Engineering saccharomycetes with high yield of rosmarinic acid and construction method thereof

Abstract

The invention relates to a construction method of high-yield rosmarinic acid engineering saccharomycetes, which comprises the following steps of: introducing synthetic route related enzymes of rosmarinic acid into starting saccharomycetes, knocking out endogenous phenylpyruvate synthetase of a strain, and introducing pre-benzoic acid transaminase and longroot acid dehydratase into the starting saccharomycetes. According to the invention, a pre-benzoate transaminase expression cassette is introduced into yeast cells, so that the yeast can synthesize the longroot acid, synthesis routes of phenylalanine, tyrosine and derivatives thereof can be reconstructed by taking the longroot acid as a center, and meanwhile, endogenous synthesis routes of phenylalanine and tyrosine in the yeast cells are knocked out; some by-products in an endogenous way are eliminated, and the yield of the rosmarinic acid is increased. On the basis, the related enzyme combination of the rosmarinic acid synthesis route is further optimized, the content of the rosmarinic acid is increased to nearly 1g / L at most, and a road is tamped for the biosynthesis industrialization of the rosmarinic acid.

Description

Technical Field

[0001] The invention belongs to the field of metabolic engineering, and particularly relates to a high-yield rosmarinic acid engineering yeast and a construction method thereof. Background Art

[0002] Rosmarinic acid (RA) is a natural antioxidant derived from plants, mainly found in plants of the Lamiaceae and Boraginaceae families. It has multiple biological activities such as anti-inflammatory and anti-tumor effects. It is widely used in food, health products, cosmetics and other fields.

[0003] Currently, the main source of RA is to directly extract it from plants that can synthesize RA, such as sage, using the aqueous enzymatic method. However, the long growth cycle of plants and the great influence of climate limit the efficient production of RA. With the elucidation of the RA biosynthetic pathway, microbial cell factories provide a more feasible solution for the efficient production of RA.

[0004] like Figure 1 As shown in Figure 1, the precursors of RA biosynthesis in plants are aromatic amino acids derived from the shikimate pathway, namely phenylalanine and tyrosine. Phenylalanine can synthesize p-coumaric acid (HCA), which is then hydroxylated at the 3rd position to synthesize caffeic acid (CA). The 4-hydroxyphenylpyruvic acid (4-HPP) formed by tyrosine deamination 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 of RA synthesis. In addition, due to the poor substrate recognition specificity of RAS, which is responsible for RA synthesis, p-HCA-CoA and 4-HPL can also combine under the action of RAS to form structural analogs of RA. Among them, products lacking 3-OH and 3′-OH modifications can further synthesize RA under the action of P450 enzymes.

[0005] At present, there have been studies on the production of RA by fermentation using Escherichia coli and Saccharomyces cerevisiae. However, in microorganisms, phenylpyruvic acid (PPY), the precursor of phenylalanine synthesis, can be synthesized into phenyllactic acid (PA) under the catalysis of LDH, an essential enzyme in the 4-HPL synthesis pathway. PA can also be recognized by RAS and then combined with CA or p-HCA to form a byproduct with a missing 4′-OH modification on the benzene ring ( Figure 2 ).

[0006] Therefore, it is necessary to carry out necessary metabolic engineering transformation of microorganisms to increase the yield of target products and reduce the production of by-products. Summary of the invention

[0007] To solve the above problems, the present invention provides a method for constructing a high-yield rosmarinic acid engineered yeast, comprising the steps of introducing enzymes related to the synthesis pathway of rosmarinic acid into a starting yeast, and also comprising the steps of knocking out endogenous phenylpyruvate synthase in the strain, and introducing prephenate aminotransferase and roentgenate dehydratase into the starting yeast.

[0008] In a specific embodiment, the amino acid reference sequence of the phenylpyruvate synthase is shown as SEQ ID NO:1.

[0009] In a specific embodiment, the amino acid sequence of the prephenate aminotransferase is selected from SEQ ID NO: 2, 6, 8;

[0010] The amino acid sequence of the longanate dehydratase is selected from SEQ ID NO: 4, 10.

[0011] In a specific embodiment, the method further comprises the steps of knocking out key genes in the tyrosine synthesis pathway and introducing pyruvic acid dehydrogenase.

[0012] In a specific embodiment, the key gene of the tyrosine biosynthesis pathway is ARO8 and / or ARO9, and the amino acid reference sequences are shown in SEQ ID NOs: 19 and 20, respectively;

[0013] The amino acid sequence of the longanate dehydrogenase is selected from SEQ ID NO: 12, 13.

[0014] In a specific embodiment, the method further comprises the step of further enhancing the expression of the prephenate aminotransferase.

[0015] In a specific embodiment, the method further comprises the step of regulating the expression level of TYR1.

