Yeast engineering strain for producing apigenin and application thereof

Abstract

The invention discloses a yeast engineering bacterium for producing apigenin and application thereof. The yeast engineering bacterium comprises a yeast genome, a whole-cell catalytic synthesis apigenin module, a heterologous apigenin biosynthesis path and an NADPH supply module, the whole-cell catalytic synthesis apigenin module comprises an FNSI coding gene and an FNSII coding gene; the heterologous apigenin biosynthesis path comprises a TAL coding gene, a 4CL coding gene, a CHS coding gene, a CHI coding gene and an FNSII coding gene; the NADPH supply module comprises a G6PD coding gene and a PGD coding gene; the method disclosed by the invention has the characteristics of greenness and safety, and lays a foundation for synthesizing apigenin in a yeast cell factory.

Description

Technical Field

[0001] The present application relates to the field of bioengineering technology, and in particular to an engineered yeast strain for producing apigenin and its application. Background Art

[0002] Apigenin is a flavonoid compound with multiple biological activities and potential biological effects, including antioxidant, anti-fat and anti-cancer activities. Apigenin can prevent oxidative damage by interacting with a variety of redox signaling molecules, and is used to treat liver cancer and prevent and treat Alzheimer's disease. In addition, apigenin can induce inflammatory antibodies or reduce the release of inflammatory factors to achieve anti-inflammatory effects. According to in vivo and in vitro experiments, apigenin has achieved many achievements in anti-tumor effects such as colon cancer, gastric cancer, breast cancer, and lung cancer, and is currently widely used in the health care products industry. In addition to being added to health care products, apigenin can also be added as a raw material in the cosmetics industry due to its antioxidant and other biological activities. At present, apigenin is mainly extracted from plant tissues, and this extraction method is limited by factors such as the plant growth cycle and living environment. Summary of the invention

[0003] The present application provides a yeast engineering bacterium for producing apigenin and its application, so as to at least solve the above technical problems existing in the prior art.

[0004] According to the first aspect of the present application, a yeast engineering bacterium for producing apigenin is provided, wherein the yeast engineering bacterium comprises:

[0005] Yeast genome, whole-cell catalytic apigenin synthesis module, heterologous apigenin biosynthetic pathway and NADPH supply module;

[0006] The whole-cell catalytic synthesis of apigenin module includes an FNSI encoding gene and an FNSII encoding gene;

[0007] The heterologous apigenin biosynthetic pathway includes TAL encoding genes, 4CL encoding genes, CHS encoding genes, CHI encoding genes and FNSII encoding genes;

[0008] The NADPH supply module includes a G6PD encoding gene and a PGD encoding gene; wherein,

[0009] The nucleotide sequence of the FNSI encoding gene is a nucleotide sequence having a homology greater than a preset value to the nucleotide sequence shown in Genbank accession number AY230247.1;

[0010] The nucleotide sequence of the FNSII encoding gene is a nucleotide sequence having a homology greater than a preset value to the nucleotide sequence shown in Genbank accession number KC521362.1;

[0011] The nucleotide sequence of the TAL encoding gene is a nucleotide sequence having a homology greater than a preset value to the nucleotide sequence shown in Genbank accession number KR095307.1;

[0012] The nucleotide sequence of the CHS encoding gene is a nucleotide sequence having a homology greater than a preset value to the nucleotide sequence shown in Genbank accession number JF694272.1;

[0013] The nucleotide sequence of the CHI encoding gene is a nucleotide sequence having a homology greater than a preset value to the nucleotide sequence shown in Genbank accession number KF765782.1;

[0014] The nucleotide sequence of the G6PD encoding gene is a nucleotide sequence having a homology greater than a preset value to the nucleotide sequence shown in Genbank accession number 2912994;

[0015] The nucleotide sequence of the PGD encoding gene is a nucleotide sequence having a homology greater than a preset value with the nucleotide sequence shown in Genbank accession number 2906913.

