WO2023164985A1 - Genetically engineered bacterium for synthesizing p-coumaric acid and derivative thereof, method for constructing same and use thereof - Google Patents

Abstract

A genetically engineered bacterium for synthesizing p-coumaric acid and derivatives thereof, a method for constructing same and use thereof. The genetically engineered bacterium for synthesizing p-coumaric acid comprises a chassis strain into which a p-coumaric acid synthesis gene has been introduced, and the chassis strain has also been subjected to any one of or a combination of at least two of the following genetic modifications: (1) deleting aro8 and aro9; (2) deleting tdh1, tdh2, tdh3, and overexpressing GAPB; (3) deleting idh1 or idh2, and simultaneously overexpressing YHM2 and IDP2; (4) deleting pgi1, pfk1, or pfk2, and simultaneously overexpressing ZWF1, GND1, RPE1, RKI1, TKL1, and TAL1; (5) overexpressing POS5; or (6) deleting gdh2. Efficient synthesis of p-coumaric acid and derivatives thereof is realized.

Description

A genetically engineered bacterium for synthesizing p-coumaric acid and its derivatives and its construction method and application technical field

The application belongs to the technical field of synthetic biology, and relates to a genetically engineered bacterium for synthesizing p-coumaric acid and its derivatives, as well as its construction method and application.

Background technique

P-coumaric acid (pCA), also known as p-hydroxycinnamic acid, has antioxidant, antibacterial and anti-mutagenic activities, and is also a precursor or intermediate of many important compounds and drugs. For example, p-coumaric acid is decarboxylated by decarboxylase to generate pCA Hydroxystyrene - an important chemical raw material, used to produce many important industrial polymers; p-coumaric acid can further form derivatives such as caffeic acid, ferulic acid and vanillin, which are widely used in medicine and food , agriculture and other fields. There are generally three ways to obtain p-coumaric acid and its derivatives: 1) plant extraction method, which is obtained by plant extraction, but there are problems such as low content, unstable supply of raw materials, and high price; 2) chemical synthesis method, but there are high pollution, 3) Enzymatic preparation, the product is completely consistent with the nature of the natural extraction product, but there are problems such as high raw material cost and unstable performance of the enzyme catalyst.

With the rapid development of metabolic engineering and synthetic biology, the construction of microbial cell factories (MCFs) to simulate the metabolic pathways in plants provides a new idea for the synthesis of high value-added p-coumaric acid and its derivatives. A typical aromatic amino acid derivative, p-coumaric acid can be generated from phenylalanine under the catalysis of phenylalanine ammonia lyase and cinnamic acid hydroxylase, and can also be catalyzed by tyrosine ammonia lyase Produced directly from tyrosine. The Nielsen team of Chalmers University of Technology in Sweden has done a lot of excellent work in the field of constructing p-coumaric acid microbial cell factories. The p-coumaric acid production of the Saccharomyces cerevisiae strain constructed after the upper tank fermentation reached 12.5g/L (see : Liu Q, Yu T, Li X, et al. Rewiring carbon metabolism in yeast for high level production of aromatic chemicals [J]. Nat Commun, 2019, 10(1): 4976.). In addition, Escherichia coli, cyanobacteria and Pseudomonas have all been successfully used as host bacteria for the synthesis of p-coumaric acid, but their yields are much lower than that of Saccharomyces cerevisiae. At the same time, Saccharomyces cerevisiae, as a biologically safe strain, has a clear genetic background and mature high-density fermentation technology, and is an ideal chassis cell for the synthesis of natural products. However, limited by the understanding of the existing metabolic regulation system, especially for this kind of heterologous metabolic pathways for the synthesis of aromatic amino acid derivatives, the intermediate products are numerous and complex, the supply of precursors is insufficient, the feedback inhibition of key enzymes, The transcriptional regulation of regulatory proteins on synthetic pathways and the transport efficiency of transporters are still not effectively resolved. The metabolic transformation ideas used in the reports have focused on overexpressing rate-limiting enzymes, knocking out competitive pathways, and lifting feedback inhibition, etc., in order to achieve the purpose of increasing metabolic flux to product synthesis pathways. However, there is still the problem of metabolic flux escape, which becomes a bottleneck to further increase the production of p-coumaric acid and its derivatives.

To sum up, how to provide a new transformation strategy that can further increase the production of p-coumaric acid and its derivatives in engineering bacteria is one of the problems that need to be solved in the field of synthesis of p-coumaric acid and its derivatives.

Contents of the invention

This application provides a genetically engineered bacterium for synthesizing p-coumaric acid and its derivatives and its construction method and application. This application designs a unique metabolic transformation strategy to construct a genetically engineered bacterium for synthesizing p-coumaric acid, and grows through coupling strains And product synthesis, combined with reducing force to drive strain growth and product synthesis, so that the growth of the strain produces a metabolic driving force, and the driving force can only be offset by the metabolic flow into the product synthesis metabolism, which significantly increases the production of p-coumaric acid. At the same time, through By transferring the genes related to the synthesis of p-coumaric acid derivatives, various p-coumaric acid derivatives can be further synthesized.

In the first aspect, the present application provides a genetically engineered bacterium for synthesizing p-coumaric acid. Acid hydroxylase gene, cytochrome B5 gene and cytochrome P450 reductase 2 gene chassis strain;

Wherein, the chassis strain has also been genetically modified by any one or at least two combinations of the following:

(1) Deletion of aromatic aminotransferase I (aro8) and aromatic aminotransferase II (aro9);

(2) Deletion of NAD-dependent 3-phosphate dehydrogenase (tdh1, tdh2, tdh3), overexpression of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase gene (GAPB);

(3) Deletion of NAD-dependent isocitrate dehydrogenase 1 (idh1) or isocitrate dehydrogenase 2 (idh2), while overexpressing the transporter gene (YHM2) and NADP-dependent isocitrate dehydrogenase gene ( IDP2);

(4) Deletion of phosphoglucose isomerase (pgi1), phosphofructokinase 1 (pfk1) or phosphofructokinase 2 (pfk2), and overexpression of glucose-6-phosphate dehydrogenase (ZWF1) gene, 6-phosphogluconate Dehydrogenase (GND1) gene, ribulose 5-phosphate epimerase (RPE1) gene, 5-phosphoribose-ketoisomerase (RKI1) gene, transketolase (TKL1) gene and transaldolase ( TAL1) gene;

(5) Overexpression of NADH kinase gene (POS5); or

(6) Deletion of the glutamate dehydrogenase gene (gdh2).

