Microorganism for reconstructing metabolic pathway of aromatic amino acid as well as construction method and application of microorganism
By introducing pre-phenyl transaminase and ronalate dehydrase in yeast, the metabolic pathways of phenylalanine and tyrosine are reconstructed, the problem of competition in metabolic pathways is solved, the yield of target products is increased and by-products are eliminated.
Patent Information
- Application Number
- CN202510158906.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-13
- Publication Date
- 2025-05-23
AI Technical Summary
In microorganisms such as yeast, the metabolic pathways of phenylalanine and tyrosine compete with the synthesis pathways of other aromatic compounds, affecting the synthesis of the target product.
By introducing a gene expression cassette of pre-phenyl transaminase into yeast cells, it produces arosenic acid and knocking out a phenylphenyl pyruvate synthetase, it introduces arosenic acid dehydratase to reconstruct the synthesis pathway of phenylalanine and tyrosine.
A synthetic pathway directing metabolic flow to the target product is achieved, the yield of target aromatic compounds is increased, and by-products are eliminated.
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Figure CN120026047A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of metabolic engineering, and in particular relates to a microorganism for reconstructing an aromatic amino acid metabolic pathway, a construction method and an application thereof. Background Art
[0002] Metabolic engineering of microorganisms can enable them to synthesize target products, thus realizing the use of microorganisms as "cell factories" to produce the compounds we want. Yeast is one of the commonly used chassis microorganisms for metabolic engineering.
[0003] Microorganisms need complex metabolic networks to maintain their survival and growth in their life activities, and some metabolic pathways will compete with the synthesis of target products. Metabolic engineering is to reconstruct some metabolic pathways in microorganisms to synthesize target products and convert as much as possible into target products.
[0004] However, in microorganisms such as yeast, there are some substances that must be synthesized to maintain life activities, such as essential amino acids such as phenylalanine (L-Phe) and tyrosine (L-Tyr). In addition to the metabolic pathways for the production of the corresponding essential amino acids, there are also other metabolic product synthesis pathways ( Figure 1 ). In the synthesis of some aromatic compounds, these metabolic pathways may seriously affect the synthesis of the target product.
[0005] Therefore, it is necessary to reconstruct the synthesis pathway of phenylalanine and / or tyrosine in yeast through metabolic engineering to direct the metabolic flow to the synthesis pathway of the target product as much as possible. Summary of the invention
[0006] To solve the above problems, the present invention provides a method for reconstructing the aromatic amino acid metabolic pathway in a microorganism, comprising the step of generating rutin acid in the microorganism, wherein the molecular formula of rutin acid is as shown in Formula I,
[0007]
[0008] In a specific embodiment, the microorganism produces arginine by introducing a gene expression cassette of prephenate aminotransferase into the microbial cell.
[0009] In a specific embodiment, the amino acid sequence of the prephenate aminotransferase is selected from one or more combinations of SEQ ID NO: 2, 6, 8, or isozymes thereof.
[0010] In a specific embodiment, the method further comprises the steps of knocking out phenylpyruvate synthase and introducing galenate dehydratase in the microbial cell.
[0011] In a specific embodiment, the amino acid sequence of the longanate dehydratase is selected from one or more combinations of SEQ ID NOs: 4 and 10, or isozymes thereof.
[0012] In a specific embodiment, the microorganism is yeast.
[0013] The present invention also provides a chassis bacterium obtained by reconstructing the aromatic amino acid metabolic pathway through the above method.
[0014] The present invention also provides application of the above-mentioned bottom plate bacteria in producing 4-hydroxyphenyllactic acid.
[0015] The present invention also provides a method for constructing an engineered strain for producing a target aromatic compound, comprising the step of introducing a synthetic pathway of the target aromatic compound into the above-mentioned base bacteria.
[0016] In a specific embodiment, the target aromatic compound is a derivative of phenylalanine and / or tyrosine.
[0017] In a specific embodiment, the target aromatic compound is selected from one or more combinations of rosmarinic acid, 4-hydroxymandelic acid, tyrosol and dopamine.
[0018] The present invention introduces a prephenate aminotransferase expression cassette into yeast cells, so that yeast can synthesize tyrosine and reconstruct the synthesis pathway of phenylalanine, tyrosine and their derivatives with tyrosine as the center. The chassis bacteria constructed by this method can be used to introduce the synthesis pathway of some aromatic target products, increase the yield of these target products, and eliminate by-products. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Schematic diagram of the phenylalanine and tyrosine metabolic pathways in yeast.
[0020] Figure 2 Schematic diagram of the RA synthesis pathway.
[0021] Figure 3 This is the peak diagram for metabolite detection in RA09.
[0022] Figure 4 Schematic diagram of the synthesis of phenyllactic acid-derived by-products in the RA synthesis pathway.