[0016] In one embodiment, the nucleotide sequence of the FNSII encoding gene is the nucleotide sequence shown in Genbank accession number KC521362.1;

[0017] The nucleotide sequence of the TAL encoding gene is a nucleotide sequence with a nucleotide sequence homology greater than a preset value as shown in Genbank accession number KR095307.1;

[0018] The nucleotide sequence of the CHS encoding gene is the nucleotide sequence shown in Genbank accession number JF694272.1;

[0019] The nucleotide sequence of the CHI encoding gene is the nucleotide sequence shown in Genbank accession number KF765782.1;

[0020] The nucleotide sequence of the G6PD encoding gene is the nucleotide sequence shown in Genbank accession number 2912994;

[0021] The nucleotide sequence of the PGD encoding gene is the nucleotide sequence shown in Genbank Accession No. 2906913.

[0022] In one embodiment, the FNSI is flavone synthase I, the FNSII is flavone synthase II; the TAL is tyrosine ammonia lyase; the 4CL is 4-coumaroyl-CoA ligase; the CHS is chalcone synthase; the CHI is chalcone isomerase; the G6PD is glucose-6-phosphate dehydrogenase; and the PGD is 6-phosphogluconate dehydrogenase.

[0023] In one embodiment, the yeast in the yeast genome is Yarrowia lipolytica or Saccharomyces cerevisiae.

[0024] In one possible implementation manner, the preset value is 80%.

[0025] According to a second aspect of the present application, a method for producing apigenin is provided, comprising:

[0026] The whole-cell catalytic synthesis module of apigenin was integrated into yeast to obtain the first yeast subgenome that can convert naringenin into apigenin;

[0027] integrating a heterologous apigenin biosynthetic pathway into a yeast subgenome to obtain a second yeast subgenome producing apigenin;

[0028] Integrating the NADPH supply module into the second yeast sub-genome to obtain an engineered yeast strain;

[0029] The yeast engineering bacteria was fermented for 72 hours using YYG medium as a substrate to obtain apigenin.

[0030] In one embodiment, the YYG medium contains 8-10 g / L yeast extract, 7-9 g / L yeast nitrogen base without amino acids, and 50 g / L glucose.

[0031] In one embodiment, the carbon-nitrogen molar ratio in the YYG medium is 10:1-250:1, and the initial pH value is 2-7.

[0032] In one embodiment, 0-1.25 mM L-tyrosine or naringenin is added to the YYG medium at different time points within a preset time period and fermented for 72-96 hours.

[0033] By utilizing the technical solution of the present application, a higher yield of apigenin is obtained, and green and safe production of apigenin can be achieved. Compared with the prior art, the yeast engineered bacteria of the present invention can achieve efficient and stable synthesis of the apigenin biosynthesis pathway in yeast.

[0034] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present application, nor is it intended to limit the scope of the present application. Other features of the present application will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] By reading the detailed description below with reference to the accompanying drawings, the above and other purposes, features and advantages of the exemplary embodiments of the present application will become readily understood. In the accompanying drawings, several embodiments of the present application are shown in an exemplary and non-limiting manner, wherein:

[0036] In the drawings, the same or corresponding reference numerals represent the same or corresponding parts.

[0037] Figure 1 The biosynthetic route of apigenin produced by yeast engineering bacteria in the embodiment of the present application is shown;

[0038] Figure 2 A schematic diagram of the implementation process of the method for producing apigenin in an embodiment of the present application is shown. DETAILED DESCRIPTION

[0039] In order to make the purpose, features, and advantages of the present application more obvious and easy to understand, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present application.

[0040] In order to make the purpose, technical solutions and advantages of the present application clearer, the present application will be further described in detail below in conjunction with the accompanying drawings. The described embodiments should not be regarded as limiting the present application. All other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of this application.

[0041] In the following description, reference is made to “some embodiments”, which describe a subset of all possible embodiments, but it can be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0042] In the following description, the terms "first\second" involved are merely used to distinguish similar objects and do not represent a specific ordering of the objects. It can be understood that "first\second" can be interchanged with a specific order or sequence where permitted, so that the embodiments of the present application described herein can be implemented in an order other than that illustrated or described herein.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein are only for the purpose of describing the embodiments of this application and are not intended to limit this application.