In this application, tyrosine ammonia lyase gene (FjTAL), phenylalanine ammonia lyase gene (AtPAL2), cinnamic acid hydroxylase gene (AtC4H), cytochrome B5 gene (CYB5) and Cytochrome P450 reductase 2 gene (AtATR2), construct p-coumaric acid synthesis pathway, and design metabolic modification strategy: (1) Coupling strain growth and product synthesis with nitrogen source, through deletion of aromatic aminotransferase I (aro8) and Aroma aminotransferase II (aro9), so that when the strain grows in a medium with phenylalanine and tyrosine as nitrogen sources, it can only decompose The deamination reaction of phenylalanine and tyrosine by ammonia enzyme to obtain nitrogen source for growth, thus coupling the growth of strain and product synthesis; (2) driving the growth of strain and product synthesis through reducing force, increasing intracellular NADPH Reducing power driving force to increase product yield; can significantly increase the yield of p-coumaric acid and its derivatives.

Preferably, the chassis strain includes any one of yeast, Escherichia coli, Bacillus subtilis or microalgae.

Preferably, the yeast comprises Saccharomyces cerevisiae.

Preferably, the Saccharomyces cerevisiae comprises Saccharomyces cerevisiae Lab001 (CEN.PK 113-5D/MATa ura3-52can1Δ::cas9-natNT2TRP1LEU2HIS3).

In this application, deletion of protein or enzyme can be done by knocking out the corresponding gene, RNA interference, gene down-regulation or removal of enzyme activity and other methods.

Preferably, the chassis strain is genetically modified by any one or combination of at least two of the following:

(1) Knock out the genes of aryl aminotransferase I (aro8) and aryl aminotransferase II (aro9);

(2) Knock out the NAD-dependent glyceraldehyde-3-phosphate dehydrogenase gene (tdh1, tdh2, tdh3), and overexpress the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase gene (GAPB);

(3) Knockout of NAD-dependent isocitrate dehydrogenase 1 (idh1) or isocitrate dehydrogenase 2 gene (idh2), while overexpressing the transporter gene (YHM2) and NADP-dependent isocitrate dehydrogenase (IDP2);

(4) Knock out the phosphoglucose isomerase gene (pgi1), phosphofructokinase 1 (pfk1) or phosphofructokinase 2 (pfk2), and overexpress the glucose-6-phosphate dehydrogenase (ZWF1) gene, 6-phosphate Gluconate dehydrogenase (GND1) gene, ribulose 5-phosphate epimerase (RPE1) gene, 5-phosphoribose-ketoisomerase (RKI1) gene, transketolase (TKL1) gene and transaldolase Enzyme (TAL1) gene;

(5) Overexpression of NADH kinase gene (POS5); or

(6) Knock out the glutamate dehydrogenase gene (gdh2).

Preferably, the chassis strain is modified for growth coupling and/or for improving reducing power.

Preferably, said growth-coupled modification comprises deletion of aryl aminotransferase I (aro8) and aryl aminotransferase II (aro9).

Preferably, the improvement of reducing power includes any one or a combination of at least two of the following methods:

(1) Deletion of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase genes (tdh1, tdh2, tdh3), overexpression of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase genes (GAPB);

(2) Deletion of NAD-dependent isocitrate dehydrogenase 1 (idh1) or isocitrate dehydrogenase 2 (idh2), while overexpressing the transporter gene (YHM2) and NADP-dependent isocitrate dehydrogenase gene ( IDP2);

(3) Deletion of phosphoglucose isomerase (pgi1), phosphofructokinase 1 (pfk1) or phosphofructokinase 2 (pfk2), and overexpression of glucose-6-phosphate dehydrogenase (ZWF1) gene, 6-phosphogluconate Dehydrogenase (GND1) gene, ribulose 5-phosphate epimerase (RPE1) gene, 5-phosphoribose-ketoisomerase (RKI1) gene, transketolase (TKL1) gene and transaldolase ( TAL1) gene;

(4) Overexpression of NADH kinase gene (POS5); or

(5) Deletion of the glutamate dehydrogenase gene (gdh2).

Preferably, the nucleic acid sequence of the tyrosine ammonia lyase gene includes the sequence shown in SEQ ID NO.1.

Preferably, the nucleic acid sequence of the phenylalanine ammonia lyase gene includes the sequence shown in SEQ ID NO.2.

Preferably, the nucleic acid sequence of the cinnamic acid hydroxylase gene includes the sequence shown in SEQ ID NO.3.

Preferably, the nucleic acid sequence of the cytochrome P450 reductase 2 gene includes the sequence shown in SEQ ID NO.4.

Preferably, the nucleic acid sequence of the cytochrome B5 gene includes the sequence shown in SEQ ID NO.5.

Preferably, the nucleic acid sequence of the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase gene includes the sequence shown in SEQ ID NO.6.

Preferably, the chassis strain further includes a gene for synthesizing p-coumaric acid derivatives.

In the present application, inserting a gene for synthesizing p-coumaric acid derivatives into the p-coumaric acid-synthesizing genetically engineered bacteria can further efficiently synthesize p-coumaric acid derivatives.

Preferably, the p-coumaric acid derivatives include any one or a combination of at least two of caffeic acid, p-hydroxystyrene or resveratrol.