[0023] Figure 5 Schematic diagram of the metabolic pathway for reconstructing the phenylalanine metabolic pathway and eliminating byproducts.
[0024] Figure 6 The production of RA and 4-HPL, and the growth conditions of different RA-synthesizing strains.
[0025] Figure 7 Peak profiles for metabolite detection in RA14 and RA54.
[0026] Figure 8 Schematic diagram of the construction of synthetic strains of 4-HMA, 4-HPL, tyrosol and dopamine.
[0027] Fig. 9 The yields of the corresponding target products in the synthetic strains of 4-HMA, 4-HPL, tyrosol and dopamine. DETAILED DESCRIPTION
[0028] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.
[0029] 1. Strains used
[0030] The starting strain used in the present invention is the high-p-coumaric acid-producing Saccharomyces cerevisiae strain QL11 constructed in the laboratory in the early stage.
[0031] 2. Rosmarinic acid synthesis strain
[0032] The synthetic pathway of rosmarinic acid (RA) is as follows: Figure 2 As shown in the figure, phenylpyruvate is synthesized into p-coumaric acid (HCA) through the phenylalanine pathway, and then hydroxylated at the 3rd position to synthesize caffeic acid (CA); on the other hand, it forms 4-hydroxyphenylpyruvic acid (4-HPP) through the tyrosine pathway, which can be converted into 4-hydroxyphenylactic acid (4-HPL) under the catalysis of reductase, and then hydroxylated at the 3rd position under the action of hydroxylase to form salvianic acid A (SAA). CA and SAA are direct precursors for RA synthesis. In addition, since the RAS substrate recognition specificity responsible for RA synthesis is poor, p-HCA-CoA and 4-HPL can also combine under the action of RAS to form structural analogs of RA.
[0033] Using CRISPR-Cas9 gene editing tools, the RA synthesis pathway genes were inserted into the genome of the QL11 strain for integrated expression, resulting in strain RA09, which achieved heterologous production of RA with a yield of 8.0 mg / L. A large amount of byproducts accumulated in the fermentation product ( Figure 3 )
[0034] Based on strain RA09, P450 enzymes from Salvia miltiorrhiza were further introduced to promote further hydroxylation modification of the intermediate products 4-HPL-CA and 4-HPL-HCA. The RA production in the resulting strain RA14 was increased to 51.2 mg / L, but by-products still accumulated significantly at this time.
[0035] Research specific reasons such as Figure 4 As shown in the figure, PPA also has other metabolic pathways, which can generate phenyllactic acid (PA) under the action of lactate reductase (LDH). PA can also combine with CA or p-HCA to form a by-product with the 4′-OH modification missing on the benzene ring.
[0036] To overcome this defect, we designed a new phenylalanine synthesis pathway by knocking out endogenous phenylpyruvate synthase (PHA). At the same time, we introduced prephenylate aminotransferase (PAT) to convert PPA into arogenate (arogenate, structural formula as shown in Formula I), and arogenate dehydratase (ADT) to convert arogenate into phenylalanine ( Figure 5 ).
[0037]
[0038] 3. Reconstruction of aromatic amino acid metabolic pathway to efficiently synthesize RA
[0039] Phenylpyruvate synthase PHA2 (amino acid sequence as shown in SEQ ID NO: 1) was knocked out in strain RA14 to obtain strain RA44. Then, prephenate aminotransferase (PAT) and adenosine dehydratase (ADT) from different sources were respectively introduced into RA44 to obtain strains RA46, RA48, RA50, RA52, RA53, and RA54, respectively.
[0040] Among them, RmPAT from Rhizobium meliloti (amino acid sequence as shown in SEQ ID NO:2, and the nucleic acid sequence after yeast codon optimization as shown in SEQ ID NO:3) and AtADT from Arabidopsis thaliana (amino acid sequence as shown in SEQ ID NO:4, and the nucleic acid sequence after yeast codon optimization as shown in SEQ ID NO:5) were transferred into RA46;
[0041] PhPAT from Petunia hybrida (amino acid sequence shown in SEQ ID NO:6, nucleic acid sequence after yeast codon optimization shown in SEQ ID NO:7) and AtADT from Arabidopsis thaliana were transferred into RA48;
[0042] AtPAT from Arabidopsis thaliana (amino acid sequence as shown in SEQ ID NO:8, nucleic acid sequence after yeast codon optimization as shown in SEQ ID NO:9) and AtADT from Arabidopsis thaliana were transferred into RA50;
[0043] RmPAT from Rhizobium meliloti and PhADT from Petunia hybrida were transferred into RA52 (the amino acid sequence is shown in SEQ ID NO: 10, and the nucleic acid sequence after yeast codon optimization is shown in SEQ ID NO: 11);
[0044] PhPAT from Petunia hybrida and PhADT from Petunia hybrida were transferred into RA53;
[0045] AtPAT from Arabidopsis thaliana and PhADT from Petunia hybrida were transferred into RA52.
