of PcpcBA, bauA, and mcr-C.

[0048] Comparative Example 2 The difference from Example 1 is that the fused n33'd gene was not constructed, and the N3D gene fragment (nucleotide sequence as shown in SEQ ID No. 12) and the F3'H gene fragment (nucleotide sequence as shown in SEQ ID No. 13) were used directly.

[0049] Example 2: Identification of dihydroquercetin content in engineered bacterial culture medium Wild-type Synechococcus PCC7002 and the positive homozygous strain 19DHQ obtained in Example 1 were cultured in seawater after filtration and sterilization under the conditions of 35°C and 1% CO2.

[0050] Comparative Examples 3-4 The difference from Example 2 is that positive homozygous strains (19DHQ1 and 19DHQ2) obtained from Comparative Example 1 and Comparative Example 2 were used for culturing.

[0051] Experiment 1 Different growth stages (OD) of Examples 2 and Comparative Examples 3-4 were taken respectively.730 Algal culture media (centrifuged to remove algal bodies) at times 1, 2, 3 and 4 respectively were used to extract dihydroquercetin from the culture media by methanol extraction, and then the content of dihydroquercetin (DHQ) in the culture media was determined by HPLC-MS.

[0052] The yield of dihydroquercetin in 19DHQ, 19DHQ1, and 19DHQ2 culture media at different logarithmic growth phases was determined by comparing the characteristic peak areas of a 100 μg / ml dihydroquercetin standard with HPLC-MS peak chromatograms. The results are shown in the table below. Figure 4 As shown in the figure, the dihydroquercetin content of 19DHQ was significantly higher than that of 19DHQ1 and 19DHQ2 at different growth stages, indicating that the malonyl-CoA synthesis module and the construction of the fused n33'd gene played a synergistic role in improving the dihydroquercetin yield. 730 When the concentration reached 4.0, the dihydroquercetin content reached 225 mg / L, which was nearly 10 times and nearly 4 times higher than that of 19DHQ1 and 19DHQ2, respectively. This achieved the goal of efficient dihydroquercetin production by engineered cyanobacteria cultured in seawater. The significant difference between 19DHQ and 19DHQ2 verified that the fusion gene was transcribed to form a fusion protein, which played a promoting role in dihydroquercetin synthesis. Furthermore, after two months of continuous culture in seawater, the dihydroquercetin yield remained at the same level, demonstrating that 19DHQ has a stable dihydroquercetin production capacity.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A genetically engineered bacterium that produces high levels of dihydroquercetin, characterized in that, It includes a malonyl-CoA synthesis module, a tyrosine ammonia-lyase expression module, a naringenin synthesis module, and a dihydroquercetin post-modification module; The malonyl-CoA synthesis module includes a first promoter, a gene encoding β-alanine-pyruvate transaminase, and a gene encoding malonyl-CoA reductase, wherein the gene encoding β-alanine-pyruvate transaminase; The tyrosine ammonia-lyase expression module includes a second promoter and a gene encoding tyrosine ammonia-lyase; The naringenin synthesis module includes a third promoter, a gene encoding 4-coumaroyl-CoA ligase, a gene encoding chalcone synthase, and a gene encoding chalcone isomerase. The dihydroquercetin post-modification module includes a fourth promoter and a gene encoding the fusion protein N33'H, which is obtained by fusing the C-terminal α-helix domain of α-ketoglutarate-3-oxygenase with the N-terminal α-helix domain of flavonoid 3' hydroxylase. The nucleotide sequence of the fusion protein N33'H is shown in SEQ ID No.

11.

2. The genetically engineered bacterium according to claim 1, characterized in that, The first promoter, the second promoter, the third promoter, and the fourth promoter are each independently selected from any one of promoter PcpcBA, promoter Prbcl, promoter PpsbA, or promoter PpsaA; The gene sequence of the promoter PcpcBA is shown in SEQ ID No. 1, the gene sequence of the promoter Prbcl is shown in SEQ ID No. 4, the gene sequence of the promoter PpsbA is shown in SEQ ID No. 6, and the gene sequence of the promoter PpsaA is shown in SEQ ID No.

10. Preferably, the first promoter is promoter PcpcBA; Preferably, the second promoter is the promoter Prbcl; Preferably, the third promoter is promoter PpsbA; Preferably, the fourth promoter is promoter PpsaA.

3. The genetically engineered bacterium according to claim 1, characterized in that, The gene sequence encoding β-alanine-pyruvate transaminase is shown in SEQ ID No. 2, and the gene sequence encoding malonyl-CoA reductase is shown in SEQ ID No.

3.

4. The genetically engineered bacterium according to claim 1, characterized in that, The gene sequence encoding tyrosine ammonia-lyase is shown in SEQ ID No.

5.

5. The genetically engineered bacterium according to claim 1, characterized in that, The gene sequence encoding 4-coumaryl-CoA ligase is shown in SEQ ID No. 7, the gene sequence encoding chalcone synthase is shown in SEQ ID No. 8, and the gene sequence encoding chalcone isomerase is shown in SEQ ID No.

9.

6. The method for preparing the genetically engineered bacteria according to any one of claims 1 to 4, characterized in that, This involves introducing a vector containing a malonyl-CoA synthesis module, a tyrosine ammonia-lyase expression module, a naringenin synthesis module, and a dihydroquercetin post-modification module into a host cell to obtain genetically engineered bacteria.

7. The preparation method according to claim 6, characterized in that, The gene encoding the N33'H fusion protein in the dihydroquercetin post-modification module was obtained by fusion PCR of the gene encoding α-ketoglutarate-3-oxygenase and the gene encoding flavonoid 3' hydroxylase. Preferably, the gene sequence encoding α-ketoglutarate-3-oxygenase is shown in SEQ ID No. 12, and the gene sequence encoding brass 3' hydroxylase is shown in SEQ ID No.

13.

8. The preparation method according to claim 6, characterized in that, The host cell includes at least one of cyanobacteria, green algae, or true eye algae; Preferably, the cyanobacteria are selected from at least one of the genera Synechococcus, Synechococcus, Cryptococcus, Anabaena, Nostoc, Oscillatoria, Chlorella, Aggregatibium, Bifidobacteria, or Diplocoidea. Preferably, the green algae are selected from the genera *Chlamydomonas* and / or *Chlorella*. Preferably, the true eye-point algae is from the genus *Micrococcus*. Preferably, the carrier comprises a shuttle plasmid; Preferably, the shuttle plasmid includes pAQE19.

9. The use of the genetically engineered bacteria according to any one of claims 1 to 5 or the genetically engineered bacteria prepared by the preparation method according to any one of claims 6 to 8 in the synthesis of dihydroquercetin.

10. A method for synthesizing dihydroquercetin, characterized in that, This includes culturing the genetically engineered bacteria according to any one of claims 1 to 4 or obtaining the genetically engineered bacteria by the preparation method according to any one of claims 5 to 8.

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