WO2025123425A1 - Recombinant yeast strain for synthesizing aromatic compound, and construction method for and use of recombinant yeast strain - Google Patents

Abstract

Disclosed in the present invention are a recombinant yeast strain for synthesizing an aromatic compound, and a construction method for and use of the recombinant yeast strain. According to the recombinant yeast strain for synthesizing the aromatic compound, Saccharomyces cerevisiae QL35 is used as a chassis, heterologously introduced phosphoketolase replaces endogenous non-oxidized phosphopentose pathway of Saccharomyces cerevisiae cells, a necessary substrate, namely, erythrose 4-phosphate, of the aromatic compound is synthesized, and acetyl phosphate is generated at the same time. Pyruvate kinase is further knocked out, the decomposition of phosphoenolpyruvic acid into pyruvic acid is blocked, and at the moment, the recombinant strain can only be converted into acetyl coenzyme A by means of acetyl phosphate to drive the growth of cells. Finally, a metabolic pathway is transformed into coupling product production and cell growth, and a recombinant yeast strain with high aromatic compound yield is obtained by means of adaptive evolution. The strain reduces the production cost by using glucose and an inorganic salt culture medium, and the growth-coupled recombinant yeast strain has the advantage of maintaining stable genetic character in fermentation process.

Description

A recombinant yeast strain for synthesizing aromatic compounds and its construction method and application Technical Field

The present invention relates to the technical field of synthetic biology, and in particular to a recombinant yeast strain for synthesizing aromatic compounds, and a construction method and application thereof.

Background Art

Aromatic compounds are organic compounds containing benzene rings in chemical molecules, including a series of compounds derived from functional groups such as side chain-linked alcohols and carboxyl groups. For example, protocatechuic acid (Pca) and coumaric acid (Cou) not only have antioxidant, antibacterial, and detoxifying effects, but also have wide applications in material synthesis, feed production, food preservation, and other fields. Among them, protocatechuic acid can be used as an intermediate product to synthesize important organic chemical raw materials such as catechol, muconic acid, and adipic acid; coumaric acid has strong conductivity and can be used in liquid crystal photosensitive materials. In addition, coumaric acid is also widely used in agriculture as a plant growth promoter and long-acting fungicide. However, the extraction of aromatic compounds from natural resources has problems such as low production efficiency, cumbersome separation and purification steps, and inability to prepare in large quantities, which restrict the development of related industries.

In recent years, the production of aromatic compounds by microbial fermentation has attracted widespread attention due to its relatively low cost, strong controllability, and no restrictions on the production environment. Although the engineered strains currently used in fermentation processes can produce higher concentrations of aromatic compounds, the yield of aromatic compounds needs to be further improved. In addition, some industrial strains have unstable genetic traits due to factors such as plasmid expression or substrate feedback inhibition. At the same time, the fermentation process requires the addition of additional aromatic amino acids or gene expression inducers to the culture medium, which increases the cost of industrial production.

Summary of the invention

Based on this, the purpose of the present invention is to provide a recombinant yeast strain for synthesizing aromatic compounds and its construction method and application, aiming to reduce the production cost of the existing engineered strains for synthesizing aromatic compounds by fermentation and further improve the quality of the aromatic compounds. further increase the production of these compounds.

The technical solution of the present invention is as follows:

In a first aspect of the present invention, a recombinant yeast strain for synthesizing aromatic compounds is provided, wherein the recombinant yeast strain for synthesizing aromatic compounds is based on Saccharomyces cerevisiae QL35 and undergoes one of the following modifications (a) to (f):

(a) Overexpressed gene 3DSD;

(b) Overexpression of the 3DSD gene and knockout of the TKL1 and TKL2 genes;

(c) overexpressing the 3DSD gene and knocking out the TKL1, TKL2, and TAL1 genes; or, overexpressing the 3DSD gene and knocking out the TKL1, TKL2, TAL1, and NQM1 genes;

(d) overexpression of the gene 3DSD and knockout of genes TKL1, TKL2, TAL1, NQM1, PYK1, and PYK2;

(e) overexpressing the 3DSD gene, knocking out the TKL1, TKL2, TAL1, NQM1, PYK1 and PYK2 genes, and overexpressing at least one of the ICL1 and MLS1 genes and PCK1 and UdhA genes in the glyoxylate cycle pathway;

(f) Overexpressing the 3DSD gene, knocking out the TKL1, TKL2, TAL1, NQM1, PYK1 and PYK2 genes, and overexpressing the ICL1 and MLS1 genes on the glyoxylate cycle pathway and at least one of the PCK1 and UdhA genes; and then performing adaptive evolution.

Optionally, the recombinant yeast strain for synthesizing aromatic compounds is based on Saccharomyces cerevisiae QL35 and is further transformed as follows after the transformation and evolution of (f):

Knockout of genes AtPAL2, AtC4H and AtATR2, and overexpression of gene 3DSD; Knockout of gene FjTAL, and overexpression of gene 3DSD; or,

The gene 3DSD was knocked out, and the genes AtATR2, AtC4H and AtATR2 were overexpressed.

Optionally, the gene 3DSD is a codon-optimized gene 3DSD, the nucleotide sequence of the codon-optimized gene 3DSD is as shown in SEQ ID NO: 1, or the nucleotide sequence of the codon-optimized gene 3DSD has at least 70% homology with the nucleotide sequence shown in SEQ ID NO: 1.

Optionally, the gene UdhA is a codon-optimized gene UdhA, the nucleotide sequence of the codon-optimized gene UdhA is as shown in SEQ ID NO: 2, or the nucleotide sequence of the codon-optimized gene UdhA has at least 70% homology with the nucleotide sequence shown in SEQ ID NO: 2.

The second aspect of the present invention provides a method for constructing a recombinant yeast strain for synthesizing aromatic compounds, wherein the construction method comprises:

Using Saccharomyces cerevisiae QL35 as the chassis, the strain was gene-edited using the CRISPR/Cas9 gene editing system according to one of the following (a) to (f):

(a) Overexpressed gene 3DSD;

(b) Overexpression of the 3DSD gene and knockout of the TKL1 and TKL2 genes;

(c) overexpressing the 3DSD gene and knocking out the TKL1, TKL2, and TAL1 genes; or, overexpressing the 3DSD gene and knocking out the TKL1, TKL2, TAL1, and NQM1 genes;

(d) overexpression of the gene 3DSD and knockout of genes TKL1, TKL2, TAL1, NQM1, PYK1, and PYK2;

(e) overexpressing the 3DSD gene, knocking out the TKL1, TKL2, TAL1, NQM1, PYK1 and PYK2 genes, and overexpressing at least one of the ICL1 and MLS1 genes and PCK1 and UdhA genes in the glyoxylate cycle pathway;

(f) Overexpressing the 3DSD gene, knocking out the TKL1, TKL2, TAL1, NQM1, PYK1 and PYK2 genes, and overexpressing the ICL1 and MLS1 genes on the glyoxylate cycle pathway and at least one of the PCK1 and UdhA genes; and then performing adaptive evolution.

