A coumaric acid biosensor and its application

Abstract

This invention discloses a coumaric acid biosensor and its application. The coumaric acid biosensor is based on the coding gene of the CarR mutant, P... lpp1.6 Promoter, gene encoding green fluorescent protein, and P that controls green fluorescent protein expression car The promoter mutant is located on the expression vector pTrc99a. The biosensor of this invention enables real-time and sensitive monitoring of coumaric acid concentration in recombinant *E. coli* strains containing a coumaric acid biosensor. It can be combined with high-throughput mutagenesis and screening systems, facilitating rapid and efficient screening of high-yielding coumaric acid engineered strains, and providing technical support for the efficient microbial fermentation production of coumaric acid and its derivatives. Constructing a coumaric acid biosensor based on transcriptional regulatory factors provides a new strategy for high-throughput screening.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, specifically to a coumaric acid biosensor and its applications. Background Technology

[0002] p-Coumaric acid is a phenolic acid compound with important biological and pharmacological effects. It is also a precursor to many phenylpropanoid compounds (such as polyphenols, flavonoids, and certain polyketides). The demand for p-coumaric acid derivatives is growing rapidly. In the past decade, the biosynthesis of p-coumaric acid has been achieved in various microorganisms through the rational rearrangement of microbial chassis metabolic networks, including *Escherichia coli*, *Saccharomyces cerevisiae*, *Corynebacterium glutamicum*, and *Pseudomonas*. Most notably, Liu et al. achieved a p-coumaric acid production level of 12.5 g / L in 96 hours by reconnecting carbon metabolism and precisely adjusting the metabolic flux of the p-coumaric acid biosynthetic pathway in yeast. However, due to a lack of in-depth understanding of the intrinsic complexity of cellular metabolic networks and the relationship between enzyme structure and function, efficient biosynthesis of p-coumaric acid through traditional metabolic engineering strategies (such as push-pull blockade and modularization) remains challenging. In particular, the high cytotoxicity of p-coumaric acid and the low catalytic activity of tyrosine ammonia-lyase severely limit its overproduction. While irrational engineering strategies such as laboratory adaptive evolution, random mutagenesis, and directed evolution can circumvent these cognitive limitations, the capacity of such irrational mutant libraries is enormous, and the limited number of screening options makes it difficult to identify mutants with the desired traits. Therefore, developing high-throughput methods for isolating high-yield p-coumaric acid strains is imperative.

[0003] Currently, several coumaric acid biosensors have been designed based on PadR, a naturally occurring transcriptional regulator of coumaric acid, both domestically and internationally. Li et al. and Jiang et al. constructed coumaric acid biosensor systems using BsPadR from *Bacillus subtilis* and BaPadR from *Bacillus amyloliquefaciens*, respectively, and optimized their dynamic performance by altering PadR expression intensity, site-directed mutagenesis, and hybridization promoter construction. However, these biosensors still suffer from problems such as high fluorescence leakage expression levels, small dynamic range, or the presence of putative genes (yveF and yveG) on the effector promoter. Overall, rapid screening and isolation of high-yielding coumaric acid strains remains significantly limited. Discovering novel transcription factors is crucial to expanding the availability of these biosensor toolsets. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a CarR mutant.

[0005] A second objective of this invention is to provide a coumaric acid biosensor.

[0006] A third objective of this invention is to provide a recombinant Escherichia coli strain containing the aforementioned p-coumaric acid biosensor.

[0007] The fourth objective of this invention is to provide the application of the above-mentioned recombinant Escherichia coli strain screening for high-coumaric acid production strains.

[0008] The technical solution of this invention is summarized as follows:

[0009] A CarR mutant, wherein CarR is a transcriptional regulatory factor derived from Acetobacterium woodii, the amino acid sequence of CarR is shown in SEQ ID NO.1, and the CarR mutant is a CarR mutant in which arginine at position 29 is deleted and isoleucine at position 30 is mutated to glycine.

[0010] A coumaric acid biosensor is based on the coding gene of the aforementioned CarR mutant, P... lpp1.6 Promoter, gene encoding green fluorescent protein, and P that controls green fluorescent protein expression car The promoter mutant is located on the expression vector pTrc99a (commercial);

[0011] The nucleotide sequence of the gene encoding the CarR mutant is shown in SEQ ID No. 3;

[0012] P lpp1.6 The nucleotide sequence of the promoter is shown in SEQ ID No. 6;

[0013] The nucleotide sequence of the gene encoding green fluorescent protein is shown in SEQ ID No. 4;

[0014] P controls the expression of green fluorescent protein car The nucleotide sequence of the promoter mutant is shown in SEQ ID No. 5;

[0015] Among them, P lpp1.6 promoter and the P car The promoter mutants are in the opposite direction.

