Genetically engineered bacterium for high yield of p-coumaric acid and application of genetically engineered bacterium
By heterologously expressing the tyrosine ammonia-lyase gene in Escherichia coli BL21, a recombinant engineered strain pCA8 with a high-copy vector was constructed, solving the problem of low yield of p-coumaric acid synthesized by Escherichia coli and realizing efficient and low-cost p-coumaric acid production.
Patent Information
- Application Number
- CN202511928189.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-19
- Publication Date
- 2026-03-17
AI Technical Summary
The current technology for synthesizing coumaric acid by Escherichia coli has a low yield, which is difficult to meet market demand. In addition, traditional methods have problems such as long cycle time, high cost, and environmental pollution.
The genes for tyrosine ammonia-lyases (FjTAL and FsTAL) derived from Flavobacterium johnsonii and Flavobacterium shuntianii were heterologously expressed in Escherichia coli BL21. Recombinant engineered strains with different copy numbers were constructed, and the optimal strain pCA8 was screened out. Coumaric acid was synthesized efficiently through shake-flask fermentation.
Highly efficient production of coumaric acid was achieved, with a yield of 251.61 mg/L, significantly increasing output and reducing production costs.
Smart Images
Figure CN121674442A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the field of synthetic biology and metabolic engineering, and particularly relates to a genetically engineered bacterium with high yield of p-coumaric acid and application thereof. BACKGROUND
[0002] P-coumaric acid (p-CA), also known as p-hydroxycinnamic acid, is an important phenylpropanoid natural compound in plants. It is a key node connecting primary metabolism and phenylpropanoid secondary metabolism, and is a direct precursor of many important bioactive substances (such as flavonoids, lignans, and resveratrol). It also has significant antioxidant, antibacterial, anti-inflammatory, and anti-ultraviolet radiation biological activities, and is widely used in biomedical, food industry, cosmetics, and other fields. At present, the production methods of p-coumaric acid mainly include plant extraction, chemical synthesis, and microbial fermentation. The plant extraction method has problems such as long cycle, low yield, limitation by season and geographical environment, complex extraction process, and high cost, which is difficult to meet the growing market demand. The chemical synthesis method often involves harsh reaction conditions, expensive catalysts, and environmental pollution problems, and it is difficult to achieve green production of optically pure products. The use of microorganisms to synthesize natural compounds such as p-coumaric acid has attracted widespread attention due to its low production cost and high conversion efficiency, and has become a strategic direction to solve the supply bottleneck, with broad industrial production potential and application prospect.
[0003] In the microbial body, p-coumaric acid is mainly generated through the metabolic pathway of phenylalanine or tyrosine. Among them, tyrosine is directly used as a precursor, and tyrosine ammonia lyase (TAL) is used to catalyze deamination to generate p-coumaric acid, which is a relatively efficient path. However, at present, the yield of p-coumaric acid synthesized from scratch by using Escherichia coli as a chassis cell is at a relatively low level. Therefore, it is crucial to develop a genetically engineered bacterium capable of efficiently synthesizing p-coumaric acid. SUMMARY
[0004] The technical problem to be solved is that in view of the above existing technical problems, the purpose of the present application is to provide a genetically engineered bacterium with high yield of p-coumaric acid and application thereof. By heterologous expression of tyrosine ammonia lyase (FjTAL, FsTAL) genes from Johnsonia and Shuntianella in Escherichia coli BL21 (DE3), eight kinds of recombinant engineering bacteria (pCA1~pCA8) containing low, medium, and high copy numbers (pACYCDuet-1, pCDFDuet-1, pETCDuet-1, pRSFDuet-1) are obtained. Through shake flask fermentation screening, the optimal strain is determined to be pCA8. The strain pCA8 uses the high-copy vector pRSFDuet-1 to express the FsTAL enzyme, and can synthesize 251.61 mg / L of p-coumaric acid from scratch in M9Y medium after 48 h of fermentation.
[0005] Technical solution: A method for constructing a genetically engineered bacterium with high p-coumaric acid yield, comprising the following steps:
[0006] S1. Obtain the corresponding sequences of tyrosine ammonia-lyase FjTAL and FsTAL from Flavobacterium sources from the NCBI database, and use the method of homologous recombination to link them with different copy numbers of expression vectors pACYCDuet-1, pCDFDuet-1, pETCDuet-1, and pRSFDuet-1, respectively, to construct recombinant expression vectors;
[0007] S2. Transfer the constructed recombinant expression vectors into Escherichia coli DH5α by heat shock method, extract the recombinant plasmid, and transfer it into the chassis cell Escherichia coli BL21(DE3) by heat shock method to obtain an engineered strain pCA1~pCA8 that can express tyrosine ammonia-lyase and synthesize p-coumaric acid.
