Method for synthesizing liquiritigenin from engineered yeast from de novo

Abstract

The invention provides a method for synthesizing liquiritigenin from engineered yeast from de novo. The saccharomyces cerevisiae engineering bacterium for producing liquiritigenin is constructed by utilizing synthetic biology. By enhancing supply of a liquiritigenin precursor and coordinating the dose-effect relationship of a key rate-limiting enzyme, efficient and stable production of liquiritigenin is realized in a saccharomyces cerevisiae cell factory, and the problem of accumulation of a by-product naringenin is solved. The supply of the acetyl coenzyme A to the synthesized liquiritigenin is further improved through a peroxisome assembly strategy, the yield of the liquiritigenin is improved, and 1102.41 mg / L of liquiritigenin can be produced by the engineering bacteria through fed-batch fermentation, which is the highest yield reported at present. The invention lays a foundation for producing liquiritigenin and high-added-value derivatives thereof by microbial fermentation, and also provides a thought for microbial efficient and sustainable production of other flavonoid compounds.

Description

Technical Field

[0001] The present invention relates to a method for engineering yeast to de novo synthesize liquiritigenin, and relates to the technical field of genetic engineering. Background Art

[0002] Liquiritigenin, also known as 7,4'-dihydroxyflavanone, belongs to flavanone in the flavonoid skeleton and is naturally present in edible plant families such as Leguminosae, Bryaceae, and Asparagaceae. Liquiritigenin is widely used in a variety of nutritional health products, cosmetics, and traditional medicines, and has various biological and pharmacological activities such as anti-inflammatory, antioxidant, liver protection, anti-cancer, and anti-depressant effects. It can also be used as a medicine for treating COVID-19. Liquiritigenin is also an important platform precursor for the production of high-value-added flavonoid derivatives such as liquiritin, licorice chalcone, biochanin A, and isoflavones, and has high medicinal and commercial value.

[0003] Currently, people mainly extract liquiritigenin from artificially cultivated licorice. Liquiritigenin is mainly distributed in the roots of licorice. The plant extraction method is to slice and crush the roots of licorice, extract with 70% or 95% ethanol, and assist with ultrasound and microwave. However, the extraction efficiency of the plant method is relatively low, there are more impurities in the extract, and the subsequent purification steps are relatively complex. In addition, wild licorice resources in China are scarce, and 80% rely on imports. In order to reduce trade pressure, China has begun to try to artificially cultivate licorice, but the liquiritigenin content varies greatly among different licorice varieties; moreover, the growth of plants is restricted by the environment, and seasonal and environmental changes lead to huge arable land pressure, which severely limits the preparation and production of liquiritigenin. In addition, liquiritigenin can also be obtained by chemical synthesis. During the chemical synthesis of liquiritigenin, a large amount of glacial acetic acid and organic extractants are used, and oil bath and water bath heating are used for a long time, which consumes a large amount of energy and harms the environment, and does not conform to the concept of green manufacturing, so it is not widely used in industrial manufacturing.

[0004] In recent years, with the development of synthetic biology, constructing microbial cell factories to synthesize natural products using renewable resources has provided new ideas for the production of liquiritigenin. Compared with plant extraction and chemical synthesis methods, the biosynthesis of flavonoid compounds by microbial cell factories has many advantages, such as mild conditions, low cost, simple operation, environmental friendliness, low energy demand, and large-scale production. Currently, the biosynthetic pathways of most flavonoid compounds have been analyzed, and researchers have constructed complete pathways of various plant-derived flavonoid compounds in Escherichia coli or Saccharomyces cerevisiae. Among them, Saccharomyces cerevisiae, as a eukaryotic model microorganism, has rapid reproduction, simple genetic manipulation, and a complete protein expression and translation system. Compounds such as intracellular endogenous aromatic amino acids and acetyl coenzyme A are important precursors for the synthesis of flavonoid compounds such as liquiritigenin, and are considered to be suitable chassis hosts for the synthesis of flavonoid compounds.