[0016] A recombinant Escherichia coli strain containing one of the aforementioned p-coumaric acid biosensors.

[0017] The above-mentioned screening of recombinant Escherichia coli strains was applied to high-coumaric acid-producing strains.

[0018] The above application includes the following steps: mutagenesis of the above recombinant Escherichia coli strain to obtain mutant strains, fermentation culture, sorting by flow cytometry, and selecting cells with rising green fluorescent protein signals, which are the high-yield paracoumaric acid strains.

[0019] Advantages of this invention:

[0020] This invention discloses a p-coumaric acid biosensor that links p-coumaric acid concentration with fluorescence signal intensity, making the p-coumaric acid concentration of the target strain visible. This enables real-time and sensitive monitoring of p-coumaric acid concentration in recombinant *E. coli* strains containing a p-coumaric acid biosensor. The biosensor can be combined with high-throughput mutagenesis and screening systems, facilitating rapid and efficient screening of high-yield p-coumaric acid engineered strains and providing technical support for the efficient fermentation production of p-coumaric acid and its derivatives by microorganisms. Constructing a p-coumaric acid biosensor based on transcriptional regulatory factors provides a new strategy for high-throughput screening (rapid identification of target mutants). Attached Figure Description

[0021] Figure 1 Example graphs of a coumaric acid biosensor;

[0022] Figure 2 The fluorescence signal response curve of the coumaric acid biosensor is shown.

[0023] Figure 3 The graph shows the coumaric acid production of each selected strain. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0025] Example 1

[0026] A CarR mutant, wherein CarR is a transcriptional regulatory factor derived from Acetobacterium woodii, the amino acid sequence of CarR is shown in SEQ ID NO.1, and the CarR mutant is a CarR mutant in which arginine at position 29 is deleted and isoleucine at position 30 is mutated to glycine.

[0027] Example 2

[0028] The construction of a coumaric acid biosensor (control) includes the following steps:

[0029] The CarR encoding gene was codon optimized according to the codon preference of E. coli, and the gene was synthesized by Qingke Company to obtain the plasmid pUC57-CarR containing the CarR encoding gene (SEQ ID No.2).

[0030] Using plasmid pUC57-CarR as a template, primers F1 (SEQ ID No. 7) and R1 (SEQ ID No. 8) were used to amplify the nucleotide fragment (SEQ ID No. 2) of the CarR encoding gene using conventional PCR.

[0031] The P-type primer controlling green fluorescent protein expression was obtained using a splicing method with primers F2 (SEQ ID No. 9) and R2 (SEQ ID No. 10). car Nucleotide fragment of the promoter mutant (SEQ ID No. 5);

[0032] P was obtained by splicing F3 (SEQ ID No. 11) and R3 (SEQ ID No. 12). lpp1.6 Promoter nucleotide fragment (SEQ ID No. 6);

[0033] Using the pCL-PesaS-eGFP plasmid reported in the literature as a template (see Gu, F et al. (2020). Quorumsensing-based dual-function switch and its application in solving two keymetabolic engineering problems), primers F4 and R4 were used to amplify the green fluorescent protein encoding gene egfp (SEQ ID No. 4) using conventional PCR methods;

[0034] Using the Escherichia coli vector pTrc99a as a template (commercially available), primers F5 (SEQ ID No. 15) and R5 (SEQ ID No. 16), F6 (SEQ ID No. 17) and R6 (SEQ ID No. 18) were used to amplify the pTrc99a-1 and pTrc99a-2 plasmid backbones sequentially using conventional PCR methods.

[0035] Using efficient and seamless cloning technology, the four fragments and two plasmid backbones obtained above were assembled. Figure 1 ), where P lpp1.6 The promoter controls the expression of the CarR coding gene (and also the coding gene of the CarR mutant), P car Promoter mutants control the expression of green fluorescent protein (reporter protein).

[0036] P lpp1.6 promoters and P car The promoter mutants are in the opposite direction.

[0037] After screening and verification, a coumaric acid biosensor (control) (essentially a plasmid) pCA-Bio was finally obtained and confirmed by company sequencing.