[0008] The tyrosine ammonia-lyase FjTAL in the above step S1 is derived from Flavobacterium johnsoniae, and the amino acid sequence of FjTAL is shown in SEQ ID NO. 1.
[0009] The tyrosine ammonia-lyase FsTAL in the above step S1 is derived from Flavobacterium columnare, and the amino acid sequence of FsTAL is shown in SEQ ID NO. 2.
[0010] The above genetically engineered bacterium with high p-coumaric acid yield is applied in the production of p-coumaric acid.
[0011] The above method for producing p-coumaric acid comprises the following steps:
[0012] S1. Inoculate the above constructed engineered strain on an LB plate, place it in a constant temperature incubator at 25~37℃, and invert it for overnight culture. After reactivation, pick a single colony and inoculate it into a flask containing fresh liquid LB medium, and culture it at 25~37℃, 180~220 rpm for 7~8 h as a primary seed liquid;
[0013] S2. Inoculate the primary seed liquid into LB liquid medium, and culture it at 25~37℃, 180~220 rpm overnight as a secondary seed liquid;
[0014] S3. Inoculate the secondary seed liquid into M9Y medium, and culture it at 25~37℃, 180~220 rpm until OD 600 0.6~0.8; add IPTG, and induce expression and culture at 25~37℃, 180~220 rpm for 40~48 h.
[0015] Preferably, the inoculation amount of the primary seed liquid in step S2 is 1~2%.
[0016] Preferably, the inoculation amount of the secondary seed liquid in step S3 is 1~2%.
[0017] Preferably, the final concentration of IPTG in step S3 is 0.3~1 mM.
[0018] Beneficial effects:
[0019] Based on the known pathway of p-coumaric acid synthesis, this invention utilizes homologous recombination technology to construct tyrosine ammonia-lyase (TAL) from two different Flavobacterium sources on vectors with different copy numbers. The optimal recombinant vector combination, pRSFDuet-FsTAL, was screened to effectively drive the conversion of tyrosine to the target product, p-coumaric acid. The genetically engineered bacteria pCA1-pCA8 constructed in this invention can efficiently produce p-coumaric acid via fermentation using glucose as a substrate. Experimental verification revealed that the optimal strain, pCA8, can efficiently express the tyrosine ammonia-lyase FsTAL from Flavobacterium hunnanium using the high-copy plasmid pRSFDuet-1. Its p-coumaric acid yield is significantly higher than that of low-copy or medium-copy vectors or other strains containing FjTAL from Flavobacterium johnsonii. After 48 hours of shake-flask culture and HPLC detection, the engineered bacteria pCA8 achieved a de novo p-coumaric acid yield of 251.61 mg / L using glucose as a substrate. Attached Figure Description
[0020] Figure 1 This is a plasmid map of the recombinant expression vector pRSFDuet-FsTAL in Example 8;
[0021] Figure 2 Gel electrophoresis images of engineered bacteria pCA1~pCA8;
[0022] Figure 3 The graph shows the production of p-coumaric acid by engineered bacteria pCA1~pCA8. Detailed Implementation
[0023] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments:
[0024] Example 1
[0025] In this embodiment, the target gene is FjTAL, the vector is pACYCDuet-1, and the construction method of the engineered strain pCA1 / pACYCDuet-FjTAL (hereinafter referred to as pCA1) and the method of producing p-coumaric acid by shake-flask fermentation using engineered strain pCA1 include the following steps:
[0026] S1. PCR amplification and purification of gene fragments: Specific primers with homologous arms were designed. Using the FjTAL gene fragment as a template, primer pair FjTAL-F / FjTAL-R was used to amplify FjTAL. The primer sequences are shown in Table 1, and the amplification system and conditions are shown in Tables 2-3. The amplification products were excised and purified to obtain the target gene fragment.