[0044] It should be understood that in the various embodiments of the present application, the size of the serial number of each implementation process does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0045] With the development of synthetic biology and metabolic engineering, it has become possible to produce natural compounds using microbial cell factories. Yeast is a unicellular fungus and an important model organism that combines multiple advantages such as clear genetic background, short fermentation cycle and high biosafety. Therefore, optimizing the apigenin biosynthesis pathway in yeast and obtaining apigenin-producing yeast engineering bacteria is one of the important ways to achieve efficient, green and safe production of apigenin.

[0046] The following is an introduction to an engineered yeast strain for producing apigenin and its application provided by the present application in conjunction with the accompanying drawings.

[0047] The present application provides a yeast engineering bacterium for producing apigenin, wherein the yeast engineering bacterium comprises:

[0048] Yeast genome, whole-cell catalytic apigenin synthesis module, heterologous apigenin biosynthetic pathway and NADPH supply module;

[0049] The whole-cell catalytic synthesis of apigenin module includes an FNSI encoding gene and an FNSII encoding gene;

[0050] The heterologous apigenin biosynthetic pathway includes TAL encoding genes, 4CL encoding genes, CHS encoding genes, CHI encoding genes and FNSII encoding genes;

[0051] The NADPH supply module includes a G6PD encoding gene and a PGD encoding gene; wherein,

[0052] The nucleotide sequence of the FNSI encoding gene is a nucleotide sequence having a homology greater than a preset value to the nucleotide sequence shown in Genbank accession number AY230247.1;

[0053] The nucleotide sequence of the FNSII encoding gene is a nucleotide sequence having a homology greater than a preset value to the nucleotide sequence shown in Genbank accession number KC521362.1;

[0054] The nucleotide sequence of the TAL encoding gene is a nucleotide sequence having a homology greater than a preset value to the nucleotide sequence shown in Genbank accession number KR095307.1;

[0055] The nucleotide sequence of the CHS encoding gene is a nucleotide sequence having a homology greater than a preset value to the nucleotide sequence shown in Genbank accession number JF694272.1;

[0056] The nucleotide sequence of the CHI encoding gene is a nucleotide sequence having a homology greater than a preset value to the nucleotide sequence shown in Genbank accession number KF765782.1;

[0057] The nucleotide sequence of the G6PD encoding gene is a nucleotide sequence having a homology greater than a preset value to the nucleotide sequence shown in Genbank accession number 2912994;

[0058] The nucleotide sequence of the PGD encoding gene is a nucleotide sequence having a homology greater than a preset value with the nucleotide sequence shown in Genbank accession number 2906913.

[0059] The default value is 80%.

[0060] It can be understood that in the above-mentioned yeast engineering bacteria, the nucleotide sequence of the FNSI encoding gene is the same as the sequence shown in Genbank Accession No. AY230247.1 or the homology is greater than 80% and the nucleotide sequence has the same function for apigenin synthesis; the nucleotide sequence of the FNSII encoding gene is the same as the sequence shown in Genbank Accession No. KC521362.1 or the homology is greater than 80% and the nucleotide sequence has the same function for apigenin synthesis; the nucleotide sequence of the TAL encoding gene is the same as the sequence shown in Genbank Accession No. KR095307.1 or the homology is greater than 80% and the nucleotide sequence has the same function for apigenin synthesis; the nucleotide sequence of the CHS encoding gene is the same as the sequence shown in Genbank Accession No. The nucleotide sequence is identical to or has a homology greater than 80% with the sequence shown in the accession number JF694272.1 and has the same function as apigenin synthesis; the nucleotide sequence of the CHI encoding gene is identical to or has a homology greater than 80% with the sequence shown in the Genbank accession number KF765782.1 and has the same function as apigenin synthesis; the nucleotide sequence of the G6PD encoding gene is identical to or has a homology greater than 80% with the sequence shown in the Genbank accession number 2912994 and has the same function as apigenin synthesis; the nucleotide sequence of the PGD encoding gene is identical to or has a homology greater than 80% with the sequence shown in the Genbank accession number 2906913 and has the same function as apigenin synthesis.