[0046] The growth of each strain, as well as the content of RA and 4-HPL were detected. The results are as follows Figure 6 As shown in Table 1, strain RA44 was unable to synthesize essential amino acids due to the knockout of the phenylalanine synthesis pathway, and the growth of bacterial cells was greatly affected. After the phenylalanine synthesis pathway was reconstructed, the growth of RA46, RA48, RA50, RA52, RA53, and RA54 returned to normal. In addition, the contents of RA and 4-HPL in the phenylalanine-reconstructed strains were greatly increased. The metabolites of RA14 and RA54 were detected, and the results are as follows Figure 7 As shown, byproducts of PA-related pathways were almost eliminated.
[0047] Table 1 The contents of RA and 4-HPL in each strain
[0048]
[0049] 4. Synthesis of other aromatic compounds based on reconstruction of phenylalanine synthesis pathway
[0050] In the above experiments, we found that not only the synthesis of phenylalanine was restored, but also the content of tyrosine precursor 4-HPL was greatly increased. It is speculated that this pathway can improve the synthesis of tyrosine pathway derivatives.
[0051] We selected the active tyrosine derivatives mentioned above, tyrosol, 4-hydroxyphenyllactic acid (4-HPL), 4-hydroxymandelic acid (4-HMA) and dopamine for testing to verify the feasibility of this strategy.
[0052] Using QL11 as the starting strain, the p-coumaric acid pathway genes AtC4H and AtPAL were knocked out to obtain strain RA152.
[0053] Based on strain RA152, NuHmaS was expressed to obtain strain RA153, which can synthesize 4-hydroxymandelic acid (4-HMA); based on strain RA152, LpLDHY52A mutant was expressed to obtain strain RA154, which can synthesize 4-hydroxyphenyllactic acid (4-HPL); based on strain RA152, ARO10 was expressed to efficiently synthesize tyrosol (Saccharomyces cerevisiae itself has the ARO10 gene and can synthesize tyrosol, and the ARO10 expressed here is the second copy); based on strain RA152, BvCYP76AD5 and PpDODC were expressed to obtain strain PA46, which can synthesize dopamine.
[0054] Based on the above strains synthesizing different products, the following modifications were performed: 1) PHA2 was knocked out to obtain related defective strains as controls; 2) PHA2 was knocked out and AtPAT and PhADT were expressed to obtain arogenate pathway reconstructed strains (such as Figure 8 ).
[0055] The growth of the above strains and related products were tested, and the results were as follows Fig. 9 As shown, the content of each product was greatly increased.
[0056] It should be noted that the present invention uses QL11 as the starting strain for the purpose of illustration only, and other yeast strains can still be used to achieve the purpose of reconstructing the phenylalanine synthesis pathway of the present invention and improve the synthesis of related target products. Therefore, specific yeast strains should not be used to limit the scope of protection of the present invention.
[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for reconstructing the aromatic amino acid metabolic pathway in a microorganism, characterized in that: The method comprises the step of generating galangin acid in the microorganism, wherein the molecular formula of the galangin acid is as shown in Formula I.
2. The method according to claim 1, characterized in that By introducing a gene expression box of prephenate aminotransferase into the microbial cells, the microorganism produces arginine.
3. The method according to claim 2, characterized in that The amino acid sequence of the prephenate aminotransferase is selected from one or more combinations of SEQ ID NOs: 2, 6, 8, or isozymes thereof.
4. The method according to claim 1, characterized in that: The method also includes the steps of knocking out phenylpyruvate synthase in the microbial cell and introducing galenate dehydratase.
5. The method according to claim 4, characterized in that The amino acid sequence of the longanate dehydratase is selected from one or more combinations of SEQ ID NOs: 4 and 10, or isozymes thereof.
6. The method according to any one of claims 1 to 5, characterized in that The microorganism is yeast.
7. A bottom plate fungus, characterized in that: The aromatic amino acid metabolic pathway is reconstructed by the method according to any one of claims 1 to 6.
8. A method for constructing an engineered strain for producing a target aromatic compound, characterized in that: The method comprises the steps of introducing a synthetic pathway of the target aromatic compound into the bottom plate fungus described in claim 7.
9. The method according to claim 8, characterized in that The target aromatic compound is phenylalanine and / or tyrosine or a derivative thereof, or an intermediate in the synthesis pathway of phenylalanine and / or tyrosine or a derivative thereof.
10. The method according to claim 9, characterized in that The target aromatic compound is selected from one or more combinations of rosmarinic acid, 4-hydroxymandelic acid, 4-hydroxyphenyllactic acid, tyrosol and dopamine.