[0005] In the structure of liquiritigenin molecule, there is a deoxy group structure on its unilateral ring. This structural feature leads to complex de-group operations during the synthesis of liquiritigenin. Therefore, during the synthesis of liquiritigenin using microbial cell factories, by-products such as naringenin often accumulate. In addition, from the principle of mass conservation, the formation of 1 molecule of flavonoid skeleton requires the consumption of 3 molecules of malonyl coenzyme A, and malonyl coenzyme A is directly synthesized in large quantities from acetyl coenzyme A in the cytoplasm. The large-scale synthesis of liquiritigenin in microbial cells will seriously disrupt the steady-state balance of acetyl coenzyme A / malonyl coenzyme A in the cells, which may not only lead to slow cell growth but also result in low yield due to insufficient supply of acetyl coenzyme A. Therefore, to date, the heterologous synthesis of liquiritigenin using microbial cell factories still has problems such as low yield and difficulties in downstream product separation. Summary of the Invention

[0006] The present invention provides an engineered Saccharomyces cerevisiae strain that can efficiently heterologously synthesize liquiritigenin, and the proportion of by-products is controlled below 5%. At the same time, the present invention also provides an engineered peroxisome assembly method, which significantly improves the supply level of intracellular acetyl coenzyme A by optimizing the metabolic pathway in yeast cells, thereby enhancing the production capacity of liquiritigenin.

[0007] The technical solution of the present invention is as follows:

[0008] In a first aspect of the present invention, there is provided an engineered bacterium, characterized in that it heterologously expresses phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, 4-coumaroyl-CoA ligase, chalcone synthase, chalcone reductase, chalcone isomerase, cytochrome P450 reductase and cytochrome B5;

[0009] The amino acid sequence of the phenylalanine ammonia-lyase derived from Arabidopsis thaliana is SEQ ID NO:1;

[0010] The amino acid sequence of the cinnamic acid 4-hydroxylase derived from Arabidopsis thaliana is SEQ ID NO:2;

[0011] The amino acid sequence of the 4-coumaroyl-CoA ligase derived from Arabidopsis thaliana is SEQ ID NO:3;

[0012] The amino acid sequence of the chalcone synthase derived from Pharbitis nil is SEQ ID NO:4;

[0013] The amino acid sequence of the chalcone reductase derived from Glycine max is SEQ ID NO:5;

[0014] The amino acid sequence of the chalcone isomerase derived from Glycine max is SEQ ID NO:6;

[0015] The amino acid sequence of the cytochrome P450 reductase derived from Arabidopsis thaliana is SEQ ID NO:7;

[0016] The nucleotide sequence encoding the endogenous cytochrome B5 enzyme of Saccharomyces cerevisiae is SEQ ID NO:8;

[0017] Those skilled in the art can use conventional technical means in the art to enable the exogenous expression of the above enzymes in the engineered strain.

[0018] In a specific embodiment, in order to achieve the efficient synthesis of liquiritin, the engineered strain also expresses the following multiple proteins: tyrosine ammonia-lyase and shikimate kinase, the mutant ARO4 of yeast endogenous K229L and the mutant ARO7 G141S , and knocking out phenylpyruvate decarboxylase (ARO10) to enhance the metabolic flux of the shikimate pathway and increase the supply of the synthetic precursor p-coumaric acid.

[0019] The amino acid sequence of the tyrosine ammonia-lyase derived from Flavobacterium johnsoniae is SEQ ID NO:9;

[0020] The amino acid sequence of the shikimate kinase derived from Escherichia coli is SEQ ID NO:10;

[0021] The nucleotide sequence encoding the endogenous ARO4 of Saccharomyces cerevisiae K229L is SEQ ID NO:11;

[0022] The nucleotide sequence encoding the endogenous ARO7 of Saccharomyces cerevisiae G141S is SEQ ID NO:12.

[0023] In a specific embodiment, in order to eliminate the accumulation of the by-product naringenin, in the engineered strain, the chalcone reductase derived from soybean is heterologously expressed with a gene copy number of 1 to 5 to enhance the contact frequency between the key rate-limiting enzyme chalcone reductase and chalcone synthase and reduce the accumulation of by-products.