Optionally, the construction method further includes:

After the transformation and evolution of (f), the following transformation is performed:

Knockout of genes AtPAL2, AtC4H and AtATR2, and overexpression of gene 3DSD; Knockout of gene FjTAL, and overexpression of gene 3DSD; or,

The gene 3DSD was knocked out, and the genes AtATR2, AtC4H and AtATR2 were overexpressed.

The third aspect of the present invention provides a recombinant yeast strain for synthesizing aromatic compounds as described above. And/or use of the recombinant yeast strain for synthesizing aromatic compounds obtained by the construction method as described above in the synthesis of aromatic compounds.

A fourth aspect of the present invention provides a method for synthesizing an aromatic compound, wherein the recombinant yeast strain for synthesizing an aromatic compound as described above and/or the recombinant yeast strain for synthesizing an aromatic compound obtained by the construction method of the present invention as described above is fermented to synthesize the aromatic compound.

Optionally, the aroma compound comprises protocatechuic acid and/or p-coumaric acid.

Optionally, the culture medium used for the fermentation includes glucose and/or ethanol.

Beneficial effects: The present invention uses a metabolic pathway design and transformation strategy to transform Saccharomyces cerevisiae QL35 as a chassis to obtain a recombinant yeast strain that overexpresses the gene 3DSD, which can synthesize aromatic compounds. Then, based on this, the metabolic pathway is transformed to obtain a new pathway for synthesizing erythrose-4-phosphate in the recombinant yeast strain, which blocks the phosphoenolpyruvate decomposition pathway, strengthens the glyoxylate cycle pathway, and indirectly activates the tricarboxylic acid cycle to supply the basic needs of strain growth; at the same time, it reduces unnecessary carbon loss and energy consumption, and drives carbon flow to the biosynthesis of aromatic compounds. In order to maintain growth, the recombinant yeast strain in the present invention must synthesize sufficient acetyl phosphate and convert it into acetyl coenzyme A for cell use. In this process, a large amount of aromatic compound synthesis substrates are produced at the same time; in order to relieve substrate feedback inhibition, the recombinant yeast strain drives the aromatic compound synthesis pathway to achieve high yield, forming a yeast strain growth and product synthesis coupling. The recombinant yeast strain provided by the present invention can synthesize aromatic compounds with high yield using glucose as a carbon source, and at the same time, it is not necessary to add additional aromatic amino acids or gene expression inducers in the culture medium to further reduce costs, and the growth of coupled strains during long-term fermentation can maintain stable genetic traits.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the transformation pedigree of the recombinant yeast strains for synthesizing aromatic compounds in an embodiment of the present invention.

FIG. 2 is a schematic diagram of the metabolic pathway of the recombinant yeast strain for synthesizing aromatic compounds in an embodiment of the present invention.

Figure 3 is a result diagram of evaluating the ability of strain Ar01 to synthesize protocatechuic acid and p-coumaric acid in Example 1 of the present invention, wherein A is a statistical result diagram of protocatechuic acid and p-coumaric acid yields and growth OD values, and B is a peak diagram of liquid chromatography analysis of protocatechuic acid and p-coumaric acid standards, and extraction and purification samples of fermentation broth of Saccharomyces cerevisiae QL35 and strain Ar01.

FIG4 is a graph showing the statistical results of protocatechuic acid and p-coumaric acid production and growth OD values of strains Ar02, Ar03, and Ar04 in Example 2 of the present invention.

5 is a graph showing the results of evaluating the ability of strains Ar05, Ar06 and Ar07 to synthesize protocatechuic acid and p-coumaric acid in Example 2 of the present invention, wherein A is a graph showing the yield of protocatechuic acid and p-coumaric acid, and B is a graph showing the growth curve results.

In FIG. 6 , A is a statistical result diagram of the protocatechuic acid and p-coumaric acid yields and growth OD values of strain Ar08 in Example 3 of the present invention in different carbon source culture media, and B is a growth curve result diagram of strains Ar08 and Ar04.

7 is a graph showing the results of evaluating the ability of the evolved strains Evo1, Evo2, and Evo3 to synthesize protocatechuic acid and p-coumaric acid in Example 4 of the present invention, wherein A is a growth curve graph and B is a graph showing the statistical results of protocatechuic acid and p-coumaric acid production.

8 is a graph showing the results of evaluating the ability of strains ME01 and ME02 to synthesize protocatechuic acid and p-coumaric acid in Example 5 of the present invention, wherein A is a graph showing the statistical results of protocatechuic acid and p-coumaric acid production and growth OD values, and B is a graph showing the growth curve results.

FIG. 9 is a graph showing the statistical results of p-coumaric acid production and growth OD value of strain ME03 in Example 6 of the present invention.

DETAILED DESCRIPTION

The present invention provides a recombinant yeast strain for synthesizing aromatic compounds and a preparation method and application thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

In the present invention, the English abbreviations in normal form represent enzymes or proteins, and the English abbreviations in italics represent genes. For example, TKL1 represents transketolase, and TKL1 represents a gene encoding transketolase (i.e., a transketolase gene);

NQM1 means transaldolase, and NQM1 means a gene encoding transaldolase (ie, a transaldolase gene).

The embodiment of the present invention provides a recombinant yeast strain for synthesizing aromatic compounds, wherein the recombinant yeast strain for synthesizing aromatic compounds is based on Saccharomyces cerevisiae QL35, and the strain QL35 overexpresses a codon-optimized phosphoketolase (BbXFPK) gene from Bifidobacterium (i.e., gene BbXFPK, whose Genbank accession number is AY518216), and performs one of the following modifications (a) to (f):

(a) Overexpression of the gene 3DSD (i.e., protocatechuate synthase gene);

(b) Overexpression of the 3DSD gene and knockout of the TKL1 and TKL2 genes (TKL1 and TKL2 are transketolase genes);

(c) overexpressing the 3DSD gene and knocking out the TKL1, TKL2 and TAL1 genes (TAL1 is a transaldolase gene); or, overexpressing the 3DSD gene and knocking out the TKL1, TKL2, TAL1 and NQM1 genes (TAL1 and NQM1 are transaldolase genes);

(d) overexpression of the gene 3DSD, and knockout of the genes TKL1, TKL2, TAL1, NQM1, PYK1, and PYK2 (genes PYK1 and PYK2 are pyruvate kinase genes);

(e) overexpressing the gene 3DSD, knocking out the genes TKL1, TKL2, TAL1, NQM1, PYK1 and PYK2, and overexpressing at least one of the genes ICL1 and MLS1 (genes ICL1 and MLS1 are isocitrate lyase gene and malate synthase gene, respectively) and genes PCK1 and UdhA (genes PCK1 and UdhA are phosphoenolpyruvate carboxykinase gene and transhydrogenase gene, respectively) in the glyoxylate cycle pathway;

(f) Overexpressing the 3DSD gene, knocking out the TKL1, TKL2, TAL1, NQM1, PYK1 and PYK2 genes, and overexpressing at least one of the genes PCK1 and UdhA on the glyoxylate cycle pathway; and then performing adaptive evolution.