[0061] In the above-mentioned yeast engineered bacteria, the amino acid sequence of the protein encoded by the FNSI gene is the same as or has a homology greater than 80% to the sequence shown in Genbank Accession No. AAP57393.1 and has the same function for apigenin synthesis; the amino acid sequence of the protein encoded by the FNSII gene is the same as or has a homology greater than 80% to the sequence shown in Genbank Accession No. AGK85254.1 and has the same function for apigenin synthesis; the amino acid sequence of the protein encoded by the TAL gene is the same as or has a homology greater than 80% to the sequence shown in Genbank Accession No. AKE50828.1 and has the same function for apigenin synthesis; the amino acid sequence of the protein encoded by the CHS gene is the same as the sequence shown in Genbank Accession No. The amino acid sequence of the protein encoded by the CHI gene is identical to or has a homology greater than 80% with the sequence shown in AED02599.1 and has the same function as the amino acid sequence for apigenin synthesis; the amino acid sequence of the protein encoded by the CHI gene is identical to or has a homology greater than 80% with the sequence shown in Genbank accession number AGZ04578.1 and has the same function as the amino acid sequence for apigenin synthesis; the amino acid sequence of the protein encoded by the G6PD gene is identical to or has a homology greater than 80% with the sequence shown in Genbank accession number 2912994 and has the same function as the amino acid sequence for apigenin synthesis; the amino acid sequence of the protein encoded by the PGD gene is identical to or has a homology greater than 80% with the sequence shown in Genbank accession number XP_500938.1 and has the same function as the amino acid sequence for apigenin synthesis.

[0062] The yeast engineered bacteria for producing apigenin provided in the present application have the FNSI encoding gene and the FNSII encoding gene of the whole-cell catalytic synthesis of apigenin module, heterologous apigenin biosynthesis pathway genes, and NADPH supply module genes integrated in the yeast genome, and the yeast chassis cell is any one of the eukaryotic organisms of the genus Saccharomyces cerevisiae, such as Yarrowia lipolytica and Saccharomyces cerevisiae. Preferably, the yeast chassis cell is Yarrowia lipolytica, which has the characteristics of strong stress tolerance, broad substrate utilization spectrum, sufficient supply of acetyl-CoA and reducing power, etc., is an ideal chassis for biosynthesis, and is conducive to the production of apigenin.

[0063] In some embodiments, the yeast genome is integrated with the FNSI encoding gene and the FNSII encoding gene of the whole-cell catalytic synthesis of apigenin module, the heterologous apigenin biosynthesis pathway genes, and the NADPH supply module genes, and the yeast chassis cell is any one of the yeast eukaryotic organisms such as Yarrowia lipolytica and Saccharomyces cerevisiae. Preferably, the yeast chassis cell is Yarrowia lipolytica, which has the characteristics of strong stress tolerance, broad substrate utilization spectrum, sufficient supply of acetyl-CoA and reducing power, etc., is an ideal chassis for biosynthesis, and is conducive to the production of apigenin.

[0064] In some embodiments, the FNSI is flavone synthase I, the FNSII is flavone synthase II; the TAL is tyrosine ammonia lyase; the 4CL is 4-coumaroyl-CoA ligase; the CHS is chalcone synthase; the CHI is chalcone isomerase; the G6PD is glucose-6-phosphate dehydrogenase; and the PGD is 6-phosphogluconate dehydrogenase.

[0065] In some embodiments, the whole-cell catalytic synthesis modules of apigenin are PcFNSI and EbFNSII; the heterologous apigenin biosynthesis pathway includes FjTAL, Eb4CL, EbCHS, MsCHI, and EbFNSII; and the NADPH supply module includes single overexpressed ScPOS5 and co-overexpressed YlG6PD and YlPGD.

[0066] Among them, the nucleotide sequence of Eb4CL is shown in Table 1;

[0067] Nucleotide sequence of Eb4CL

[0068]

[0069] like Figure 2 As shown, the present application embodiment provides a method for producing apigenin, the method comprising:

[0070] S101, integrating the whole-cell catalytic synthesis module of apigenin into yeast to obtain the first yeast subgenome that can convert naringenin into apigenin;

[0071] It can be understood that in the present application, the FNSI and FNSII encoding genes in the whole-cell catalytic synthesis of apigenin module are respectively integrated into the yeast genome to obtain a yeast engineering bacterium that can transform naringenin to produce apigenin, namely the first yeast subgenome, and the best one is determined to be EbFNSII.