[0024] The second aspect of the present invention provides an engineered peroxisome assembly strategy. In order to increase the supply of acetyl-CoA in yeast cells and achieve further production increase of liquiritin. It is characterized in that one or a combination of multiple yeast endogenous peroxisome biogenesis factors (PEX) are overexpressed in the engineered strain, including PEX5, PEX7, PEX13, PEX14, PEX17, PEX4, PEX2, PEX10, PEX12, PEX8, PEX1, PEX6, PEX15, PEX3, PEX19 and PEX11, to enhance peroxisome assembly and increase the supply of acetyl-CoA.

[0025] The nucleotide sequences of the endogenous PEX of each of the encoded Saccharomyces cerevisiae are SEQ ID NO: 13 to SEQ ID NO: 28.

[0026] In a specific embodiment, in order to further enhance the transport and synthesis of peroxisomal acetyl-CoA, the carnitine acetyl-CoA transferase (CAT2) and acetyl-CoA synthetase (ACS1) endogenous to yeast are also overexpressed in the engineered strain.

[0027] The nucleotide sequence of the encoded carnitine acetyl-CoA transferase endogenous to Saccharomyces cerevisiae is SEQ ID NO: 29.

[0028] The nucleotide sequence of the encoded acetyl-CoA synthetase endogenous to Saccharomyces cerevisiae is SEQ ID NO: 30.

[0029] In a specific embodiment, any of the above-mentioned engineered bacteria is cultured, and the culture product is collected to obtain liquiritin.

[0030] Through metabolic engineering and synthetic biology means, the present invention significantly enhances the supply capacity of precursors p-coumaric acid and acetyl-CoA in the yeast engineered bacteria for producing liquiritin; at the same time, by combining multi-copy expression of the exogenous rate-limiting enzyme chalcone reductase, the efficient de novo synthesis of liquiritin in the microbial cell factory is successfully achieved, and the proportion of fermentation by-products is strictly controlled below 5%. This yeast engineered strain not only improves the production efficiency of liquiritin, but also ensures the rapidity and environmental friendliness of the production process, provides a potentially efficient method for the large-scale industrial production of liquiritin, and also provides important references and ideas for the sustainable microbial production of other flavonoid compounds, and has broad application prospects. Description of the Drawings

[0031] Figure 1 Schematic diagram of the synthesis pathway of liquiritin in the engineered bacteria;

[0032] Figure 2 Product detection diagram of the engineered bacteria LIG01 producing liquiritin;

[0033] Figure 3 Titer test results of the engineered bacteria LIG02 and schematic diagram of the shikimic acid metabolic pathway;

[0034] Figure 4 Titer test results of the engineered bacteria LIG02-LIG06 with increased copy number of the key enzyme CHR gene;

[0035] Figure 5 Grouping diagram of yeast endogenous PEX and titer test results of the engineered bacteria LIG07-LIG22;

[0036] Figure 6Titer test results of engineered strain LIG23-LIG34 with combined overexpression of PEX;

[0037] Figure 7 Titer test results of engineered strain LIG35 for enhancing acetyl-CoA transport. Specific implementation manners

[0038] The following further describes the specific implementation manners of the present invention in detail in combination with embodiments. The following embodiments are used to illustrate the present invention but not to limit the scope of the present invention.

[0039] Example 1 Construction of an engineered yeast strain for producing liquiritigenin

[0040] 1.1 Construction of engineered strain LIG01

[0041] In this example, Saccharomyces cerevisiae SynV was used as the starting strain. By the method of homologous recombination in Saccharomyces cerevisiae, the genes encoding Arabidopsis phenylalanine ammonia-lyase AtATR (the amino acid sequence of this enzyme is shown in SEQ ID NO:1), Arabidopsis cinnamate 4-hydroxylase AtC4H gene (the amino acid sequence of this enzyme is shown in SEQ ID NO:2), Arabidopsis 4-coumaroyl-CoA ligase At4CL gene (the amino acid sequence of this enzyme is shown in SEQ ID NO:3), Arabidopsis cytochrome P450 reductase AtATR gene (the amino acid sequence of this enzyme is shown in SEQ ID NO:7), and Saccharomyces cerevisiae cytochrome B5 enzyme CYB5 gene (the nucleotide sequence encoding this enzyme is shown in SEQ ID NO:8) were integrated into the HO locus of the Saccharomyces cerevisiae genome together. Among them, the AtC4H gene was fused to the C-terminus of the At4CL gene through a flexible linker GGGS. The genes encoding Pharbitis nil chalcone synthase PhCHS (the amino acid sequence of this enzyme is shown in SEQ ID NO:4), Glycine max chalcone reductase GmCHR gene (the amino acid sequence of this enzyme is shown in SEQ ID NO:5), and Glycine max chalcone isomerase GmCHI gene (the amino acid sequence of this enzyme is shown in SEQ ID NO:6) were integrated into the YPRCτ3 locus of the Saccharomyces cerevisiae genome together. After screening and verification on a defective plate, the engineered strain LIG01 capable of producing liquiritigenin was constructed.