Preferably, the synthetic genes of caffeic acid include 4-hydroxyphenylacetate 3-hydroxylase gene (HPAB) and flavin oxidoreductase gene (HPAC), or 4-hydroxyphenylacetate 3-hydroxylase gene (HPAB) Hydroxylase gene (HPAB) and xanthine reductase gene (VLMR).

Preferably, the synthesis gene of p-hydroxystyrene includes phenolic acid decarboxylase gene (BAPDA).

Preferably, the resveratrol synthesis gene includes a stilbene synthase gene (VvSTS) and a ligase gene (4CL1).

Preferably, the nucleic acid sequence of the 4-hydroxyphenylacetate 3-hydroxylase gene includes the sequence shown in SEQ ID NO.7.

Preferably, the nucleic acid sequence of the flavin oxidoreductase gene includes the sequence shown in SEQ ID NO.8.

Preferably, the nucleic acid sequence of the xanthine reductase gene includes the sequence shown in SEQ ID NO.9.

Preferably, the nucleic acid sequence of the phenolic acid decarboxylase gene includes the sequence shown in SEQ ID NO.10.

Preferably, the nucleic acid sequence of the stilbene synthase gene includes the sequence shown in SEQ ID NO.11.

Preferably, the nucleic acid sequence of the ligase gene includes the sequence shown in SEQ ID NO.12.

In a second aspect, the present application provides a method for constructing a genetically engineered bacterium for synthesizing p-coumaric acid described in the first aspect, the construction method comprising:

The CRISPR/Cas9 gene editing system was used to edit the genes of the chassis strain, including the transfer of tyrosine ammonia lyase gene, phenylalanine ammonia lyase gene, cinnamate hydroxylase gene, cytochrome B5 gene and cytochrome P450 reduction Enzyme 2 gene, and perform gene editing in any one of the following ways or a combination of at least two:

(1) Deletion of aromatic amino acid transferase I (aro8) and aromatic amino acid transferase II (aro9);

(2) Deletion of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (tdh1, tdh2, tdh3), overexpression of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase gene (GAPB);

(3) Deletion of NAD-dependent isocitrate dehydrogenase 1 (idh1) or isocitrate dehydrogenase 2 (idh2), while overexpressing the transporter gene (YHM2) and NADP-dependent isocitrate dehydrogenase gene ( IDP2);

(4) Deletion of phosphoglucose isomerase (pgi1), phosphofructokinase 1 (pfk1) or phosphofructokinase 2 (pfk2), and overexpression of glucose-6-phosphate dehydrogenase (ZWF1) gene, 6-phosphogluconate Dehydrogenase (GND1) gene, ribulose 5-phosphate epimerase (RPE1) gene, 5-phosphoribose-ketoisomerase (RKI1) gene, transketolase (TKL1) gene and transaldolase ( TAL1) gene;

(5) Overexpression of NADH kinase gene (POS5); or

(6) Deletion of the glutamate dehydrogenase gene (gdh2);

The genetically engineered bacterium for synthesizing p-coumaric acid is obtained.

Preferably, the chassis strain includes any one of yeast, Escherichia coli, Bacillus subtilis or microalgae.

Preferably, the yeast comprises Saccharomyces cerevisiae.

Preferably, the Saccharomyces cerevisiae comprises Saccharomyces cerevisiae Lab001 (CEN.PK 113-5D/MATa ura3-52can1Δ::cas9-natNT2TRP1LEU2HIS3).

Preferably, the chassis strain is gene-edited in any one of the following ways or a combination of at least two:

(1) Knock out the genes of aromatic amino acid transferase I (aro8) and aromatic amino acid transferase II (aro9);

(2) Knock out the NAD-dependent glyceraldehyde-3-phosphate dehydrogenase gene (tdh1, tdh2, tdh3), and overexpress the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase gene (GAPB);

(3) Knockout of the NAD-dependent isocitrate dehydrogenase 1 gene (idh1) or isocitrate dehydrogenase 2 gene (idh2), while overexpressing the transporter gene (YHM2) and NADP-dependent isocitrate dehydrogenation Enzyme gene (IDP2);

(4) Knock out the phosphoglucose isomerase gene (pgi1), phosphofructokinase 1 (pfk1) or phosphofructokinase 2 (pfk2), and overexpress the glucose-6-phosphate dehydrogenase (ZWF1) gene, 6-phosphate Gluconate dehydrogenase (GND1) gene, ribulose 5-phosphate epimerase (RPE1) gene, 5-phosphoribose-ketoisomerase (RKI1) gene, transketolase (TKL1) gene and transaldolase Enzyme (TAL1) gene;

(5) Overexpression of NADH kinase gene (POS5); or

(6) Knock out the glutamate dehydrogenase gene (gdh2).

In a third aspect, the present application provides the application of the p-coumaric acid-synthesizing genetically engineered bacteria described in the first aspect in the production of p-coumaric acid and/or p-coumaric acid derivatives.

Preferably, the p-coumaric acid derivatives include any one or a combination of at least two of caffeic acid, p-hydroxystyrene or resveratrol.

In a fourth aspect, the present application provides a method for preparing p-coumaric acid and/or p-coumaric acid derivatives, the method comprising:

Fermentation is carried out by using the genetically engineered bacterium for synthesizing p-coumaric acid described in the first aspect.

Compared with the prior art, the present application has the following beneficial effects:

This application designs a unique metabolic transformation strategy to couple strain growth and product synthesis with nitrogen source, and drive strain growth and product synthesis through reducing force, so that the growth of the strain can generate metabolic driving force, and the driving force can only enter the product synthesis metabolism through metabolic flow With the growth of the strain, the product can be continuously synthesized, so as to realize the efficient synthesis of p-coumaric acid and its derivatives.

Description of drawings

Figure 1 is a schematic diagram of the strategy model for constructing recombinant yeast strains.

Fig. 2 is a graph showing the results of p-coumaric acid (pCA) synthesized by fermentation of strain LW001.

Fig. 3 is a graph showing the growth of strain Lab001 in different culture media.