[0038] To ensure sequence accuracy during amplification, a commercially available, high-efficiency, ultra-fidelity DNA polymerase was used for the PCR reaction. The 50 μL PCR amplification system consisted of: 1 μL Phanta Max Super-Fidelity DNA polymerase, 25 μL 2×PhantaMax Buffer, 4 μL 2.5 mM dNTP Mix, 2 μL 10 μM upstream primer, 2 μL 10 μM downstream primer, 1 μL template, and ddH2O to a final volume of 50 μL.

[0039] The PCR amplification conditions were: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 60℃ annealing for 10s, 72℃ extension for 2min, 35 cycles; 72℃ extension for 5min.

[0040] Employing commercially available, highly efficient, seamless cloning based on recombinases The technique was used for plasmid assembly. The recombination reaction system consisted of 5 μL of 2×2X MultiF Seamless Assembly Mix, 2 μL of Lexnase II, 0.03 pmol of vector backbone, 0.06 pmol of insert fragment, and ddH2O to a final volume of 10 μL. The recombination reaction conditions were as follows: the recombination reaction system was prepared on ice and reacted at 50°C for 30 min. The ligated system was then transferred to DH5α competent cells, mixed, and incubated on ice for 20 min. A heat shock at 42°C for 60 s was performed, followed immediately by an ice incubation of 2-3 min. 900 μL of LB broth was added, and the cells were incubated at 37°C for 1 h. After centrifugation at 8000 rpm for 2 min, a portion of the supernatant was discarded, and approximately 100 μL of the bacterial resuspended cells was plated onto a plate containing 100 mg / L ampicillin and incubated overnight at 37°C. After single colonies grew on the plate, colony PCR and sequencing were performed to identify and sequence the colonies. Positive transformants were selected, transferred to shake-tube culture, and then extracted using a plasmid extraction kit to obtain pCA-Bio.

[0041] Example 3

[0042] The construction of a coumaric acid biosensor includes the following steps:

[0043] Using pCA-Bio obtained in Example 2 as a template, linear plasmids were obtained by amplification with high-fidelity Phanta Max Super-Fidelity DNA polymerase using primers F7 (SEQ ID No. 19) and R7 (SEQ ID No. 20). This one-step, efficient, and seamless cloning method was then employed. The technology was used to assemble the plasmids obtained above, and after screening and verification, the biosensor plasmid pCA-Bio-R29 / I30G was finally obtained and confirmed by company sequencing.

[0044] The biosensor plasmid pCA-Bio-R29 / I30G contains the coding gene for the CarR mutant (SEQ ID No. 3).

[0045] Biosensor plasmids pCA-Bio and pCA-Bio-R29 / I30G were introduced into wild-type Escherichia coli W3110 to obtain recombinant strains containing different biosensors.

[0046] Using exogenous p-coumaric acid concentration as the x-axis and relative fluorescence intensity as the y-axis, the correlation between fluorescence intensity and p-coumaric acid concentration in recombinant strains containing different biosensors was plotted. The results are as follows: Figure 2 As shown, a better-performing coumaric acid biosensor was obtained by molecularly modifying the CarR transcription regulator.

[0047] Example 4: Construction of a recombinant Escherichia coli strain containing a p-coumaric acid biosensor

[0048] 1. Gene editing methods

[0049] This invention employs the CRISPR / Cas9 gene editing method, following the guidelines in the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome editing. Metabolic engineering, 2015, 31:13-21.). The pREDCas9 plasmid carries the pGRB elimination system, the λ phage Red recombination system, and the Cas9 protein expression system, along with spectinomycin resistance (working concentration: 100 mg / L), and is cultured at 32°C. The pGRB plasmid is used to express the gRNA-Cas9 binding region sequence and the terminator sequence, along with ampicillin resistance (working concentration: 100 mg / L), and is cultured at 37°C.

[0050] The specific steps of this method are as follows:

[0051] 1.1 Construction of pGRB plasmid

[0052] Using the CHOPCHOP website tools, a suitable target sequence (PAM: 5'-NGG-3') was identified. Two reverse complementary primers were designed and synthesized. The two primers were annealed to form a DNA double strand, which was then ligated to the pGRB plasmid vector using a seamless cloning enzyme. The entire ligated system was transferred to DH5α competent cells, mixed, and incubated on ice for 20 min. The cells were then heat-shocked at 42°C for 60 s, immediately followed by an ice incubation for 2-3 min. 900 μL of LB liquid medium was added, and the cells were incubated at 37°C for 1 h. After centrifugation at 8000 rpm for 2 min, a portion of the supernatant was discarded, and approximately 100 μL of the bacterial resuspended cells was plated onto a plate containing 100 mg / L ampicillin and incubated overnight at 37°C. After single colonies grew on the plate, colony PCR and sequencing were performed to identify and sequence the colonies. Positive transformants were selected, transferred to shake-tube culture, and extracted using a plasmid extraction kit.