[0042] The promoters and terminators used are as follows:

[0043] GAL7p-AtPAL-PGI1t; GAL2p-AtC4H-L-At4CL-TPS1t; GAL1p-AtATR-CYC1t; GAL10p-CYB5-ADH1t; GAL2p-PHCHS-PGI1t; GAL1p-GmCHR-CYC1t; GAL10p-GmCHI-ADH1t;

[0044] Continue to refer to Figure 1 , -phenylalanine -Phe is converted to p-coumaric acid p-CA by the action of phenylalanine ammonia-lyase AtPAL and cinnamic acid 4-hydroxylase AtC4H. Among them, cytochrome P450 reductase AtATR and cytochrome B5 enzyme CYB5 are used to optimize the oxidation efficiency of AtC4H. Subsequently, 4-coumaric acid coenzyme A ligase At4CL converts p-CA, which is a key precursor, into p-coumaric acid coenzyme A p-CA-CoA. Under the combined action of chalcone synthase PhCHS and chalcone reductase GmCHR, p-CA-CoA and three molecules of malonyl-CoA condense to form isoliquiritigenin IsoLIG, and IsoLIG is further cyclized to liquiritigenin by the action of chalcone isomerase GmCHI.

[0045] Ferment the engineered strain LIG01 and perform UPLC analysis on the fermentation products. The analysis results are as Figure 2 shown. LIG01 can produce liquiritigenin, but the titer is only 42.83 mg / L. In addition, it is confirmed by LC-MS negative mode detection that there is a product liquiritigenin with a molecular weight of [M-H] - = 271.1, and at the same time, a by-product naringenin with a molecular weight of [M-H] - = 255.1 is also detected.

[0046] 1.2 Construction of engineered strain LIG02

[0047] Refer to Figure 3 a. During the fermentation of the engineered strain LIG01, 0.3 mM p-coumaric acid p-CA is added externally, and the titer of liquiritigenin is further increased to 73.34 mg / L, indicating that the supply of the precursor p-coumaric acid p-CA is significantly insufficient during the synthesis of liquiritigenin. Therefore, further strengthen the metabolic flux of the shikimate pathway and increase the supply of the synthetic precursor p-coumaric acid, as Figure 3 shown in b. On the basis of LIG01, the gene of tyrosine ammonia-lyase FjTAL from Flavobacterium johnsoniae (the amino acid sequence of this enzyme is as shown in SEQ ID NO:9), the gene of shikimate kinase EcaroL from Escherichia coli (the amino acid sequence of this enzyme is as shown in SEQ ID NO:10), and the mutant ARO4 of yeast endogenous originK229L (The nucleotide sequence of the gene encoding ARO4 K229L is shown in SEQ ID NO:11) and the mutant ARO7 G141S gene (encoding ARO7 G141S whose nucleotide sequence is shown in SEQ ID NO:12) were integrated into the ARO10 locus of the Saccharomyces cerevisiae genome. At the same time as the gene integration, the endogenous phenylpyruvate decarboxylase ARO10 of yeast was knocked out.

[0048] The promoters and terminators used are as follows:

[0049] GAL7p-FjTAL-PGI1t; GAL2p-EcaroL-TPS1t; GAL1p-AR04K299L-CYC1t; GAL10p-ARO7G141S-ADH1t;

[0050] The engineered strain LIG02 was fermented, and the fermentation product was analyzed by UPLC. The analysis results are as Figure 3 shown in a. The titer of liquiritigenin in LIG02 was increased to 69.74 mg / L. During the fermentation of LIG02, 0.3 mM p-coumaric acid p-CA was further added externally, and the titer of liquiritigenin did not increase further, indicating that the precursor supply was sufficient.