[0016] In a specific embodiment, the enzymes involved in the synthesis pathway of rosmarinic acid include lactate reductase, acetyl CoA ligase, rosmarinic acid synthase, and P450 hydroxylase and / or flavin-dependent hydroxylase.

[0017] In a specific embodiment, the amino acid sequence of the acetyl-CoA ligase is shown in SEQ ID NO: 15

[0018] In a specific embodiment, the enzymes related to the synthesis pathway of rosmarinic acid include two P450 hydroxylases, and the amino acid sequences are selected from SEQ ID NO: 16, 17, 18 and 26.

[0019] The present invention also provides a high-yield rosmarinic acid engineered yeast constructed by the method.

[0020] The present invention introduces a prephenate aminotransferase expression cassette into yeast cells, so that yeast can synthesize rosmarinic acid, and can reconstruct the synthesis pathway of phenylalanine, tyrosine and their derivatives with rosmarinic acid as the center, while knocking out the endogenous phenylalanine and tyrosine synthesis pathways in yeast cells, eliminating some by-products in the endogenous pathways, and increasing the yield of rosmarinic acid. On this basis, the present invention further optimizes the relevant enzyme combination of the rosmarinic acid synthesis pathway, and increases the content of rosmarinic acid to nearly 1g / L, which paves the way for the industrialization of the biosynthesis of rosmarinic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the RA synthesis pathway.

[0022] Figure 2 Schematic diagram of the synthesis of phenyllactic acid-derived by-products in the RA synthesis pathway.

[0023] Figure 3 This is the peak diagram for metabolite detection in RA09.

[0024] Figure 4 Schematic diagram of the metabolic pathway for reconstructing the phenylalanine metabolic pathway and eliminating byproducts.

[0025] Figure 5 The production of RA and 4-HPL, and the growth conditions of different RA-synthesizing strains.

[0026] Figure 6 Peak profiles for metabolite detection in RA14 and RA54.

[0027] Figure 7 Schematic diagram of the reconstructed metabolic pathway for the tyrosine pathway, as well as the production of RA and 4-HPL and growth conditions in different RA-synthesizing strains.

[0028] Figure 8 Schematic diagram of the metabolic pathways after the modification of the phenylalanine and tyrosine synthesis pathways; the production of RA and 4-HPL in different RA synthesis strains, and the growth conditions; and the peak diagrams of some product detections of RA69 and RA77. DETAILED DESCRIPTION

[0029] 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.

[0030] 1. Strains used

[0031] 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.

[0032] 2. Rosmarinic acid synthesis strain

[0033] Using the CRISPR-Cas9 gene editing tool, the gene expression cassettes of enzymes related to the RA synthesis pathway (flavin-dependent hydroxylases HpaB and HpaC, lactate reductase LDH, acetyl CoA ligase At4CL1, rosmarinic acid synthase MoRAS, P450 hydroxylase CbCYP98A14, amino acid sequences are shown in SEQ ID NO: 21-26, respectively) were inserted into the genome of the QL11 strain for integrated expression, and the strain RA09 was obtained, which achieved heterologous production of RA with a yield of 8.0 mg / L. A large amount of by-products accumulated in the fermentation product ( Figure 3 ).

[0034] Based on strain RA09, a P450 enzyme from Salvia miltiorrhiza (amino acid sequence as shown in SEQ ID NO: 7) was 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] To overcome this defect, we designed a new phenylalanine synthesis pathway by knocking out endogenous phenylpyruvate synthase (PHA) and introducing prephenylate aminotransferase (PAT) to convert PPA into arogenate and arogenate dehydratase (ADT) to convert arogenate into phenylalanine ( Figure 4 ).

[0036] 3. Reconstruction of aromatic amino acid metabolic pathway to efficiently synthesize RA

[0037] 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.

[0038] 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;

[0039] 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;

[0040] 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;

[0041] 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);

[0042] PhPAT from Petunia hybrida and PhADT from Petunia hybrida were transferred into RA53;

[0043] AtPAT from Arabidopsis thaliana and PhADT from Petunia hybrida were transferred into RA52.

[0044] The growth of each strain, as well as the content of RA and 4-HPL were detected. The results are as follows Figure 5 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 6 As shown, byproducts of PA-related pathways were almost eliminated.

[0045] Table 1 RA and 4-HPL content in each strain

[0046]

[0047] 4. Further optimization of RA synthesis pathway

[0048] Further optimization based on RA54.

[0049] After the reconstruction of the phenylalanine metabolic pathway, although the accumulation of PA-related by-products was avoided, the accumulation of 4-HPL, the synthetic substrate of RA, was significantly increased at the same time, which was presumably due to the imbalance of phenylalanine and tyrosine metabolic flows in the downstream branch pathway of prephenic acid.