The embodiment of the present invention uses a metabolic pathway design and transformation strategy to transform Saccharomyces cerevisiae QL35 as a chassis to obtain a recombinant yeast strain that overexpresses 3DSD, which can synthesize aromatic compounds. Then, based on this, the metabolic pathway is transformed to obtain a recombinant yeast strain that reshapes the new pathway for Saccharomyces cerevisiae to synthesize erythrose 4-phosphate (E4P), blocks the phosphoenol pyruvate (PEP) decomposition pathway, strengthens the glyoxylate cycle pathway, and indirectly activates the tricarboxylic acid cycle to supply the basic needs of strain growth; at the same time, it reduces unnecessary carbon loss and energy consumption. The recombinant yeast strain provided by the present invention can synthesize aromatic compounds with high yield using glucose as a carbon source, and does not need to add additional aromatic amino acids and gene expression inducers to the culture medium, which can further reduce costs. The details are as follows:

As shown in Figure 1, the recombinant yeast strain obtained by transformation according to (a) is recorded as strain Ar01. In the present invention, by overexpressing 3DSD, strain Ar01 capable of simultaneously synthesizing protocatechuic acid and p-coumaric acid was constructed. After fermentation, the yields of protocatechuic acid and p-coumaric acid of strain Ar01 were 421.3 mg/L and 120.8 mg/L, respectively.

As shown in FIG1 , according to step (b), based on strain Ar01, genes TKL1 and TKL2 (Genbank accession numbers are 856188 and 852414, respectively) were knocked out, and the resulting recombinant yeast strain was designated as strain Ar02.

As shown in FIG. 1 , in one modification method according to (c), based on strain Ar02, gene TAL1 (Genbank accession number: 851068) was knocked out, and the resulting recombinant yeast strain was recorded as strain Ar03.

As shown in FIG1 , in another modification method according to (c), on the basis of strain Ar03, the gene NQM1 (Genbank accession number is 852934) is knocked out, and the resulting recombinant yeast strain is recorded as strain Ar04. It can also be said that strain Ar04 is obtained by knocking out genes TKL1, TKL2, TAL1 and NQM1 on the basis of strain Ar01. Strain Ar04 can only synthesize E4P, an essential substrate for aromatic compound metabolism, by cleaving fructose-6-phosphate (F6P) through the BbXFPK function derived from Bifidobacterium breve overexpressed in the chassis strain QL35, that is, strain Ar04 can only synthesize E4P, an essential substrate for aromatic compound metabolism, through the BbXFPK pathway, but not through the non-oxidative pentose phosphate pathway. Specifically, as shown in FIG2 , wild-type Saccharomyces cerevisiae needs to undergo multiple steps of reaction in the non-oxidative pentose phosphate pathway to obtain E4P, while the BbXFPK pathway only requires F6P to undergo a one-step reaction to obtain E4P. It can be seen that the strain Ar04 obtained by the transformation according to method (c) can shorten the steps of biosynthesis of aromatic compounds and reduce carbon loss.

In addition, as shown in Figure 2, the acetyl phosphate (AcP) product catalyzed by BbXFPK can be converted into acetyl coenzyme A (AcCoA) by phosphotransacetylase (CkPTA) from Clostridium kluyveri. Acetyl coenzyme A can enter the glyoxylate cycle to provide the substrate required for cell growth, thereby achieving the purpose of coupling the synthesis of aromatic compounds and strain growth.

According to (d), based on the strain Ar04, the genes PYK1 and PYK2 were knocked out, and the recombinant yeast strain obtained was able to block the decomposition of PEP into pyruvate (Pyr), thereby abolishing the competitive metabolic pathway for PEP synthesis.

As shown in Figure 1, in one method of transformation according to (e), the genes PYK1 and PYK2 are knocked out on the basis of strain Ar04, and the genes ICL1 and MLS1 and genes PCK1 and UdhA on the glyoxylate cycle pathway are overexpressed to obtain a recombinant yeast strain for aromatic compound synthesis and growth coupling, which is recorded as strain Ar08. In an embodiment of the present invention, as shown in Figure 2, by knocking out the genes PYK1 and PYK2 (Genbank accession numbers are 851193 and 854529, respectively) in strain Ar04, PEP cannot be catalyzed to generate pyruvate (Pyr), blocking the decomposition of PEP into pyruvate, that is, abolishing the competitive metabolic pathway for PEP synthesis. At the same time, the isocitrate lyase gene ICL1 (Genbank accession number 856794) and malate synthase gene MLS1 (Genbank accession number 855606) on the endogenous glyoxylate cycle pathway of yeast cells were overexpressed to increase the expression levels of isocitrate lyase and malate synthase, strengthen the glyoxylate cycle pathway, and the intermediates in the pathway were transported to the mitochondria through transporters to enter the tricarboxylic acid (TCA) cycle. The gene PCK1 (Genbank accession number 853972) was further overexpressed to increase the expression level of phosphoenolpyruvate carboxykinase (PCK1), recycle the excess oxaloacetate in the cytoplasm, convert it into PEP, and relieve substrate feedback inhibition. The gene UdhA from Escherichia coli (E. coli) was overexpressed to increase the expression level of transhydrogenase (UdhA), dynamically regulate the balance of NADPH and NADH in the recombinant yeast cells, and then construct strain Ar08.