Fig. 4 is a graph showing the growth of strains Lab001, LW002 and SV001 in M-Delft medium.

Figure 5 is a diagram of the fermentation results of strains LW001 and LW002.

Figure 6 is a diagram of the fermentation results of strain LW003.

Figure 7 is a diagram of the fermentation results of strain LW004.

Figure 8 is a diagram of the fermentation results of strain LW005.

Figure 9 is a diagram of the fermentation results of strain LW006.

Fig. 10 is a model diagram of the metabolic transformation strategy of reducing power of the strain.

Figure 11 is a diagram of the fermentation results of strain LW007.

Fig. 12 is a graph showing the results of spot plate experiments on strain LW007.

Figure 13 is a diagram of the fermentation results of strains LW008 and LW009.

Figure 14 is a diagram of the fermentation results of strains LW010, LW011, LW012 and LW013.

Fig. 15 is a graph showing the fermentation results of strain LW013 under the addition of different concentrations of phenylalanine.

Detailed ways

In order to further illustrate the technical means and effects adopted by the present application, the present application will be further described below in conjunction with the embodiments and accompanying drawings. It can be understood that the specific implementation manners described here are only used to explain the present application, but not to limit the present application.

If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field, or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products commercially available through regular channels.

In the examples of this application, Saccharomyces cerevisiae was used as the chassis cell as an example to construct genetically engineered bacteria for the synthesis of p-coumaric acid and its derivatives. The starting strain used was Lab001, which was obtained by transforming wild-type Saccharomyces cerevisiae CEN.PK113-5D Uracil (Ura) auxotrophic Saccharomyces cerevisiae, and can express the cas9 protein system for gene editing, the Cas9 gene is integrated at the site can1, can refer to the literature Mans R., van Rossum H.M., Wijsman M., et al.CRISPR /Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae[J]. Fems Yeast Research, 2015(2): 2.. The Saccharomyces cerevisiae genome was edited using the CRISPR/CAS9 method. For specific methods, please refer to the literature Mans R., van Rossum H.M., Wijsman M., et al. CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae[J]. Fems Yeast Research, 2015(2): 2. Saccharomyces cerevisiae transformation method refers to the literature (Gietz RD, Woods R A. Transformation of yeast by the LiAc/ss carrier DNA/PEG method [J]. Methods in Molecular Biology, 2006, 313: 107-120.). The experimental methods not specifying the specific conditions in the examples were carried out according to conventional conditions, the conditions described in "Molecular Cloning: A Laboratory Guide" (New York: Cold Spring Harbor laboratory Press, 2001). The strain strategy model diagram constructed in this application is shown in Fig. 1 .

The exogenous genes of Saccharomyces cerevisiae used in the examples of the present application are as follows: the tyrosine ammonia lyase gene FjTAL (SEQ ID NO.1) derived from Flavobacterium johnsoniae, the phenylalanine ammonia lyase gene AtPAL2 derived from Arabidopsis thaliana (SEQ ID NO.2), cinnamic acid hydroxylase gene AtC4H (SEQ ID NO.3) and cytochrome P450 reductase 2 gene AtATR2 (SEQ ID NO.4), derived from the glyceraldehyde-3- Phosphate dehydrogenase gene GAPB (SEQ ID NO.6), derived from 4-hydroxyphenylacetate 3-hydroxylase gene HPAB (SEQ ID NO.7) of Pseudomonas aeruginosa, derived from flavin oxidation of Salmonella enterica The reductase gene HPAC (SEQ ID NO.8) is derived from the NADPH-specific xanthine reductase gene VLRM (SEQ ID NO.9) of Vibrio harveyi, and the phenolic acid decarboxylase gene BAPDA (SEQ ID NO.9) derived from Bacillus amyloliquefaciens .10), derived from the stilbene synthase gene VvSTS (SEQ ID NO.11) of Vitis vinifera, and the ligase gene 4CL1 (SEQ ID NO.12). The base sequences of all genes can be obtained from the NCBI database, and after codon optimization, they will be synthesized by a gene synthesis company and used as templates for PCR amplification. All promoters and terminators used are derived from the Saccharomyces cerevisiae CEN.PK113-5D genome, wherein the promoters are as follows: CCW12 gene promoter (SEQ ID NO.13) CCW12p, TDH3 gene promoter (SEQ ID NO.14) TDH3p, TEF1 gene promoter (SEQ ID NO.15) TEF1p, TIP gene promoter (SEQ ID NO.16) TIPp, tHXT7 gene promoter (SEQ ID NO.17) tHXT7p, PGK1 gene promoter (SEQ ID NO. 18) PGK1p; the terminator is as follows: FBA1 gene terminator (SEQ ID NO.19) FBA1t, ADH1 gene terminator (SEQ ID NO.20) ADH1t, DIT1 gene terminator (SEQ ID NO.21) DIT1t, PYK1 gene terminator Son (SEQ ID NO.22) PYK1t, TDH2 gene terminator (SEQ ID NO.23) TDH2t. Saccharomyces cerevisiae CEN.PK113-5D genome was used as the amplification template for the amplification of endogenous sequences, and the specific sequences were consistent with those recorded in the NCBI database.

The medium used in this application includes: YPD medium: 1% yeast extract, 2% peptone, 2% glucose (different carbon sources can be replaced according to needs, such as YPE medium to replace glucose with ethanol). Delft medium: 1L medium contains the following components, (NH ₄ ) ₂ SO ₄ 7.5g, KH ₂ PO ₄ 14.4g, MgSO ₄ ·7H ₂ O 0.5g, glucose 20g, trace element mixture 2mL and vitamin mixture solution 1mL, among them, trace element mixed solution: FeSO ₄ 7H ₂ O 3.0g/L, ZnSO ₄ 7H ₂ O 4.5g/L, CaCl ₂ 2H ₂ O 4.5g/L, MnCl ₂ 4H ₂ O 1g /L, CoCl ₂ 6H ₂ O 300mg/L, CuSO ₄ 5H ₂ O 300mg/L, Na ₂ MoO ₄ 2H ₂ O 400mg/L, H ₃ BO ₃ 1g/L, KI 100mg/L, Na ₂ EDTA·2H ₂ O 19g/L; vitamin mixture: D-biotin 50mg/L, vitamin B5 1.0g/L, vitamin B1 1.0g/L, vitamin B6 1.0g/L, niacin 1.0g/L, 4 -Aminobenzoic acid 0.2g/L, inositol 25g/L. M-Delft medium: replace 7.5g/L (NH ₄ ) ₂ SO ₄ in Delft medium with 9.9g/L K ₂ SO ₄ . SD medium is synthetic complete medium; SC-U medium is SD medium without uracil.