[0050] Therefore, based on RA54, the present invention further reconstructs the tyrosine synthesis pathway, knocks out the key genes ARO8 and ARO9 of the tyrosine synthesis pathway (the amino acid sequences are shown in SEQ ID NOs: 19 and 20, respectively), introduces adenosine dehydrogenase ADH, and introduces a second copy of PAT to obtain strains RA69 and RA70 (introducing AtADH from Arabidopsis thaliana (the amino acid sequence is shown in SEQ ID NO: 12) and MtADH from Medicago truncatula (the amino acid sequence is shown in SEQ ID NO: 13)).

[0051] The results are as follows Figure 7 As shown, the accumulation of substrate 4-HPL was effectively reduced in RA69.

[0052] On the basis of RA69, the expression level of TYR1 (TYR1 is an endogenous tyrosine pathway gene of Saccharomyces cerevisiae, encoding prephenate dehydrogenase, which can convert prephenate into 4-hydroxyphenylpyruvate (4-HPP)) was regulated, and TYR1 was expressed with promoters of different strengths to construct strains RA74-77.

[0053] Among them, the promoter sequence used by RA77 is shown in SEQ ID NO:14.

[0054] The results are as follows Figure 8 As shown, the RA production in RA77 can reach 472.8 mg / L, which is significantly improved.

[0055] Further studies have found that the substrate p-HCA or CA must first be converted into the corresponding p-HCA-CoA or CA-CoA under the action of 4CL before it can bind to the other side substrate 4-HPL. When the synthesis efficiency of p-HCA-CoA or CA-CoA matches that of 4-HPL, the RA production efficiency is the highest. It is necessary to control the catalytic efficiency of 4CL to promote the synthesis of RA. On this basis, At4CL1 was replaced with At4CL2 (amino acid sequence shown in SEQ ID NO: 15) to obtain strain RA93, and the RA yield can be further increased to 595.7 mg / L.

[0056] A second copy of a P450 enzyme from Salvia miltiorrhiza (amino acid sequence as shown in SEQ ID NO: 16) was introduced to replace the original p-coumarate hydroxylase HpaBC, promoting the conversion of the intermediate product to the final product RA, thereby obtaining strain RA102. The RA yield in strain RA102 can be increased to 674.6 mg / L.

[0057] On the basis of strain RA102, P450 enzymes from different plant sources were further expressed in combination, including LeCYP98A6 from Lithospermum officinale, PcCYP98A112 and PcCYP98A113 from Salvia officinalis, etc. Among them, the RA yield in strain RA125 was significantly improved to 972.8 mg / L, and the plant P450 enzymes expressed in combination were PcCYP98A113 and LeCYP98A6 (amino acid sequences are shown in SEQ ID NOs: 17 and 18, respectively).

[0058] 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 constructing a high-yield rosmarinic acid engineered yeast, comprising the steps of introducing enzymes related to the synthesis pathway of rosmarinic acid into a starting yeast, characterized in that: The method also includes the steps of knocking out endogenous phenylpyruvate synthase of the strain, and introducing prephenate aminotransferase and pyruvate dehydratase into the starting yeast.

2. The method according to claim 1, characterized in that The amino acid reference sequence of the phenylpyruvate synthase is shown in SEQ ID NO:

1.

3. The method according to claim 1, characterized in that The amino acid sequence of the prephenate aminotransferase is selected from SEQ ID NO: 2, 6, 8; The amino acid sequence of the longanate dehydratase is selected from SEQ ID NO: 4, 10.

4. The method according to claim 1, characterized in that: It also includes the steps of knocking out key genes in the tyrosine synthesis pathway and introducing pyruvate dehydrogenase.

5. The method according to claim 4, characterized in that The key genes of the tyrosine biosynthesis pathway are ARO8 and / or ARO9, and the amino acid reference sequences are shown in SEQ ID NOs: 19 and 20, respectively; The amino acid sequence of the longanate dehydrogenase is selected from SEQ ID NO: 12, 13.

6. The method according to claim 4, characterized in that The method also includes a step of further enhancing the expression of the prephenate aminotransferase.

7. The method according to claim 6, characterized in that The method further comprises the step of regulating the expression level of TYR1.

8. The method according to any one of claims 1 to 7, characterized in that The enzymes involved in the synthesis of rosmarinic acid include lactate reductase, acetyl CoA ligase, rosmarinic acid synthase, and P450 hydroxylase and / or flavin-dependent hydroxylase.

9. The method according to claim 8, characterized in that The amino acid sequence of the acetyl-CoA ligase is shown in SEQ ID NO:

15.

10. The method according to claim 8, characterized in that The enzymes related to the synthesis pathway of rosmarinic acid include two P450 hydroxylases, and the amino acid sequences are selected from SEQ ID NOs: 16, 17, 18 and 26.

11. A high-yield rosmarinic acid engineered yeast, characterized in that: The method is constructed by any one of claims 1 to 10.

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