[0051] 1.3 Construction of engineered strains LIG03-LIG06

[0052] As Figure 1As shown in the figure, in the synthesis pathway of liquiritigenin, only when chalcone synthase and chalcone reductase are co-expressed and cooperate with each other and catalyze synergistically can the target product liquiritigenin be successfully synthesized. When there is only chalcone synthase in the synthesis pathway or the synergistic catalytic efficiency of the two is low, the synthesis or the accumulation of by-product naringenin will occur. Based on the engineering bacterium LIG02, the GmCHR gene encoding soybean chalcone reductase was overexpressed in multiple copies in the range of copy numbers 1-5, thereby constructing engineering strains LIG03-LIG06. Among them, based on LIG02, the gene encoding soybean chalcone reductase GmCHR was integrated into the HO site of the Saccharomyces cerevisiae genome to obtain the engineering strain LIG03; based on LIG03, the gene encoding soybean chalcone reductase GmCHR was integrated into the ARO10 site of the Saccharomyces cerevisiae genome to obtain the engineering bacterium LIG04; based on LIG04, the gene encoding soybean chalcone reductase GmCHR was integrated into the YHRCΔ14 site of the Saccharomyces cerevisiae genome to obtain the engineering bacterium LIG05; based on LIG05, the gene encoding soybean chalcone reductase GmCHR was integrated into the GAL80 site of the Saccharomyces cerevisiae genome to obtain the engineering bacterium LIG06. Gradually enhance the expression level of the key rate-limiting enzyme chalcone reductase GmCHR, increase the contact frequency with chalcone synthase PhCHS, and reduce the synthesis of by-product naringenin.

[0053] The promoters and terminators used are as follows:

[0054] GAPp-GmCHR5-TPS1t (integrated at the HO site and ARO10 site); GAL1p-GmCHR5-CYC1t (integrated at the YHRCΔ14 site and GAL80 site);

[0055] The engineering strains LIG03-LIG06 were fermented, and the fermentation products were analyzed by UPLC. The analysis results are as Figure 4 shown. As the copy number of GmCHR increases, the titer of liquiritigenin also increases. In the engineering bacterium LIG05 with a GmCHR copy number of 4, the titer of liquiritigenin is the highest, reaching 111.27 mg / L. At this time, the accumulation of by-product naringenin is also significantly reduced, accounting for less than 5% (9.77 mg / L).

[0056] Example 2 Regulation of peroxisome assembly

[0057] Peroxisomes are the only organelles for fatty acid β-oxidation in yeast and are the main source of acetyl-CoA. PEX proteins are components of peroxisomes and are responsible for regulating the assembly of peroxisomes. For example, they are involved in the import of matrix proteins and the regulation of quantity and size. By regulating the assembly of peroxisomes, the synthesis of acetyl-CoA is further promoted, providing sufficient acetyl-CoA and malonyl-CoA for the efficient synthesis of liquiritin. According to different functions (as shown in Figure 5 Figure a), PEX derived from yeast is divided into 4 groups (groups A, B, C, and D): PEX belonging to group A is responsible for matrix protein recruitment; PEX belonging to group B is involved in peroxisome biosynthesis; PEX belonging to group C is involved in peroxisome proliferation and lysis; PEX belonging to group D is responsible for regulating the size and quantity of peroxisomes.