[0072] S102, integrating the heterologous apigenin biosynthetic pathway into the yeast subgenome to obtain a second yeast subgenome that produces apigenin;

[0073] Then, the related genes FjTAL, Eb4CL, EbCHS, MsCHI and EbFNSII in the apigenin biosynthesis pathway were integrated into the yeast genome to obtain the apigenin-producing yeast engineered bacteria, i.e., the second yeast sub-genome.

[0074] S103, integrating the NADPH supply module into the second yeast sub-genome to obtain an engineered yeast strain;

[0075] The NADPH supply module was integrated into the yeast genome in S2 to obtain an engineered yeast strain that produces apigenin.

[0076] S104, fermenting the yeast engineering bacteria with YYG medium as a substrate for 72 hours to obtain apigenin.

[0077] The obtained apigenin-producing yeast engineered bacteria were fermented and optimized, and apigenin was obtained by fermenting for 72 hours using YYG medium as a substrate.

[0078] In some embodiments, the YYG medium contains 8-10 g / L yeast extract, 7-9 g / L yeast nitrogen base without amino acids, and 50 g / L glucose.

[0079] In some embodiments, the carbon-nitrogen molar ratio in the YYG medium is 10:1-250:1, and the initial pH value is 2-7.

[0080] In some embodiments, 0-1.25 mM L-tyrosine or naringenin is added to the YYG medium at different time points within a preset time period and fermented for 72-96 hours.

[0081] As a specific example 1, the compatibility of flavonoid synthases from different sources with Yarrowia lipolytica in the whole-cell catalytic synthesis of apigenin module:

[0082] 1. Use high-fidelity enzyme amplification to construct PcFNSI and EbFNSII.

[0083] 2. Construct the integration vectors pINA1317-PcFNSI and pINA1317-EbFNSII respectively.

[0084] 3. The recovered functional module fragments PcFNSI and EbFNSII were transformed into Yarrowia lipolytica Polf-Δku70 respectively to construct yeast engineered bacteria YLAPI-1 and YLAPI-2.

[0085] 4. Apigenin was obtained by fermenting YYG medium containing 9 g / L yeast extract, 8 g / L yeast nitrogen source without amino acids and 50 g / L glucose with naringenin as the only precursor substrate for 72 h.

[0086] As Example 2, the construction of heterologous apigenin biosynthesis pathway and the biosynthesis of apigenin by yeast engineering bacteria:

[0087] 1. Use high-fidelity enzyme amplification to construct multi-copy integration site upstream homologous arm rDNA (up), multi-copy integration site downstream homologous arm rDNA (down), promoter TEFp, genes FjTAL, Eb4CL, EbCHS, MsCHI, EbFNSII, terminators LIP1t1, MIG1t1, LIP1t2, MIG1t2, LIP2t, and screening marker HisG-URA3-HisG (HUH).

[0088] 2. Construct the integration vectors pGGAselect-Naringenin-M1, pUC19-EbFNSII-M2, and pUC19-HUH respectively.

[0089] 3. The recovered functional module fragments Naringenin-M1, EbFNSII-M2 and screening marker module fragment HUH-M3 were co-transformed into Yarrowia lipolytica Polf-Δku70 to obtain the yeast engineered strain YLAPI-3.

[0090] 4. Apigenin was obtained by fermenting YYG medium containing 9 g / L yeast extract, 8 g / L yeast nitrogen source without amino acids and 50 g / L glucose for 72 h.

[0091] As Example 3, the construction of NADPH supply module for apigenin biosynthesis and the production of apigenin by yeast engineering bacteria:

[0092] 1. Use high-fidelity enzyme amplification to construct multi-copy integration site upstream homologous arm rDNA (up), multi-copy integration site downstream homologous arm rDNA (down), promoters TEFp, FBAp, genes YlG6PD, YlPGD, ScPOS5, terminators XPR2t, LIP2t and screening markers HisG-URA3-HisG and LEU.