The gRNA knockout plasmids used in this application are as follows: PQC030, PQC033, PQC034, PQC073 and PQC135, construction method reference: Liu Q, Yu T, Li X, et al. Rewiring carbon metabolism in yeast for high level production of aromatic chemicals [J]. Nature Communications, 2019, 10(1): 4976.

Example 1 Construction of Saccharomyces cerevisiae Genetically Engineered Bacteria for Synthesizing p-coumaric Acid

In order to obtain a yeast chassis strain capable of synthesizing p-coumaric acid, Saccharomyces cerevisiae Lab001 was used as the starting strain, and tyrosine ammonia lyase genes FjTAL and phenylalanine ammonia lyase were inserted into chromosome XI-3 and XII-2 sites Gene AtPAL2, cinnamic acid hydroxylase gene AtC4H, cytochrome P450 reductase 2 gene AtATR2 and cytochrome B5 gene CYB5 were used to obtain the chassis strain LW001. The specific experimental process includes the following steps:

(1) Using Gibson DNA assembly technology to construct a DNA repair fragment A with the upper and lower homology arms of S. DNA repair fragment B of the upper and lower homology arms of yeast chromosome XII-2 and genes AtC4H, CYB5 and AtATR2: XII-2UP&DIT1t&AtC4H&TIPp&tHXT7p&CYB5&PYK1t&TDH2t&AtATR2&PGK1p&XII-2DN, and then, use the constructed plasmid PQC073 as the CRISPR/C for constructing genetically engineered strains AS9 system gRNA knockout plasmids (all gRNA knockout plasmids used in this application contain Ura tags);

(2) Construction of genetically engineered strains by means of lithium acetate heat shock transformation

1) Preparation of Saccharomyces cerevisiae Competence: Pick the bacterial liquid from the glycerol tube in which the parent live bacteria are preserved, streak it on the YPD solid medium for activation, pick the single clone on the solid medium and inoculate it into 1mL YPD medium at 30°C Cultivate overnight, take an appropriate amount of seed bacterial liquid and transfer it to 20mL YPD medium, make the initial OD ₆₀₀ = 0.1, stop culturing at 30°C until OD ₆₀₀ = 0.6, collect the bacterial liquid and centrifuge at 4°C, 4000×g 5min, remove the supernatant and add 20mL of pre-cooled sterile water to resuspend the bacteria, then centrifuge again at 4°C and 4000×g for 5min, remove the supernatant and add 1mL of 0.1M lithium acetate to resuspend the bacteria, and Transfer to a 1.5mL EP tube, then centrifuge again at 4°C and 4000×g for 5min, remove the supernatant and add 200μL of 0.1M lithium acetate to resuspend the bacteria, and divide the resulting bacterial solution into four 1.5mL EP tubes Centrifuge again at 4°C and 4000×g for 5 minutes, remove the supernatant, and prepare the competent state for use;

2) Heat shock transformation: Add heat shock transformation buffer (1μg DNA repair fragment, 2μg knockout plasmid, 240μL 50% w/v polyethylene glycol 3500, 36μL 1.0M lithium acetate, 25μL 2.0mg /mL salmon sperm DNA, add sterile water to a total volume of 360 μL) and mix thoroughly; place the mixed system in a 30°C water bath for 30 min, and then transfer to a 42°C water bath for heat shock for 25 min. After the heat shock, 4000 Collect the bacteria by centrifugation at ×g for 5 minutes, resuspend the bacteria with sterile water, spread them on a suitable solid medium, and cultivate them in a 30°C incubator. After the colonies grow, pick a single clone to verify whether the genetically engineered bacteria have been constructed. success. Successfully verified engineered bacteria need to complete the removal of the gRNA knockout plasmid on a solid medium supplemented with 5-fluoroorotic acid (5-FOA) and Ura, and then use the same method and steps to carry out the next round of gene renovation;

(3) Verification of the phenotype of the chassis strain LW001 by shake flask fermentation experiments

The liquid seed medium is YPD medium; the fermentation medium is M-Delft liquid medium. The specific steps of the fermentation experiment are as follows: Pick a single clone strain on the strain activation plate and inoculate it into 2mL (10mL shaker tube) liquid seed medium Culture in medium until the OD ₆₀₀ is close to 10, then collect the bacteria, wash the bacteria twice with sterile water and resuspend in the fermentation medium, then inoculate an appropriate amount of the resuspended bacteria into 20mL (50mL shake flask) for liquid fermentation In the culture medium, continue to cultivate at 30°C and 200rpm for 72 hours, then end the fermentation, and extract the fermentation product by ethanol extraction. The process is as follows: Take 0.6mL of the fermented bacterial liquid and 0.6mL of the extraction solvent ethanol in a 2mL centrifuge tube, and vortex to mix. After fully oscillating on the instrument for 10 minutes, centrifuge at 13,500×g for 5 minutes, take the supernatant into a new centrifuge tube as the sample to be analyzed, and use liquid chromatography-mass spectrometry system (LC-MS) for qualitative and quantitative analysis of the product: Chromatographic column: Welch Xtimate C18 (4.6×250mm, 5μm), mobile phase: mobile phase A is 10mM formic acid solution, mobile phase B is acetonitrile (chromatographic grade); column temperature: 30°C; flow rate: 0.3mL/min; The running program is shown in Table 1.