[0058] 2.1 Overexpression of PEX to construct engineering bacteria LIG07-LIG22

[0059] Refer to Figure 5 Figure b. Using homologous recombination of yeast, the promoter GAL1, PEX gene, and terminator CYC1 were assembled by fragments to construct a PEX overexpression cassette. Based on engineering bacteria LIG05, 16 PEXs derived from yeast (belonging to groups A, B, and C) were overexpressed, including PEX5, PEX7, PEX13, PEX14, PEX17, PEX4, PEX2, PEX10, PEX12, PEX8, PEX1, PEX6, PEX15, PEX3, PEX19, and PEX11 (the nucleotide sequences encoding the PEX genes are shown in SEQ ID NO:13 to SEQ ID NO:28), and engineering bacteria LIG07-LIG22 were obtained. After successful verification, the engineering bacteria LIG07-LIG22 were fermented, fermented and catalyzed at 30 °C for 7 days, the fermentation broth was collected, and the fermentation products were analyzed by UPLC. The analysis results are as shown in reference Figure 2 Figure. In group A, in the strains overexpressing PEX13, PEX2, PEX10, PEX8, PEX1, PEX6, and PEX15, the titer of liquiritin increased by 55%-124%. In group B, overexpression of PEX3 and PEX19 increased the titer of liquiritin to 232.31 and 192.62 mg / L, respectively. In group C, the engineering bacteria LIG22 overexpressing PEX11 produced 267.84 mg / L of liquiritin.

[0060] 2.2 Combined overexpression of PEX to construct engineering bacteria LIG23-LIG34

[0061] Furthermore, based on the engineered strain LIG05, the effective PEXs from the same or different groups were further combinatorially expressed to obtain the engineered strains LIG23 - LIG34; the combination methods and the test results of liquiritin titers are as shown in the reference Figure 6 as follows. Among the engineered strains LIG23 - LIG34 that combinatorially expressed PEXs, the liquiritin titers were all increased to varying degrees. Among them, the engineered strain LIG25 overexpressing PEX6 and PEX15 exhibited the highest liquiritin titer, which was 328.48 mg / L.

[0062] 2.3 Construction of the engineered strain LIG35

[0063] Reference Figure 7 , in order to further promote the transport and synthesis of acetyl - CoA in peroxisomes. Based on the engineered strain LIG25 with the highest liquiritin titer, the endogenous CAT2 from Saccharomyces cerevisiae (the nucleotide sequence encoding the CAT2 gene is as shown in SEQ ID NO:29) and ACS1 (the nucleotide sequence encoding the ACS1 gene is as shown in SEQ ID NO:30) were further overexpressed to obtain the engineered strain LIG35. The test results are as Figure 2 follows. In the engineered strain LIG35, the liquiritin titer was further increased to 403.06 mg / L, which was 12.85 - fold higher than that of the initial engineered strain LIG01.

[0064] Experimental Example 3 Fermentation and Detection of Liquiritin

[0065] The single colony of the engineered strain on the plate was inoculated into 5 mL of YPD medium and cultured overnight at 30 °C with shaking at 200 rpm to obtain the seed liquid. Then, it was inoculated into 30 mL of YPD medium at an inoculation amount with an OD 600 of 0.3 and cultured at 30 °C with shaking at 200 rpm for 7 days. After fermentation, 500 μL of the fermentation broth was taken, the same volume of ethyl acetate was added, and the organic phase was collected by vortex mixing and repeated 3 times. The collected organic phase was evaporated to dryness and redissolved with methanol, and then filtered through a 0.2 - μm organic filter membrane for high - performance liquid chromatography to detect the content of liquiritin.

[0066] Experimental Example 4 Fed - batch Fermentation for the Production of Liquiritin

[0067] Fed - batch fermentation was carried out on the engineered strain LIG35 with the highest shake - flask yield of liquiritin. A single clone from the plate was inoculated into a test tube containing 5 mL of YPD liquid medium and cultured overnight at 30 °C with shaking at 200 rpm. Then, it was transferred to a 250 - mL Erlenmeyer flask containing 50 mL of YPD liquid medium at a ratio of 10% and cultured at 30 °C with shaking at 200 rpm for 24 h to obtain the seed liquid. The seed liquid was inoculated into the fermenter, the fermentation temperature was controlled at 30 °C, the fermentation pH was controlled at 6.0, and the feeding concentration of glucose was controlled within 20 g / L.