[0027] Table 1 Primer Sequences
[0028]
[0029] Note: The underlined parts are homologous arm sequences;
[0030] Table 2 Amplification System
[0031]
[0032] Table 3 Amplification conditions
[0033]
[0034] S2. Construction of recombinant expression vector pACYCDuet-FjTAL: The recombinant expression vector pACYCDuet-1 was constructed using homologous recombination. The pACYCDuet-1 plasmid was double-digested with restriction endonucleases BamHI and HindIII, followed by agarose gel electrophoresis. The linearized vector was purified by gel extraction and recovery. The linearized vector fragment and the target gene fragment FjTAL were purified and mixed at a molar ratio of 1:2 using a homologous recombination cloning kit. The recombinant product was ligated at 50℃ for 5 min to obtain the recombinant expression vector pACYCDuet-FjTAL containing the target gene FjTAL.
[0035] S3. Construction of engineered strain pCA1: The recombinant expression vector plasmid pACYCDuet-FjTAL was transformed into Escherichia coli BL21(DE3) by heat shock method, plated on LB solid plates containing chloramphenicol, and colony PCR was performed on the grown single clones to confirm that the recombinant plasmid was transformed into BL21(DE3). The positive transformants were expanded to obtain engineered strain pCA1, which was stored at -80℃ for later use.
[0036] S4. The engineered strain pCA1 was streaked onto an LB agar plate and incubated overnight at 37°C inverted to reactivate. Single colonies of the engineered strain were picked and inoculated into shake flasks containing fresh LB (chloramphenicol) and cultured at 37°C and 200 rpm for 8 h to obtain the primary seed culture. The primary seed culture was inoculated at a ratio of 1% into LB liquid medium and incubated overnight at 37°C and 200 rpm to obtain the secondary seed culture. The secondary seed culture was then inoculated into M9Y medium and cultured at 30°C and 200 rpm until OD...600 The concentration was increased to between 0.6 and 0.8; IPTG was added to achieve a final concentration of 0.5 mM, and expression was induced at 30°C and 200 rpm for 48 h.
[0037] Example 2
[0038] In this embodiment, the target gene is FjTAL, the vector is pCDFCDuet-1, and the construction method of the engineered strain pCA2 / pCDFCDuet-FjTAL (hereinafter referred to as pCA2) and the method of producing p-coumaric acid by shake-flask fermentation using engineered strain pCA2 are the same as in Example 1.
[0039] Example 3
[0040] In this embodiment, the target gene is FjTAL, the vector is pETDuet-1, and the construction method of the engineered strain pCA3 / pETDuet-FjTAL (hereinafter referred to as pCA3) and the method of producing p-coumaric acid by shake-flask fermentation using engineered strain pCA3 are the same as in Example 1.
[0041] Example 4
[0042] In this embodiment, the target gene is FjTAL, the vector is pRSFDuet-1, and the construction method of the engineered strain pCA4 / pRSFDuet-FjTAL (hereinafter referred to as pCA4) and the method of producing p-coumaric acid by shake-flask fermentation using engineered strain pCA4 are the same as in Example 1.
[0043] Example 5
[0044] In this embodiment, the target gene is FsTAL, the vector is pACYCDuet-1, the construction method of the engineered strain pCA5 / pACYCDuet-FsTAL (hereinafter referred to as pCA5) and the method of producing p-coumaric acid by shake-flask fermentation of engineered strain pCA5 are the same as in Example 1, and the primer sequences are shown in Table 4.
[0045] Table 4 Primer Sequences
[0046]
[0047] Note: The underlined part is the homologous arm sequence.
[0048] Example 6
[0049] In this embodiment, the target gene is FsTAL, the vector is pCDFCDuet-1, and the construction method of the engineered strain pCA6 / pCDFCDuet-FsTAL (hereinafter referred to as pCA6) and the method of producing p-coumaric acid by shake-flask fermentation using engineered strain pCA6 are the same as in Example 5.
[0050] Example 7
[0051] In this embodiment, the target gene is FsTAL, the vector is pETDuet-1, and the construction method of the engineered strain pCA7 / pETDuet-FsTAL (hereinafter referred to as pCA7) and the method of producing p-coumaric acid by shake-flask fermentation using engineered strain pCA7 are the same as in Example 5.
[0052] Example 8
[0053] In this embodiment, the target gene is FsTAL, the vector is pRSFDuet-1, and the construction method of the engineered strain pCA8 / pRSFDuet-FsTAL (hereinafter referred to as pCA8) and the method of producing p-coumaric acid by shake-flask fermentation using engineered strain pCA8 are the same as in Example 5.