[0053] 1.2 Preparation of Recombinant DNA Fragments

[0054] Recombinant fragments used for knockout or integration were amplified by high-fidelity enzyme PCR, followed by overlap PCR. After verifying the correct bands by gel electrophoresis, the fragments were recovered using a DNA purification and recovery kit.

[0055] 1.3 Preparation of Electrocompetent States

[0056] Pick a single colony from the plate or transfer 10 μL of bacterial culture from a glycerol tube into an LB shaker (for colonies carrying the pREDCas9 plasmid, add 100 mg / L spectinomycin), incubate overnight, then transfer to 2×YT medium and incubate at 32°C until OD500. 600 When the concentration reaches 0.1-0.2, add IPTG to a final concentration of 0.1 mM and continue culturing until the OD value is reached. 600 Competent cells were prepared when the phosphorus content was between 0.6 and 0.7. The preparation process followed standard operating procedures.

[0057] 1.4 Electric Transfer

[0058] pGRB plasmid and recombinant DNA fragment were simultaneously electroporated into competent cells. After electroporation, the recovered bacterial culture was centrifuged and plated entirely onto LB agar plates containing ampicillin and spectinomycin. After overnight incubation at 32°C, colony PCR was performed to screen for positive transformants and the cells were preserved.

[0059] 1.5 Plasmid Elimination

[0060] Positive transformants were transferred to LB shakers containing 0.2% arabinose and spectinomycin and cultured overnight. Single colonies were obtained by dividing the plate into three sections. Single colonies were then picked and spotted onto LB plates resistant to ampicillin and spectinomycin respectively. No growth was observed on the ampicillin plate. Single colonies that grew on the spectinomycin-resistant plate were preserved and transferred to LB shakers without antibiotics. The plates were cultured overnight at 42°C. Single colonies were obtained by dividing the plate into three sections and then spotted onto LB plates resistant to spectinomycin and without antibiotics respectively. No growth was observed on the spectinomycin-resistant plate. Single colonies that grew on the without antibiotics plate were preserved.

[0061] Genes involved in the construction of strain 2

[0062] Table 1 Gene Accession Numbers

[0063]

[0064] Construction of 3. Genetically engineered bacteria producing p-coumaric acid

[0065] 3.1 Knockout of the lacIZ gene

[0066] Using Escherichia coli W3110 as a template, the upstream homologous arm fragment of lacIZ knockout was obtained by PCR with primers lacIZ-UP-F (SEQ ID NO.21) and lacIZ-UP-R (SEQ ID NO.22). The downstream homologous arm fragment of lacIZ knockout was obtained by PCR with primers lacIZ-DN-F (SEQ ID NO.23) and lacIZ-DN-R (SEQ ID NO.24). The upstream and downstream homologous arm fragments were fused by PCR with primers lacIZ-UP-F (SEQ ID NO.21) and lacIZ-DN-R (SEQ ID NO.24) to obtain the fusion fragment of lacIZ knockout.

[0067] The DNA double-stranded fragment was obtained by annealing with primers lacI-gRNA-F (SEQ ID NO.25) and lacI-gRNA-R (SEQ ID NO.26), and the fragment was ligated to the pGRB linear plasmid using the method shown in 1.1 to obtain the pGRB-lacI plasmid.

[0068] The pREDCas9 plasmid was electroporated into W3110 electrocompetent cells to obtain strain W3110-pREDCas9. To prepare W3110-pREDCas9 electrocompetent cells, the fusion fragment with lacIZ knocked out and the pGRB-lacI plasmid were simultaneously electroporated into the W3110-pREDCas9 electrocompetent cells, and strain SAA-1 was obtained according to the methods shown in 1.4 and 1.5.