As shown in Figure 1, the recombinant yeast strain obtained by transformation and adaptive evolution according to (f) is recorded as strain Evo. Based on strain Ar08, the present invention improves the growth ability of the strain and increases the yield of aromatic compounds synthesized by the strain through adaptive laboratory evolution (Adaptive laboratory evolution, referred to as adaptive evolution), thereby obtaining strain Evo (specifically Evo1, Evo2 and Evo3). The evolved strains Evo1, Evo2 and Evo3 undergo a mutation that inactivates hexokinase 2 (Hexokinase 2, HXK2, the Genbank accession number of its gene HXK2 is 852639, and its nucleotide sequence is shown in SEQ ID NO: 3), and lose the phosphorylation function of glucose, thereby eliminating the glucose effect in the growth process of brewer's yeast. Compared with strain Ar08, it has stronger growth ability and increased aromatic compound production. Evo1 can be a frameshift mutation formed by deleting the 24th base "A" of gene HXK2. Evo2 can be a gene After the 1185th base of HXK2, 20 bases (sequence: GTGCTAGAGCTGCTAGATTG) are inserted to form a frameshift mutation. Evo3 may be the 82nd base of gene HXK2, which mutates from "G" to "T" to obtain the stop codon TAG.

In some embodiments, the Evo2 strain is obtained by using Saccharomyces cerevisiae QL35 as the chassis and undergoing the transformation and evolution of (f), wherein the recombinant yeast strain is further transformed as follows on the basis of the Evo2 strain:

Knock out the genes AtPAL2 (i.e., phenylalanine ammonia lyase gene), AtC4H (i.e., cinnamate 4-hydroxylase gene), and AtATR2 (i.e., NADP ⁺ -cytochrome P450 reductase gene), and simultaneously overexpress the gene 3DSD, i.e., integrate the 3DSD gene at the site of p-coumaric acid synthesis pathway 1; then, knock out the gene FjTAL (i.e., tyrosine ammonia lyase gene), and simultaneously overexpress the gene 3DSD, i.e., integrate the 3DSD gene at the site of p-coumaric acid synthesis pathway 2; or,

The 3DSD gene was knocked out and the genes AtPAL2, AtC4H and AtATR2 were overexpressed.

Through the transformation of this embodiment, a strain producing a single aromatic compound can be constructed. Based on the Evo2 strain obtained by transformation and evolution (f), the genes AtPAL2, AtC4H and AtATR2 are knocked out, and the gene 3DSD is overexpressed at the same time; then the gene FjTAL is knocked out, and the gene 3DSD is overexpressed to construct a strain that only produces protocatechuic acid. Based on the Evo2 strain obtained by transformation and evolution (f), the gene 3DSD is knocked out, and the genes AtPAL2, AtC4H and AtATR2 are overexpressed to construct a strain that only produces p-coumaric acid.

As shown in FIG1 , after the transformation of this embodiment, the recombinant yeast strain obtained is recorded as strain ME02 or ME03. Specifically, as shown in FIG2 , on the basis of strain Evo (specifically Evo2), AtPAL2, AtC4H and AtATR2 are knocked out, and gene 3DSD is overexpressed at the same time; gene FjTAL is knocked out, and gene 3DSD is overexpressed at the same time, that is, p-coumaric acid synthesis pathway 1 and 2 are knocked out to obtain strain ME02 (it can also be understood as knocking out p-coumaric acid synthesis pathway 1 to obtain strain ME01, and on the basis of strain ME01, p-coumaric acid synthesis pathway 2 is knocked out to obtain strain ME02), which can only synthesize protocatechuic acid, and its yield reaches 2.02 g/L in shake flask fermentation (4.8 times the protocatechuic acid yield of strain Ar01), and the protocatechuic acid yield is increased to 20.5 g/L in parallel bioreactor fermentation. Based on strain Evo2, the gene 3DSD was knocked out, that is, the protocatechuic acid synthesis pathway was knocked out, and the genes AtPAL2, AtC4H and AtATR2 were overexpressed to obtain strain ME03, which can only synthesize p-coumaric acid, and its yield reached 690 mg/L in shake flask fermentation (5.7 times the p-coumaric acid yield of strain Ar01). The recombinant yeast strain provided in the invention uses glucose as a carbon source to reduce production costs and can maintain the stability of genetic shape during the fermentation process.

In some embodiments, the gene 3DSD is a codon-optimized gene 3DSD, the nucleotide sequence of the codon-optimized gene 3DSD is as shown in SEQ ID NO: 1, or the nucleotide sequence of the codon-optimized gene 3DSD has at least 70% homology with the nucleotide sequence shown in SEQ ID NO: 1.

In some embodiments, the gene UdhA is a codon-optimized gene UdhA, the nucleotide sequence of the codon-optimized gene UdhA is as shown in SEQ ID NO: 2 (the Genbank accession number of wild-type UdhA is 948461), or the nucleotide sequence of the codon-optimized gene UdhA has at least 70% homology with the nucleotide sequence shown in SEQ ID NO: 2.

The present invention also provides a method for constructing a recombinant yeast strain, wherein the method comprises:

Using Saccharomyces cerevisiae QL35 as the chassis, the chassis strain was gene edited using the CRISPR/Cas9 gene editing system according to one of the following (a)-(g):

(a) Overexpressed gene 3DSD;

(b) Overexpression of the 3DSD gene and knockout of the TKL1 and TKL2 genes;

(c) overexpressing the 3DSD gene and knocking out the TKL1, TKL2, and TAL1 genes; or, overexpressing the 3DSD gene and knocking out the TKL1, TKL2, TAL1, and NQM1 genes;

(d) overexpression of the gene 3DSD and knockout of genes TKL1, TKL2, TAL1, NQM1, PYK1, and PYK2;

(e) overexpressing the gene 3DSD, knocking out the genes TKL1, TKL2, TAL1, NQM1, PYK1 and PYK2, and overexpressing at least one of the genes ICL1 and MLS1, PCK1 and UdhA;

(f) overexpressing the gene 3DSD, knocking out the genes TKL1, TKL2, TAL1, NQM1, PYK1 and PYK2, and overexpressing at least one of the genes ICL1 and MLS1, PCK1 and UdhA; and then performing adaptive evolution;

(g) Overexpression of 3DSD gene, knockout of TKL1, TKL2, TAL1, NQM1, PYK1 and PYK2, and overexpress genes ICL1 and MLS1, PCK1 and UdhA; then perform adaptive evolution; knock out genes AtPAL2, AtC4H and AtATR2 in the evolved strain, and overexpress gene 3DSD; knock out gene FjTAL, and overexpress gene 3DSD; or,

The 3DSD gene was knocked out in the evolved strain, while the AtATR2, AtC4H and AtATR2 genes were overexpressed.