Table 1

time (min)	Mobile phase A(%)	Mobile phase B(%)
0.00	95	5
12.00	5	95
12.10	95	5
13.00	95	5

The product detection results are shown in Figure 2, and the constructed chassis strain LW001 can successfully synthesize p-coumaric acid.

Example 2 Construction of growth-coupled yeast genetically engineered bacteria for synthesis of p-coumaric acid derivatives

Using the chassis strain LW001 constructed in Example 1 as the starting strain, the aro8 and aro9 gene double-deleted strain LW002 was constructed. First, the aro8 and aro9 gene double-site gRNA knockout plasmid PL004 was constructed; DNA fragment C for repairing the Aro8 site was synthesized and repair the DNA fragment D at the Aro9 site; subsequently, the construction of bacterial strain LW002 with reference to the implementation case 1, due to the deletion of Aro8 and Aro9 genes, when the bacterial strain grows in a medium with phenylalanine and tyrosine as a nitrogen source, only Nitrogen source can be obtained by the deamination reaction of phenylalanine (Phe) and tyrosine (Tyr) by exogenously introduced tyrosine ammonia lyase (FjTAL) and phenylalanine ammonia lyase (AtPAL2) for growth.

Using the strain Lab001 as the starting strain, the aro8 and aro9 genes were knocked out to obtain the strain SV001, which was used as the experimental control strain. The growth and product synthesis coupling of the constructed strains were compared by comparing the growth of different strains in different media. The results are shown in Figure 3-Figure 5, Figure 3 is the growth of strain Lab001 in different Delft medium: Lab001 can grow in the M-Delft medium (M-Delft+Phe+Tyr) with Phe and Tyr added, It shows that it uses Phe and Tyr as nitrogen source to supply growth; Figure 4 shows the growth of different strains in Delft medium with Phe and Tyr as nitrogen source. First, the strain SV001 after knocking out aro8 and aro9 based on Lab001 cannot use Phe and Tyr as a nitrogen source for growth, and the strain LW002 introduced into the p-coumaric acid synthesis pathway at the same time restored the ability to use Phe and Tyr as a nitrogen source for growth. Further, the bacterial strain LW002 was grown in the fermentation medium M-Delft (containing 1.0 g/L Phe or containing 1.5g/L Phe) Fermentation product p-coumaric acid detection results are shown in Figure 5, strain LW002 successfully achieved the coupling of strain growth and product synthesis.

In order to obtain a yeast strain with a synthetic p-coumaric acid derivative - caffeic acid, the Gibson DNA assembly technology was first used to construct DNA repair fragments with upper and lower homology arms at the XII-5 site of Saccharomyces cerevisiae chromosome and genes HPAB and HPAC E:XII-5 UP&TDH2t&HPAC&CCW12p&TDH3p&HPAB&DIT1t&XII-5DN, then, using the chassis strain LW002 as the starting strain, transform the plasmid PQC033 and the DNA repair fragment E to obtain the strain LW003, and the detection results of the fermentation product of the strain LW003 in the fermentation medium M-Delft are shown in Figure 6 As shown, it shows that the genetically engineered strain LW003 for synthesizing caffeic acid has been successfully constructed.

In order to obtain a yeast strain with a synthetic p-coumaric acid derivative—p-hydroxystyrene (pHS), the Gibson DNA assembly technology was first used to construct a yeast strain with upper and lower homology arms at the XI-1 site of Saccharomyces cerevisiae chromosome and the gene BAPAD. DNA repair fragment F: XI-1UP&PYK1t&BAPAD&TEF1p&XI-1DN, then, using chassis strain LW002 as the starting strain, transform plasmid PQC030 and DNA repair fragment F to obtain strain LW004, and the detection results of the fermentation product of strain LW004 in the fermentation medium M-Delft are as follows As shown in Figure 7, it shows that the synthesis of p-hydroxystyrene has been successfully realized.

In order to obtain a yeast strain with a synthetic p-coumaric acid derivative—resveratrol (Resveratrol), firstly, the Gibson DNA assembly technology was used to construct the homology arms with the upper and lower downstream homology arms of the Saccharomyces cerevisiae chromosome XI-1 site and the genes 4CL1, The DNA repair fragment G of VvSTS: XI-1UP&TDH2t&VvSTSs&CCW12p&TDH3p&4CL1&DIT1t&XI-1DN, then, the chassis strain LW002 was used as the starting strain, and the plasmid PQC030 and DNA repair fragment G were transformed to obtain the strain LW005, and the fermentation product detection of the strain LW005 in the fermentation medium M-Delft The results are shown in Figure 8, indicating that the synthesis of resveratrol was successfully achieved.

Example 3 Application of Reducing Force Driven Model in Genetically Engineered Bacteria Synthesizing p-coumaric Acid Derivatives

Taking strain LW003 as an example, in the process of caffeic acid synthesis, NADPH is required to provide reducing power for cytochrome P450 reductase 2 (AtATR2), and NADH is required to provide reducing power for flavin oxidoreductase (HPAC). Considering that NADPH in Saccharomyces cerevisiae The metabolic regulation of caffeic acid is more stringent, and it is suitable as the driving force in the reducing force model to promote the synthesis of products. This application replaced the NAD-dependent HPAC with the NADP-dependent xanthine reductase (VLMR) to re-construct the caffeic acid synthesis strain: first construct the DNA repair fragment G with the upper and lower homology arms of Saccharomyces cerevisiae chromosome XII-5 and genes HPAB and VLMR: XII-5UP&TDH2t&VLMR&CCW12p&TDH3p&HPAB&DIT1t&XII-5DN; then, the chassis strain LW002 was used as the starting strain to transform the plasmid PQC033 and the DNA repair fragment G , to obtain strain LW006, construct the DNA repair fragment I with the upper and lower homology arms of Saccharomyces cerevisiae chromosome XII-5 site and the gene HPAB: XII-5UP&TDH3p&HPAB&DIT1t&XII-5DN, take the chassis strain LW002 as the starting strain, transform the plasmid PQC033 and DNA Fragment I was repaired to obtain strain LW006B, which was used as a control strain in this experiment. After fermentation test (Figure 9), compared with LW003, the production of caffeic acid in strain LW006 has decreased, but it still has the ability to synthesize caffeic acid (strain LW006B does not have coffee Acid synthesis ability).