[0054] like Figure 2 As shown, the recombinant strains pCA1 to pCA8 from Examples 1 to 8 were successfully constructed by colony PCR identification.
[0055] Indicator Test
[0056] 1. Detection of coumaric acid yield:
[0057] The fermentation broth was diluted 5-fold with ultrapure water, and the diluted sample was filtered through a 0.22 μm filter membrane for later use. The yield of p-coumaric acid was detected by HPLC at 40℃. The chromatographic column used was a ZORBAX Eclipse Plus C18 column (4.6×150 mm, 5 μm), the flow rate was 0.5 mL / min, the injection volume was 10 μL, the mobile phase A was an aqueous solution containing 0.1% formic acid, and the mobile phase B was acetonitrile containing 0.1% formic acid. The UV detection wavelength was 310 nm. The elution conditions were: 0~1 min, 5% mobile phase B; 1~20 min, 5~20% mobile phase B; 20~25 min, 20~90% mobile phase B; 25~27 min, 90% mobile phase B; 27~28 min, 90%~5% mobile phase B; 28~30 min, 5% mobile phase B.
[0058] like Figure 3 As shown, p-coumaric acid was detectable in recombinant E. coli pCA1~pCA8 induced by IPTG, with pRSFDuet-1 showing the best effect, followed by pCDFDuet-1, pACYCDuet-1, and pETDuet-1. Regarding the detection results of tyrosine ammonia-lyases from different sources, FsTAL showed better performance than p-coumaric acid. The optimal combination was pRSFDuet-FsTAL (Example 8), which could de novo synthesize 251.61 mg / L of p-coumaric acid after 48 h of culture.
[0059] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.
Claims
1. A method for constructing a genetically engineered bacterium that produces high levels of p-coumaric acid, characterized in that, The method comprises the following steps: S1. Obtain the corresponding sequences of tyrosine ammonia-lyases FjTAL and FsTAL from Flavobacterium from the NCBI database, and link them to expression vectors pACYCDuet-1, pCDFDuet-1, pETCDuet-1 and pRSFDuet-1 with different copy numbers by homologous recombination to construct recombinant expression vectors; S2. Transfer the constructed recombinant expression vectors into Escherichia coli DH5α by heat shock method, extract the recombinant plasmids, and then transfer them into the chassis cell Escherichia coli BL21(DE3) by heat shock method to obtain an engineering strain pCA1-pCA8 capable of expressing tyrosine ammonia-lyase and synthesizing p-coumaric acid.
2. The method for constructing a genetically engineered p-coumaric acid-producing bacterium according to claim 1, characterized by: The tyrosine ammonia-lyase FjTAL in step S1 is derived from Johnson Flavobacterium, and the amino acid sequence of FjTAL is shown as SEQ ID NO.
1.
3. The method for constructing a genetically engineered p-coumaric acid-producing bacterium according to claim 1, characterized by: The tyrosine ammonia-lyase FsTAL in step S1 is derived from Shangtian Flavobacterium, and the amino acid sequence of FsTAL is shown as SEQ ID NO.
2.
4. A genetically engineered bacteria with high yield of p-coumaric acid constructed by the construction method according to any one of claims 1-3.
5. The genetically engineered bacteria with high yield of p-coumaric acid according to claim 4 for use in the production of p-coumaric acid.
6. Use according to claim 5, characterized in that, The method for producing p-coumaric acid comprises the following steps: S1. Inoculate the engineering strain constructed in claim 1 on an LB plate, place it in a constant temperature incubator at 25-37℃, and incubate it overnight. After reactivation, pick a single colony and inoculate it into a flask containing fresh liquid LB medium, and incubate it at 25-37℃ and 180-220 rpm for 7-8 h as a primary seed liquid; S2. Inoculate the primary seed liquid into LB liquid medium, and incubate it at 25-37℃ and 180-220 rpm overnight as a secondary seed liquid; S3. Inoculate the secondary seed liquid into M9Y medium, cultivate at 25-37 °C, 180-220 rpm to OD 600 0.6-0.8; add IPTG, induce expression at 25-37 °C, 180-220 rpm and cultivate for 40-48 h.
7. Use according to claim 6, characterized in that: The inoculation amount of the primary seed liquid in step S2 is 1-2%.
8. Use according to claim 6, characterized in that: The inoculation amount of the secondary seed liquid in step S3 is 1-2%.
9. Use according to claim 6, characterized in that: The final concentration of IPTG in step S3 is 0.3-1 mM.