[0093] 2. Construct the integration vectors pUC19-YlG6PD-M1, pUC19-YlPGD-M2, pUC19-ScPOS5-M1, pUC19-LEU and pUC19-HUH respectively.

[0094] 3. The engineered bacteria YLAPI-3 was subjected to URA3 screening marker loss using HUH, and then the recovered functional module fragments YlG6PD-M1, YlPGD-M2 and the screening marker module fragment HUH were co-transformed into the Yarrowia lipolytica engineered bacteria YLAPI-3 to obtain the yeast engineered bacteria YLAPI-4; the recovered functional module fragment ScPOS5-M1 and the screening marker module fragment LEU were co-transformed into the Yarrowia lipolytica engineered bacteria YLAPI-3 to obtain the yeast engineered bacteria YLAPI-5.

[0095] 4. YYG medium containing 9 g / L yeast extract, 8 g / L yeast nitrogen source without amino acids and 50 g / L glucose was used as substrate for fermentation for 72 h to obtain yeast engineered bacteria YLAPI-4 and yeast engineered bacteria YLAPI-5 that biosynthesized apigenin.

[0096] As Example 4, yeast engineering bacteria efficiently produces apigenin:

[0097] The only difference between Example 4 and Example 2 is that in step 4, the precursor L-tyrosine (at a concentration of 0.5 mM) is initially added to the YYG medium, and the engineered bacteria YLAPI-3 is fermented for 96 hours to obtain apigenin.

[0098] As Example 5, yeast engineering bacteria efficiently produces apigenin:

[0099] The fifth embodiment is basically the same as the second embodiment, and the only difference is that in step 4, the precursor substance naringenin (concentration is 0.75 mM) is added to the YYG medium for 48 hours of fermentation, and the engineered bacteria YLAPI-3 is fermented for 96 hours to obtain apigenin.

[0100] As an example, yeast engineering bacteria efficiently produces apigenin:

[0101] Example 6 is basically the same as Example 3, except that in step 4, the carbon-nitrogen molar ratio of the YYG medium is adjusted to 10:1, the initial pH value is 6, and the volume of the groove shake flask is 50 mL. Under the optimal fermentation conditions, the engineered strain YLAPI-5 is added with 0.75 mM naringenin at 48 h and fermented for 72 h to obtain apigenin.

[0102] Among them, the production of apigenin by yeast engineering bacteria using culture medium as substrate was tested, and the test results are shown in Table 2.

[0103] Table 2 The yield test results of apigenin produced using culture medium as substrate

[0104]

[0105] As can be seen from Table 2, the yeast engineering bacteria YLAPI-2 of Example 1 is constructed by the whole-cell catalytic synthesis of apigenin module, and the chassis strain Yarrowia lipolytica is determined to produce apigenin. The yeast engineering bacteria YLAPI-3 of Example 2 is constructed by a heterologous apigenin biosynthesis pathway, but the apigenin production is low. The yeast engineering bacteria YLAPI-4 and YLAPI-5 of Example 3 are based on YLAPI-3, integrating different NADPH supply modules into the yeast coding genome, and the apigenin production of the yeast engineering bacteria YLAPI-5 is improved. Example 4-5 explored the engineering bacteria YLAPI-3 around the precursor addition engineering strategy, and it can be seen that the apigenin production of the engineering strain YLAPI-3 was improved under the conditions of adding 0.75mM naringenin at 48h and fermenting for 96h. As can be seen from Example 6, under the optimal fermentation conditions, the engineering strain YLAPI-5 was fermented for 72h after adding 0.75mM naringenin at 48h, which can effectively achieve the accumulation of apigenin in the fermentation broth and increase the production of apigenin.

[0106] In summary, the apigenin yield obtained by using the optimized YYG medium provided in the present application is relatively high, and green and safe production of apigenin can be achieved. Compared with the prior art, the yeast engineered bacteria of the present invention can achieve efficient and stable synthesis of the apigenin biosynthetic pathway in yeast.