The embodiment of the present invention uses a metabolic pathway design and transformation strategy to transform Saccharomyces cerevisiae QL35 as a chassis to obtain a recombinant yeast strain that overexpresses 3DSD, which can synthesize aromatic compounds. Then, based on this, the metabolic pathway is transformed to obtain a recombinant yeast strain that reshapes a new pathway for synthesizing E4P, blocks the PEP decomposition pathway, strengthens the glyoxylate cycle pathway, and indirectly activates the tricarboxylic acid cycle to supply the basic needs of strain growth; at the same time, it reduces unnecessary carbon loss and energy consumption, and drives carbon flow to the synthesis of aromatic compounds. The recombinant yeast strain provided by the present invention can synthesize higher yields of aromatic compounds (such as protocatechuic acid and coumaric acid) using glucose as a carbon source, and at the same time, there is no need to add additional aromatic amino acids (as substrates) or gene expression inducers to the culture medium, which can further reduce costs. In addition, the growth-coupled recombinant yeast strain can maintain genetic trait stability during the fermentation process.

Of course, the gene knockout method used in this embodiment can also be other technologies that can achieve the same effect, such as gene mutation, RNA interference technology, low-intensity promoter replacement, etc.

The embodiment of the present invention also provides the use of the recombinant yeast strain for synthesizing aromatic compounds as described above in the embodiment of the present invention in synthesizing aromatic compounds.

The embodiment of the present invention also provides the use of the recombinant yeast strain for synthesizing aromatic compounds obtained by the construction method described above in the embodiment of the present invention in synthesizing aromatic compounds.

The embodiments of the present invention also provide the recombinant yeast strain for synthesizing aromatic compounds as described above in the embodiments of the present invention and the recombinant yeast strain for synthesizing aromatic compounds obtained by the construction method described above in the embodiments of the present invention for use in synthesizing aromatic compounds.

The recombinant yeast strain for synthesizing aroma compounds in the embodiment of the present invention can produce aroma compounds during the fermentation process.

The embodiment of the present invention also provides a method for synthesizing an aromatic compound, which comprises the steps of:

The recombinant yeast strain synthesizing aromatic compounds as described above in the embodiment of the present invention is fermented, or The recombinant yeast strain for synthesizing aromatic compounds obtained by the construction method described above in the embodiment of the present invention is fermented, or the recombinant yeast strain for synthesizing aromatic compounds described above in the embodiment of the present invention and the recombinant yeast strain for synthesizing aromatic compounds obtained by the construction method described above in the embodiment of the present invention are fermented to synthesize the aromatic compounds. The embodiment of the present invention provides a simple method for synthesizing aromatic compounds, and the aromatic compounds can be synthesized by fermenting the recombinant yeast strain for synthesizing aromatic compounds described above in the embodiment of the present invention.

In some embodiments, the aromatic compounds include but are not limited to protocatechuic acid and/or p-coumaric acid. In other words, among the recombinant yeast strains obtained by modifying different metabolic pathways using Saccharomyces cerevisiae QL35 as the chassis, some can synthesize protocatechuic acid and p-coumaric acid at the same time, some can synthesize only protocatechuic acid, some can synthesize only p-coumaric acid, and some can synthesize other aromatic compounds.

In some embodiments, the culture medium used in the fermentation includes glucose and/or ethanol (glucose and/or ethanol as carbon source). In some specific embodiments, the culture medium includes but is not limited to a rich medium (YP) containing glucose and/or ethanol, a synthetic complete medium (SC) containing glucose and/or ethanol, an inorganic salt medium (Delft) containing glucose and/or ethanol, etc. The recombinant yeast strain provided by the present invention uses glucose or ethanol as a carbon source, and has a low cost.

The following describes it in detail through specific embodiments.

The genotype of Saccharomyces cerevisiae QL35 used in the following examples is:

MATa ura3-52 can1Δ::CAS9-natNT2 TRP1 LEU2 HIS3 gpp1ΔXII-2::(GPM1p-AtPAL2-FBA1t)+(TDH3p-AtC4H-CYC1t)+(tHXT7p-AtATR2-pYX212t)+(PGK1p-CYB5-ADH1t)X-3::(TPI1p-EcaroL-pYX212t)+(ADH1t-ARO7 ^G141S -TEF1p)+(PGK1p-ARO4 ^K229L -CYC1t)X-4::(CYC1t-ARO1-TPI1p)+(TDH3p-ARO2-ADH1t)+(TDH2t-ARO3 ^K222L -TEF1p)X-2::(GPM1p-PHA2-CYC1t)XI-3::(TEF1p-FjTAL-TDH2t) + (FBA1t-MtPDH1-TDH3p) The reference is (Liu Q, Yu T, Li X, et al. Rewiring carbon metabolism in yeast for high level production of aromatic chemicals. Nature communications, 2019, 10.1:1-13.).

The recombinant yeast strains for synthesizing aromatic compounds in the following examples were all constructed based on the CRISPR/Cas9 gene editing technology, with reference to the literature (Mans R, van Rossum HM, Wijsman M, et al. CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. FEMS Yeast Research, 2015, 15.1).

Unless otherwise specified in the following examples, all materials used are commercially available products.

Example 1

(1) Using Saccharomyces cerevisiae QL35 as the chassis, overexpressing the codon-optimized gene 3DSD (SEQ ID NO: 1), a recombinant yeast strain capable of simultaneously synthesizing protocatechuic acid and p-coumaric acid was constructed, namely strain Ar01. The specific steps are as follows:

Reference (Mikkelsen MD, Buron LD, Salomonsen B, et al. Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in a versatile yeast expression platform. Metabolic engineering, 2012, 14.2: 104-111.) Design of Saccharomyces cerevisiae XI-1 site-specific recognition gRNA primers (GAAAGATAAATGATC GCAATGCGATGTTAGTTTAG GTTTTAGAGCTAGAAATAGCAAGT, underlined to specifically recognize the site of Saccharomyces cerevisiae chromosome), the primers are used to amplify 2 μm fragments. The reaction system is the reaction system 1 shown in Table 1 below.

Table 1. Reaction system 1

The pROS10 plasmid backbone was amplified by PCR using primer F and primer R (primer F and primer R were both GATCATTTATCTTTCACTGCGGAGAAG). The reaction system was reaction system 2 shown in Table 2 below.

Table 2. Reaction system 2

The 2 μm fragments amplified by PCR and purified by the product and the pROS10 plasmid backbone were used to construct the pROS10 guide RNA plasmid specified in the present invention (which can be recorded as pROS10-xxx, xxx represents the recognition site), for example, the XI-1 site is the recognition site. Taking the XI-1 site as an example, the wild-type Saccharomyces cerevisiae genome is used as a PCR amplification template, and the primers XI-1 UP F (ATTTGTGTGAAGGAATAGTGACG) and XI-1 UP-(link xi-1dw) R (GATTTGCCAATGCCAAGAAACAATGGGCTTGGTATTCCG) are used to amplify the XI-1 upstream repair fragment; the primers XI-1DW (link xi-1 up) F (CGGAATACCAAGCCCATTGTTTCTTGGCATTGGCAAATCTCT) and XI-1 DW R (AAGAGCCGAGTCCCCATCAG) are used to amplify the XI-1 downstream repair fragment. Add 50 ng of each upstream and downstream fragments and the above-mentioned primer XI-1 UP F and primer XI-1 DW R to perform upstream and downstream fragment fusion PCR (see Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in a versatile yeast expression platform).