[0068] The composition of the fermentation medium is as follows: glucose 40 g / L, (NH4)2SO4 15 g / L, KH2PO4 8 g / L, MgSO4·7H2O 6.15 g / L, vitamin solution 12 mL / L, trace metal salt solution 10 mL / L. The composition of the vitamin solution is: biotin 0.05 g / L, calcium pantothenate 1 g / L, nicotinic acid 1 g / L, inositol 25 g / L, thiamine 1 g / L, pyridoxal 1 g / L, p-aminobenzoic acid 0.2 g / L. The composition of the trace metal salt solution is: EDTA 15 g / L, ZnSO4·7H2O 5.75 g / L, MnCl2·4H2O 0.32 g / L, CuSO4 0.5 g / L, CoCl2·6H2O 0.47 g / L, Na2MoO4·2H2O 0.48 g / L, CaCl2·2H2O 2.9 g / L, FeSO4·7H2O 2.8 g / L.

[0069] Through fed-batch fermentation, the engineered strain LIG35 can synthesize liquiritigenin at a concentration of 1102.41 mg / L.

Claims

1. An engineered bacterium for producing liquiritigenin, characterized in that, Using Saccharomyces cerevisiae SynⅤ as the starting strain, heterologously express phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, 4-coumaroyl-CoA ligase, chalcone synthase, chalcone reductase, chalcone isomerase, cytochrome P450 reductase and cytochrome B5 enzyme; The amino acid sequence of the phenylalanine ammonia-lyase derived from Arabidopsis thaliana is SEQ ID NO:1; The amino acid sequence of the cinnamic acid 4-hydroxylase derived from Arabidopsis thaliana is SEQ ID NO:2; The amino acid sequence of the 4-coumaroyl-CoA ligase derived from Arabidopsis thaliana is SEQ ID NO:3; The amino acid sequence of the chalcone synthase derived from Pharbitis nil is SEQ ID NO:4; The amino acid sequence of the chalcone reductase derived from Glycine max is SEQ ID NO:5; The amino acid sequence of the chalcone isomerase derived from Glycine max is SEQ ID NO:6; The amino acid sequence of the cytochrome P450 reductase derived from Arabidopsis thaliana is SEQ ID NO:7; The nucleotide sequence encoding the cytochrome B5 enzyme endogenous to Saccharomyces cerevisiae is SEQ ID NO:

8.

2. The engineered bacterium for producing liquiritin according to claim 1, wherein Heterologously express tyrosine ammonia-lyase and shikimate kinase in the engineered strain, overexpress the mutant ARO4 of endogenous yeast K229L and the mutant ARO7 G141S , and knockout phenylpyruvate decarboxylase (ARO10); The amino acid sequence of the tyrosine ammonia-lyase derived from Flavobacterium johnsoniae is SEQ ID NO:9; The amino acid sequence of the shikimate kinase derived from Escherichia coli is SEQ ID NO:10; The nucleotide sequence of the endogenous ARO4 of the engineered Saccharomyces cerevisiae K229L is SEQ ID NO:11; The nucleotide sequence encoding the endogenous ARO7 of Saccharomyces cerevisiae G141S is SEQ ID NO:

12.

3. The engineered bacterium for producing liquiritin according to claim 2, wherein In the said engineered strain, heterologously express the chalcone reductase derived from Glycine max with a copy number of 1 to 5.

4. A peroxisome assembly strategy, characterized in that, In the engineered bacterium for producing liquiritigenin described in claim 3, overexpress one or more yeast endogenous peroxisome biogenesis factors (PEX), including PEX5, PEX7, PEX13, PEX14, PEX17, PEX4, PEX2, PEX10, PEX12, PEX8, PEX1, PEX6, PEX15, PEX3, PEX19 and PEX11; The nucleotide sequences encoding the yeast endogenous PEX are SEQ ID NO:13 to SEQ ID NO:

28.

5. The peroxisome assembly strategy according to claim 4, characterized in that, In the said engineered strain, also overexpress the yeast endogenous carnitine acetyl-CoA transferase (CAT2) and acetyl-CoA synthetase (ACS1) to strengthen the transport and synthesis of peroxisomal acetyl-CoA; The nucleotide sequence encoding the yeast endogenous CAT2 is nucleotide sequence SEQ ID NO:29; The nucleotide sequence encoding the yeast endogenous ACS1 is nucleotide sequence SEQ ID NO:

30.

6. A method for synthesizing liquiritigenin, characterized in that, The engineered strain for producing liquiritigenin according to any one of claims 1-5 is cultured with glucose as the carbon source, and the culture product is collected to obtain liquiritigenin.

Images