[0107] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A yeast engineering bacterium for producing apigenin, characterized in that: The yeast engineering bacteria include: Yeast genome, whole-cell catalytic apigenin synthesis module, heterologous apigenin biosynthetic pathway and NADPH supply module; The whole-cell catalytic synthesis of apigenin module includes an FNSI encoding gene and an FNSII encoding gene; The heterologous apigenin biosynthetic pathway includes TAL encoding genes, 4CL encoding genes, CHS encoding genes, CHI encoding genes and FNSII encoding genes; The NADPH supply module includes a G6PD encoding gene and a PGD encoding gene; wherein, The nucleotide sequence of the FNSI encoding gene is a nucleotide sequence having a homology greater than a preset value to the nucleotide sequence shown in Genbank accession number AY230247.1; The nucleotide sequence of the FNSII encoding gene is a nucleotide sequence having a homology greater than a preset value to the nucleotide sequence shown in Genbank accession number KC521362.1; The nucleotide sequence of the TAL encoding gene is a nucleotide sequence having a homology greater than a preset value to the nucleotide sequence shown in Genbank accession number KR095307.1; The nucleotide sequence of the CHS encoding gene is a nucleotide sequence having a homology greater than a preset value to the nucleotide sequence shown in Genbank accession number JF694272.1; The nucleotide sequence of the CHI encoding gene is a nucleotide sequence having a homology greater than a preset value to the nucleotide sequence shown in Genbank accession number KF765782.1; The nucleotide sequence of the G6PD encoding gene is a nucleotide sequence having a homology greater than a preset value to the nucleotide sequence shown in Genbank accession number 2912994; The nucleotide sequence of the PGD encoding gene is a nucleotide sequence having a homology greater than a preset value with the nucleotide sequence shown in Genbank accession number 2906913.

2. The yeast engineering bacteria for producing apigenin according to claim 1, characterized in that: The nucleotide sequence of the FNSII encoding gene is the nucleotide sequence shown in Genbank accession number KC521362.1; The nucleotide sequence of the TAL encoding gene is a nucleotide sequence with a nucleotide sequence homology greater than a preset value as shown in Genbank accession number KR095307.1; The nucleotide sequence of the CHS encoding gene is the nucleotide sequence shown in Genbank accession number JF694272.1; The nucleotide sequence of the CHI encoding gene is the nucleotide sequence shown in Genbank accession number KF765782.1; The nucleotide sequence of the G6PD encoding gene is the nucleotide sequence shown in Genbank accession number 2912994; The nucleotide sequence of the PGD encoding gene is the nucleotide sequence shown in Genbank Accession No. 2906913.

3. The yeast engineering bacteria for producing apigenin according to claim 1, characterized in that: The FNSI is flavone synthase I, the FNSII is flavone synthase II; the TAL is tyrosine ammonia lyase; the 4CL is 4-coumaroyl-CoA ligase; the CHS is chalcone synthase; the CHI is chalcone isomerase; the G6PD is glucose-6-phosphate dehydrogenase; and the PGD is 6-phosphogluconate dehydrogenase.

4. The yeast engineering bacteria for producing apigenin according to claim 1, characterized in that: The yeast in the yeast genome is Yarrowia lipolytica or Saccharomyces cerevisiae.

5. The yeast engineering bacteria for producing apigenin according to claim 1, characterized in that: The preset value is 80%.

6. A method for producing apigenin, characterized in that: The method comprises: The whole-cell catalytic synthesis module of apigenin was integrated into yeast to obtain the first yeast subgenome that can convert naringenin into apigenin; integrating a heterologous apigenin biosynthetic pathway into a yeast subgenome to obtain a second yeast subgenome producing apigenin; Integrating the NADPH supply module into the second yeast sub-genome to obtain an engineered yeast strain; The yeast engineering bacteria was fermented for 72 hours using YYG medium as a substrate to obtain apigenin.

7. The method according to claim 6, characterized in that The YYG medium contains 8-10 g / L yeast extract, 7-9 g / L yeast nitrogen source without amino acids, and 50 g / L glucose.

8. The method according to claim 7, characterized in that The carbon-nitrogen molar ratio in the YYG medium is 10:1-250:1, and the initial pH value is 2-7.

9. The method according to claim 8, characterized in that 0-1.25 mM L-tyrosine or naringenin is added to the YYG medium at different time points within a preset time period, and fermented for 72-96 hours.

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