Further, different metabolic transformation strategies (Figure 10) are used to increase the driving force of intracellular NADPH reducing force to increase product yield. Strategy 1: Knock out the NAD-dependent glyceraldehyde-3-phosphate dehydrogenase genes tdh1 and tdh2 of Saccharomyces cerevisiae and tdh3, replaced by the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase gene GAPB; strategy two: Knock out the NAD-dependent isocitrate dehydrogenase gene idh1 in the mitochondria, and overexpress the transporter gene YHM2 and cytoplasm NADP-dependent isocitrate dehydrogenase gene IDP2; Strategy 3: Knockout phosphoglucose isomerase gene pgi1 and overexpress 6 genes in the PPP pathway; Strategy 4: Overexpress NADH kinase gene POS5; Strategy 5 , to knock out the glutamate dehydrogenase gene gdh2.

In order to realize strategy 1: using LW006 as the starting strain, the genes tdh1, tdh2 and tdh3 were knocked out, and the codon-optimized GAPB gene was expressed at the same time to obtain the strain LW007. The fermentation results showed (Figure 11) that although the growth of the strain was affected by However, the production of caffeic acid was significantly improved, and in order to verify whether the reducing force-driven model was successful, a spot plate experiment was designed: inoculate the monoclonal strain LW007 in YPGE liquid seed medium, and cultivate it to the logarithmic phase; collect Bacteria solution, after washing twice, use different concentrations of Phe (0, 2.5, 5, 10g/L) solutions to resuspend the bacteria, and dilute into bacteria suspensions with different OD (1, 0.1, 0.01, 0.001, 0.0001) Get 8 μ L of different bacterial suspensions and carry out spot plate experiment on SD solid medium respectively (LW003 bacterial strain is this experiment contrast bacterial strain), bacterial strain growth situation is shown in Figure 12, known Phe and its derivatives have certain toxicity to Saccharomyces cerevisiae, Moreover, the caffeic acid synthesis pathway needs to consume a large amount of NADPH; the analysis of the results shows that after the transformation of the metabolic pathway, the strain LW007 will produce a large amount of NADPH during the growth process, resulting in an imbalance of intracellular redox power and inhibiting the growth of the strain; and after adding Phe , activating the caffeic acid synthesis pathway can effectively consume NADPH, restore the balance of intracellular redox power, and rescue the growth ability of the strain. The experimental results show that, driven by the reducing power, the growth of the strain LW007 is more deeply coupled with the product synthesis , verifying the successful construction of the reducing force-driven model.

In order to realize the second strategy: using LW006 as the starting strain, the gene idh1 was knocked out to obtain the strain LW008, and the genes YHM2 and IDP2 were overexpressed at the same time to obtain the strain LW009. The fermentation phenotype results of the strain are shown in Figure 13, and the gene idh1 was knocked out The post-TCA cycle was inhibited, which affected the growth of the strain, but the caffeic acid production was improved; after overexpressing the genes YHM2 and IDP2, the growth of the strain was restored to a certain extent, and the production was further improved.

Simultaneously implement strategy 1 and strategy 2: take LW007 as the starting strain, knock out the gene idh1 and overexpress the genes YHM2 and IDP2 at the same time, and obtain the strain LW010. The fermentation phenotype results of the strain are shown in Figure 14. The growth of the strain LW010 is strongly affected The inhibition is not conducive to the synthesis of the product. Considering that the growth of the strain is inhibited but the product yield is not significantly improved when the strategy 2 is implemented alone, it is subsequently decided to implement the remaining strategies while implementing the strategy 1 to transform the strain.

Overlay implementation strategies 1, 3, 4 and 5: take LW007 as the starting strain, knock out the gene pgi1 and overexpress 6 genes in the PPP pathway (ZWF1, GND1, RPE1, RKI1, TKL1 and TAL1), and obtain the strain LW011; Further, the strain LW012 was obtained by overexpressing the gene POS5; further, the strain LW013 was obtained by knocking out the gene gdh2, and the fermentation phenotype results of the strain are shown in Figure 14. Strain LW013 exhibited excellent caffeic acid synthesis ability. As shown in Figure 15, it is the fermentation result of strain LW013 when different concentrations of phenylalanine were used as substrates. When the concentration of phenylalanine was 1g/L, the product It is high-concentration caffeic acid (the detection concentration of pCA in the sample is close to the error range of the instrument); when the concentration of phenylalanine is 4g/L, the comprehensive conversion efficiency of the product can reach more than 95% of the theoretical conversion rate.

To sum up, this application designs a unique metabolic transformation strategy, which uses nitrogen source to couple strain growth and product synthesis, and drives strain growth and product synthesis through reducing force, so that the growth of the strain generates a metabolic driving force, and the driving force can only be through the metabolic flow The method of entering into the synthetic metabolism of the product is counteracted. With the growth of the strain, the product can be continuously synthesized, so as to realize the efficient synthesis of p-coumaric acid and its derivatives.

The applicant declares that the present application illustrates the detailed method of the present application through the above-mentioned examples, but the present application is not limited to the above-mentioned detailed method, that is, it does not mean that the application must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement to the present application, the equivalent replacement of each raw material of the product of the present application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present application.