The overexpression gene module fusion is an upstream repair fragment, a promoter (a universal promoter is used, and this embodiment uses TDH3p, CCW12p, TEF1p, tHXK7p and TDH1p), a gene, and a terminator (a universal terminator is used, and this embodiment uses ADH1t, CYC1t, TDH2t, PYK1t, DIT1t, FBA1t and TPS1t) The complete expression frame of the downstream repair fragment was added to the PCR reaction system at a molar concentration ratio of 1:3:5:3:1. No primers were added to the PCR reaction system, and the first round of PCR was performed according to the procedure shown in Table 3 below. The first round of PCR product was used as a template, primers XI-1 UP F and primers XI-1 DW R were added, and the second round of PCR amplification was performed according to the following reaction procedure.

Table 3. PCR program

The repair fragment (upstream and downstream fragments fusion) or gene overexpression module (upstream fragment-promoter-gene-terminator-downstream fragment fusion) after purification of PCR amplification product and pROS10 guide RNA plasmid are operated according to the following Saccharomyces cerevisiae chemical reagent transformation method (refer to the literature Gietz RD, Schiestl RH. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature protocols, 2007, 2.1: 31-34.) to obtain a recombinant yeast strain. The specific steps are as follows: Pick fresh monoclonal yeast to 2mL YPD medium (10g/L yeast extract, 20g/L peptone, 20g/L glucose), and culture overnight at 30°C, 200rpm in a shaking table. Take an appropriate amount of seed bacterial solution and transfer it to 20mL YPD medium, so that the initial dilution bacterial solution concentration reaches OD ₆₀₀ =0.2, and culture it in a shaking table at 30°C, 200rpm until the bacterial solution concentration reaches OD ₆₀₀ =0.6. The culture medium was removed by centrifugation, and the cell pellet was resuspended with 1 mL of sterile deionized water. After centrifugation at 3000 g for 1 minute, the supernatant was removed and then 1 mL of 0.1 M lithium acetate was added to resuspend the cell pellet. After centrifugation at _{3000 g for 1 minute, the supernatant was removed to obtain yeast competent cells. The yeast competent cells were transformed into recombinant yeast by the Saccharomyces cerevisiae chemical reagent transformation method, and each transformation system was divided into a bacterial amount of OD 600} = 1 according to the transformation requirements to obtain strain Ar01 (same The corresponding recombinant yeast strains were obtained by referring to the above method in the following examples). The yeast chemical reagent transformation system is shown in Table 4 below.

Table 4. Yeast chemical reagent transformation system

(2) In order to evaluate the ability of the modified strains to synthesize protocatechuic acid and p-coumaric acid, Saccharomyces cerevisiae QL35 and strain Ar01 were fermented in shake flasks for evaluation. The specific steps of the evaluation method are as follows: a single clone was picked from the YPD plate and cultured in 2 mL DelftD (2%) medium (yeast inorganic salt medium with 2% glucose as the carbon source, references see Jensen NB, Strucko T, Kildegaard KR, et al. EasyClone: method for iterative chromosomal integration of multiple genes Saccharomyces cerevisiae. FEMS yeast research, 2014, 14.2: 238-248) to culture the seed bacterial liquid. Take an appropriate amount of seed bacterial solution and transfer it to 20 mL DelftD (2%) medium, so that the initial inoculation bacterial solution _OD600 = 0.2, 30°C, 200rpm shake flask culture, after 4 days of fermentation, take 0.5 mL of bacterial solution and add an equal volume of 100% ethanol, shake and mix thoroughly in an oscillator at 2000rpm for 10min, then centrifuge at 13000rpm for 10min, take the supernatant and filter it through a 0.22μm filter membrane, use liquid mass spectrometer to test, and perform quantitative analysis.

The quantitative analysis method is as follows: the liquid phase mass spectrometer uses a Phenomenex Kinetex C18 column (100×2.1 mm, particle size 2.6 μm) chromatographic column, the column oven is 30° C., and the injection volume is 2 μL. Mobile phase A: deionized water containing 0.1% formic acid, mobile phase B: acetonitrile containing 0.1% formic acid, and the mobile phase flow rate is 0.2 mL/min. The mass spectrometer electrospray voltage is set to 3.0 kV, the carrier gas is N ₂ (purity greater than 99%), the flow rate is 120 L/h, and the drying gas temperature is 400° C. The target product is detected in ESI negative ion mode (the charge-to-mass ratio of protocatechuic acid is [MH] ^— =153.1, and the charge-to-mass ratio of p-coumaric acid is [MH] ^— =163.1).

The statistical results of the yields of the two aromatic compounds are shown in Figure 3. The fermentation product of strain Ar01 is Yuanyuan catechu. The quantitative yields of protocatechuic acid and p-coumaric acid were 421.3 mg and 120.8 mg per liter of fermentation broth, respectively, recorded as 421.3 mg/L and 120.8 mg/L (the mg/L in the yield evaluation results of the present invention refers to how many mg of protocatechuic acid or p-coumaric acid is contained in each liter of fermentation broth).

In the following Examples 2-3, referring to the method of Example 1, the primers shown in Table 5 were used to construct the pROS10 guide RNA plasmid for knocking out the corresponding gene.

Table 5. Primer table

The underline indicates a 20-base region specifically recognized in the yeast genome.

Example 2 Knockout of the non-oxidative pentose phosphate pathway and remodeling of the E4P metabolic pathway

Referring to Example 1, the primers in Table 5 were used to construct pROS10 guide RNA plasmids for knocking out genes TKL1, TKL2, TAL1 and NQM1 in the non-oxidative pentose phosphate pathway. Using the Ar01 strain in Example 1 as the starting strain, according to the method in Example 1, the genes TKL1 and TKL2 were knocked out to construct strain Ar02; the genes TKL1, TKL2 and TAL1 were knocked out to construct strain Ar03; the genes TKL1, TKL2, TAL1 and NQM1, and constructed strain Ar04.