Claims (10)

1. A genetic engineering bacterium for synthesizing p-coumaric acid, which includes tyrosine ammonia lyase gene, phenylalanine ammonia lyase gene, cinnamic acid hydroxylase gene, cytochrome B5 gene and cytochrome P450 reductase 2 Genetic chassis strains;

   Wherein, the chassis strain has also been genetically modified by any one or at least two combinations of the following:

   (1) Deletion of aryl aminotransferase I and aryl aminotransferase II;

   (2) Deletion of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, overexpression of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase gene;

   (3) Deletion of NAD-dependent isocitrate dehydrogenase 1 or isocitrate dehydrogenase 2, while overexpressing the transporter gene and NADP-dependent isocitrate dehydrogenase 2;

   (4) Deletion of phosphoglucose isomerase gene, phosphofructokinase 1 or phosphofructokinase 2 gene, and overexpression of glucose-6-phosphate dehydrogenase gene, 6-phosphogluconate dehydrogenase gene, ribulose 5- Phosphate epimerase gene, 5-phosphoribose-ketoisomerase gene, transketolase gene and transaldolase gene;

   (5) Overexpression of NADH kinase gene; or

   (6) Deletion of glutamate dehydrogenase gene.
2. The genetically engineered bacterium for synthesizing p-coumaric acid according to claim 1, wherein the chassis strain comprises any one of saccharomyces, Escherichia coli, Bacillus subtilis or microalgae;

   Preferably, the yeast comprises Saccharomyces cerevisiae.
3. The genetic engineering bacterium of synthesizing p-coumaric acid according to claim 1, wherein, the nucleotide sequence of described tyrosine ammonia lyase gene comprises the sequence shown in SEQ ID NO.1;

   Preferably, the nucleic acid sequence of the phenylalanine ammonia lyase gene includes the sequence shown in SEQ ID NO.2;

   Preferably, the nucleic acid sequence of the cinnamic acid hydroxylase gene includes the sequence shown in SEQ ID NO.3;

   Preferably, the nucleic acid sequence of the cytochrome P450 reductase 2 gene includes the sequence shown in SEQ ID NO.4;

   Preferably, the nucleic acid sequence of the cytochrome B5 gene includes the sequence shown in SEQ ID NO.5.
4. The genetic engineering bacterium of synthesizing p-coumaric acid according to claim 1, wherein, the gene nucleic acid sequence of the glyceraldehyde-3-phosphate dehydrogenase of described NADP dependence comprises the sequence shown in SEQ ID NO.6.
5. The genetically engineered bacterium for synthesizing p-coumaric acid according to any one of claims 1-4, wherein the chassis strain also includes a gene for synthesizing p-coumaric acid derivatives;

   Preferably, the p-coumaric acid derivatives include any one or a combination of at least two of caffeic acid, p-hydroxystyrene or resveratrol.
6. The genetically engineered bacterium for synthesizing p-coumaric acid according to claim 5, wherein the synthetic gene of caffeic acid comprises 4-hydroxyphenylacetate 3-hydroxylase gene and flavin oxidoreductase gene, or 4-hydroxyphenylacetate 3-hydroxylase gene and xanthine reductase gene;

   Preferably, the synthetic gene of p-hydroxystyrene comprises a phenolic acid decarboxylase gene;

   Preferably, the resveratrol synthesis gene includes a stilbene synthase gene and a ligase gene.
7. The genetically engineered bacterium for synthesizing p-coumaric acid according to claim 6, wherein the nucleotide sequence of the 4-hydroxyphenylacetate 3-hydroxylase gene comprises the sequence shown in SEQ ID NO.7;

   Preferably, the nucleic acid sequence of the flavin oxidoreductase gene includes the sequence shown in SEQ ID NO.8;

   Preferably, the nucleotide sequence of the xanthine reductase gene comprises the sequence shown in SEQ ID NO.9;

   Preferably, the nucleotide sequence of the phenolic acid decarboxylase gene includes the sequence shown in SEQ ID NO.10;

   Preferably, the nucleic acid sequence of the stilbene synthase gene includes the sequence shown in SEQ ID NO.11;

   Preferably, the nucleic acid sequence of the ligase gene includes the sequence shown in SEQ ID NO.12.
8. A construction method of the genetically engineered bacterium of synthetic p-coumaric acid described in any one of claims 1-7, it comprises:

   The CRISPR/Cas9 gene editing system was used to edit the genes of the chassis strain, including the transfer of tyrosine ammonia lyase gene, phenylalanine ammonia lyase gene, cinnamate hydroxylase gene, cytochrome B5 gene and cytochrome P450 reduction Enzyme 2 gene, and perform gene editing in any one of the following ways or a combination of at least two:

   (1) Deletion of aryl aminotransferase I and aryl aminotransferase II;

   (2) Deletion of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, overexpression of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase gene;

   (3) Deletion of NAD-dependent isocitrate dehydrogenase 1 or isocitrate dehydrogenase 2, while overexpressing the transporter gene and NADP-dependent isocitrate dehydrogenase 2 gene;

   (4) Deletion of phosphoglucose isomerase gene, phosphofructokinase 1 gene or phosphofructokinase 2 gene, and overexpression of glucose-6-phosphate dehydrogenase gene, 6-phosphogluconate dehydrogenase gene, ribulose 5 gene - phosphate epimerase gene, 5-phosphoribose-ketoisomerase gene, transketolase gene and transaldolase gene;

   (5) Overexpression of NADH kinase gene; or

   (6) Deletion of glutamate dehydrogenase gene;

   Obtain the genetically engineered bacteria that synthesize p-coumaric acid;

   Preferably, the chassis strain includes any one of yeast, Escherichia coli, Bacillus subtilis or microalgae;

   Preferably, the yeast comprises Saccharomyces cerevisiae.
9. Application of the genetically engineered bacteria for the synthesis of p-coumaric acid described in any one of claims 1-7 in the production of p-coumaric acid and/or p-coumaric acid derivatives;

   Preferably, the p-coumaric acid derivatives include any one or a combination of at least two of caffeic acid, p-hydroxystyrene or resveratrol.
10. A method for preparing p-coumaric acid and/or p-coumaric acid derivatives, comprising:

    Fermentation is carried out by utilizing the genetically engineered bacterium for synthesizing p-coumaric acid described in any one of claims 1-7.

Images