Strain Ar04 synthesizes E4P using the BbXFPK pathway instead of the non-oxidative pentose phosphate pathway, reducing the ineffective cycle of E4P. The reconstructed metabolic pathway can directly generate E4P from F6P, synthesize 3-deoxy-D-arabino-heptulosonate 7-phosphate (3-Deoxy-D-arabino-heptulosonate 7-phosphate, DAHP) and enter the shikimic acid metabolic pathway. According to the evaluation method in Example 1, strains Ar02, Ar03 and Ar04 were fermented for 4 days using yeast inorganic salt medium, and the ability of strains Ar02, Ar03 and Ar04 to synthesize protocatechuic acid and p-coumaric acid was evaluated, and the results are shown in Figure 4. The yields of protocatechuic acid and p-coumaric acid in the fermentation product of strain Ar02 were 196.28 mg/L and 82.36 mg/L, respectively; the yields of protocatechuic acid and p-coumaric acid in the fermentation product of strain Ar03 were 186.96 mg/L and 72.35 mg/L, respectively; the yields of protocatechuic acid and p-coumaric acid in the fermentation product of strain Ar04 were 179.96 mg/L and 89.47 mg/L, respectively.

Referring to the evaluation method in Example 1, the gene BbXFPK was knocked out on the basis of strain Ar04 to obtain strain Ar05. Then, the blank plasmid was overexpressed in strain Ar05 to obtain strain Ar06, and the BbXFPK plasmid was overexpressed in strain Ar05 to obtain strain Ar07. Strains Ar05, Ar06 and Ar07 were shaken in SC medium (Synthetic complete medium) lacking three aromatic amino acids (phenylalanine, tyrosine, tryptophan), and strain Ar05 was shaken in SC medium containing three aromatic amino acids (phenylalanine, tyrosine, tryptophan) for 4 days to evaluate the yield of aromatic compounds. The results are shown in Figure 5. It can be seen that strain Ar05 lacks both the non-oxidative pentose phosphate pathway and the BbXFPK pathway and cannot grow in SC medium. However, by supplementing the above three aromatic amino acids (3AA) or back-complementing BbXFPK, the recombinant strains (Ar05+3AA and Ar07 in Figure 5) restored their growth ability. It was further demonstrated that strain Ar04 could only rely on heterologous overexpressed BbXFPK to maintain cell growth and synthesize metabolites.

Example 3 Knockout of genes PYK1 and PYK2 to increase PEP synthesis flux

Referring to the method in Example 1, the primers in Table 5 were used to construct the pROS10 guide RNA plasmid for knocking out the genes PYK1 and PYK2. Based on the strain Ar04 obtained in Example 2, the genes PYK1 and PYK2 for converting PEP to pyruvate were knocked out. The specific operation was: knocking out the gene PYK1 while overexpressing the endogenous genes ICL1, MLS1 and PCK1 of Saccharomyces cerevisiae; knocking out the gene PYK2 while overexpressing the genes The E. coli derived gene UdhA (SEQ ID NO: 2) was codon optimized to obtain strain Ar08.

Referring to the evaluation method of Example 1, strain Ar08 was fermented in YPD (2% glucose as carbon source) medium and YPED (2% ethanol and 0.2% glucose as mixed carbon source) medium, respectively, to evaluate the ability of Ar08 to synthesize protocatechuic acid and p-coumaric acid. The results of the production of protocatechuic acid and p-coumaric acid are shown in Figure 6A (which is the result of 4 days of fermentation). In addition, strains Ar04 and Ar08 were tested for their growth ability under YPD (2% glucose as carbon source) medium conditions, and their growth curves are shown in Figure 6B. It can be seen that strain Ar08 cannot grow normally when glucose is used as a carbon source, mainly because the key endogenous yeast genes related to its growth (TKL1, TKL2, TAL1, NQM1, PYK1 and PYK2) have been knocked out.

Example 4 Improving the growth ability of strain Ar08 by adaptive evolution

Take three monoclones of strain Ar08 obtained in Example 3 and perform adaptive evolution respectively. The specific operation is as follows: the above-mentioned strain uses a rich medium with ethanol and glucose mixed carbon sources (2% ethanol and 0.2% glucose are added to YP) as the starting medium. As the phenotype of the evolved strain changes, the amount of ethanol is gradually reduced and the percentage of glucose is increased for subculture. When the evolved strain no longer continues to grow in a rich medium with glucose as a carbon source (2% glucose is added to YP), it is switched to an inorganic salt medium containing 2% glucose (2% glucose is added to Delft) for subculture. The evolutionary process starts from a rich medium and gradually increases the growth environment of the strain by adjusting the medium conditions. In this process, in order to maintain growth, the strain enriches mutations that are beneficial to growth to achieve the effect of adaptive evolution. Finally, three strains, Evo1, Evo2 and Evo3, evolved, and then the whole genomes of Evo1, Evo2 and Evo3 were resequenced. The above three strains all had HXK2 gene mutations. Among them, Evo1 lost its function after the 24th base "A" was deleted from the HXK2 gene to form a frameshift mutation; Evo2 lost its function after 20 bases were inserted after the 1185th base to form a frameshift mutation; Evo3 could not express the complete HXK2 protein after the 82nd base was mutated from "G" to "T" to obtain the termination codon TAG.

Referring to the evaluation method of Example 1, the evolved strains were evaluated by shake flask fermentation using yeast inorganic salt medium. The growth ability of the evolved strains is shown in Figure 7A, and the statistical results of the aromatic compound production are shown in Figure 7B (which is the result of 4 days of fermentation). It can be seen that Evo1, Evo2 and Evo3 have stronger growth ability and ability to synthesize aromatic compounds than Ar08. The reason is that the gene HXK2 mutant strain Evo1, Evo2 and Evo3 lost their glucose phosphorylation activity, thus eliminating the glucose effect of the evolved strains. Among them, Evo2 was the strain with the highest production of two aromatic compounds (protocatechuic acid and p-coumaric acid).

Example 5 Construction of a cell factory for high production of a single aromatic compound

Referring to the method of Example 1, using the primers in Table 5, based on Evo2 of Example 4, by knocking out the 1 site of the p-coumaric acid synthesis pathway (i.e., knocking out the genes AtPAL2, AtC4H and AtATR2) and adding 1 copy of 3DSD, the strain ME01 that only produces protocatechuic acid was transformed. On the basis of the ME01 strain, the 2 sites of the p-coumaric acid synthesis pathway (i.e., knocking out the gene FiTAL gene) and adding 1 copy of 3DSD were knocked out to obtain the ME02 strain (with 3 copies of 3DSD). In the strain Evo2, 3DSD was knocked out, and the genes AtPAL2, AtC4H and AtATR2 were overexpressed at the same time, the protocatechuic acid synthesis pathway was abolished, and it was transformed into a strain that can only produce p-coumaric acid, and the strain ME03 was obtained.

According to the evaluation method in Example 1, yeast inorganic salt medium was used to evaluate the ability of strain Evo2, strain ME01 and strain ME02 to produce aromatic compounds through shake flask fermentation. The statistical results of protocatechuic acid production are shown in Figure 8 A (which is the result of 4 days of fermentation). Strain ME02 only synthesizes protocatechuic acid, and the yield is as high as 2.02 g/L (4.8 times the yield of strain Ar01). The yield and cell dry weight results of the ME02 strain in the parallel bioreactor test are shown in Figure 8 B.

According to the evaluation method in Example 1, strains Ev02 and ME03 were shaken for 4 days to evaluate their ability to synthesize aromatic compounds. The results are shown in FIG9 , which show that they can only synthesize p-coumaric acid with a yield of 690 mg/L (5.7 times that of strain Ar01).

In summary, the present invention reshapes a new pathway for yeast to synthesize E4P and blocks the PEP decomposition pathway, strengthening the glyoxylate cycle pathway to indirectly activate the TCA cycle to supply the basic needs of strain growth. At the same time, it reduces unnecessary carbon loss and energy consumption, driving carbon flow to the biosynthesis of aromatic compounds. The recombinant strain increased the production of aromatic compounds through adaptive evolution and discovered the molecular mechanism by which HXK2 inactivation can increase the production of aromatic compound synthesis, and finally constructed a recombinant yeast strain chassis cell that continuously and stably produces aromatic compounds.

It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims (10)

A recombinant yeast strain for synthesizing aromatic compounds, characterized in that the recombinant yeast strain for synthesizing aromatic compounds is based on Saccharomyces cerevisiae QL35 and undergoes one of the following modifications (a) to (f): (a) Overexpressed gene 3DSD; (b) Overexpression of the 3DSD gene and knockout of the TKL1 and TKL2 genes; (c) overexpressing the 3DSD gene and knocking out the TKL1, TKL2, and TAL1 genes; or, overexpressing the 3DSD gene and knocking out the TKL1, TKL2, TAL1, and NQM1 genes; (d) overexpression of the gene 3DSD and knockout of genes TKL1, TKL2, TAL1, NQM1, PYK1, and PYK2; (e) overexpressing the 3DSD gene, knocking out the TKL1, TKL2, TAL1, NQM1, PYK1 and PYK2 genes, and overexpressing at least one of the ICL1 and MLS1 genes and PCK1 and UdhA genes in the glyoxylate cycle pathway; (f) Overexpressing the 3DSD gene, knocking out the TKL1, TKL2, TAL1, NQM1, PYK1 and PYK2 genes, and overexpressing the ICL1 and MLS1 genes on the glyoxylate cycle pathway and at least one of the PCK1 and UdhA genes; and then performing adaptive evolution. The recombinant yeast strain for synthesizing aromatic compounds according to claim 1, characterized in that the recombinant yeast strain for synthesizing aromatic compounds is based on Saccharomyces cerevisiae QL35 and undergoes the following transformations after the transformation and evolution of (f): Knockout of genes AtPAL2, AtC4H and AtATR2, and overexpression of gene 3DSD; Knockout of gene FjTAL, and overexpression of gene 3DSD; or, The gene 3DSD was knocked out, and the genes AtATR2, AtC4H and AtATR2 were overexpressed. The recombinant yeast strain for synthesizing aromatic compounds according to claim 1, characterized in that the gene 3DSD is a codon-optimized gene 3DSD, the nucleotide sequence of the codon-optimized gene 3DSD is as shown in SEQ ID NO: 1, or the nucleotide sequence of the codon-optimized gene 3DSD has at least 70% homology with the nucleotide sequence shown in SEQ ID NO: 1. The recombinant yeast strain for synthesizing aromatic compounds according to claim 1, characterized in that the gene UdhA is a codon-optimized gene UdhA, the nucleotide sequence of the codon-optimized gene UdhA is as shown in SEQ ID NO: 2, or the nucleotide sequence of the codon-optimized gene UdhA has at least 70% homology with the nucleotide sequence shown in SEQ ID NO: 2. A method for constructing a recombinant yeast strain for synthesizing aromatic compounds, characterized in that the construction method comprises: Using Saccharomyces cerevisiae QL35 as the chassis, the strain was gene-edited using the CRISPR/Cas9 gene editing system according to one of the following (a) to (f): (a) Overexpressed gene 3DSD; (b) Overexpression of the 3DSD gene and knockout of the TKL1 and TKL2 genes; (c) overexpressing the 3DSD gene and knocking out the TKL1, TKL2, and TAL1 genes; or, overexpressing the 3DSD gene and knocking out the TKL1, TKL2, TAL1, and NQM1 genes; (d) overexpression of the gene 3DSD and knockout of genes TKL1, TKL2, TAL1, NQM1, PYK1, and PYK2; (e) overexpressing the 3DSD gene, knocking out the TKL1, TKL2, TAL1, NQM1, PYK1 and PYK2 genes, and overexpressing at least one of the ICL1 and MLS1 genes and PCK1 and UdhA genes in the glyoxylate cycle pathway; (f) Overexpressing the 3DSD gene, knocking out the TKL1, TKL2, TAL1, NQM1, PYK1 and PYK2 genes, and overexpressing the ICL1 and MLS1 genes on the glyoxylate cycle pathway and at least one of the PCK1 and UdhA genes; and then performing adaptive evolution. The construction method according to claim 5, characterized in that the construction method further comprises: After the transformation and evolution of (f), the following transformation is performed: Knockout of genes AtPAL2, AtC4H and AtATR2, and overexpression of gene 3DSD; Knockout of gene FjTAL, and overexpression of gene 3DSD; or, The gene 3DSD was knocked out, and the genes AtPAL2, AtC4H and AtATR2 were overexpressed. The recombinant yeast strain for synthesizing aromatic compounds according to any one of claims 1 to 4 and/or the method of claim 1 The use of the recombinant yeast strain for synthesizing aromatic compounds obtained by the construction method described in any one of claims 5-6 in synthesizing aromatic compounds. A method for synthesizing an aromatic compound, characterized in that it comprises the steps of: The recombinant yeast strain for synthesizing aromatic compounds according to any one of claims 1 to 4 and/or the recombinant yeast strain for synthesizing aromatic compounds obtained by the construction method according to any one of claims 5 to 6 are fermented to synthesize the aromatic compounds. The synthesis method according to claim 8, characterized in that the aromatic compound comprises protocatechuic acid and/or p-coumaric acid. The synthesis method according to claim 8 is characterized in that the culture medium used for the fermentation includes glucose and/or ethanol.