[0069] 3.2 Knockout of the tyrR gene

[0070] Using Escherichia coli W3110 as a template, the upstream homologous arm fragment of tyrR knockout was obtained by PCR with primers tyrR-UP-F (SEQ ID NO.27) and tyrR-UP-R (SEQ ID NO.28). The downstream homologous arm fragment of tyrR knockout was obtained by PCR with primers tyrR-DN-F (SEQ ID NO.29) and tyrR-DN-R (SEQ ID NO.30). The fusion fragment of tyrR knockout was obtained by PCR by fusing the upstream and downstream homologous arm fragments with primers tyrR-UP-F (SEQ ID NO.27) and tyrR-DN-R (SEQ ID NO.30).

[0071] The DNA double-stranded fragment was obtained by annealing with primers tyrR-gRNA-F (SEQ ID NO.31) and tyrR-gRNA-R (SEQ ID NO.32), and the fragment was ligated to the pGRB linear plasmid using the method shown in 1.1 to obtain the pGRB-tyrR plasmid.

[0072] SAA-1-pREDCas9 electroporation competent cells were prepared by simultaneously electroporating the fusion fragment with tyrR knockout and the pGRB-tyrR plasmid into the SAA-1-pREDCas9 electroporation competent cells, and strain SAA-2 was obtained according to the methods shown in 1.4 and 1.5.

[0073] 3.3 Integration of aroG at the tehB site S180F

[0074] Using *E. coli* W3110 as a template, the upstream homologous arm fragment of tehB knockout was obtained by PCR using primers tehB-UP-F (SEQ ID NO. 33) and tehB-UP-R (SEQ ID NO. 34), the downstream homologous arm fragment of tehB knockout was obtained by PCR using primers tehB-DN-F (SEQ ID NO. 35) and tehB-DN-R (SEQ ID NO. 36), and the aroG was obtained by PCR using primers aroG-F (SEQ ID NO. 39) and aroG-S180F-R (SEQ ID NO. 40). S180F The upstream fragment was obtained by PCR using primers aroG-S180F-F (SEQ ID NO. 41) and aroG-R (SEQ ID NO. 42) to obtain aroG. S180F The downstream fragment was obtained, and the above four fragments were fused by PCR using primers tehB-UP-F (SEQ ID NO. 33) and tehB-DN-R (SEQ ID NO. 36) to obtain ΔtehB::P trc -aroG S180F Fragment fusion.

[0075] The DNA double-stranded fragment was obtained by annealing with primers tehB-gRNA-F (SEQ ID NO.37) and tehB-gRNA-R (SEQ ID NO.38), and the fragment was ligated to the pGRB linear plasmid using the method shown in 1.1 to obtain the pGRB-tehB plasmid.

[0076] To prepare SAA-2-pREDCas9 electrocompetent states, ΔtehB::P trc -aroG S180F The fusion fragment and pGRB-tehB plasmid were simultaneously electrotransferred into SAA-2-pREDCas9 electrocompetent cells, and strain SAA-3 was obtained according to the methods shown in 1.4 and 1.5.

[0077] 3.4 Integration of tyrA at the mbhA site M53I / A354V

[0078] Using *E. coli* W3110 as a template, the upstream homologous arm fragment of mbhA knockout was obtained by PCR using primers mbhA-UP-F (SEQ ID NO.43) and mbhA-UP-R (SEQ ID NO.44), the downstream homologous arm fragment of mbhA knockout was obtained by PCR using primers mbhA-DN-F (SEQ ID NO.45) and mbhA-DN-R (SEQ ID NO.46), and the tyrA was obtained by PCR using primers tyrA-F (SEQ ID NO.49) and tyrA-M53I-R (SEQ ID NO.50). M53I / A354V The first fragment was obtained by PCR using primers tyrA-M53I-F (SEQ ID NO.51) and tyrA-A354V-R (SEQ ID NO.52). M53I / A354V The second fragment was obtained by PCR using primers tyrA-A354V-F (SEQ ID NO.53) and tyrA-R (SEQ ID NO.54). M53I / A354V The third fragment was obtained, and the above five fragments were fused by PCR using primers mbhA-UP-F (SEQ ID NO.43) and mbhA-DN-R (SEQ ID NO.46) to obtain △mbhA::P. trc -tyrA M53I / A354V Fragment fusion.

[0079] The DNA double-stranded fragment was obtained by annealing with primers mbhA-gRNA-F (SEQ ID NO.47) and mbhA-gRNA-R (SEQ ID NO.48), and the fragment was ligated to the pGRB linear plasmid using the method shown in 1.1 to obtain the pGRB-mbhA plasmid.

[0080] To prepare SAA-3-pREDCas9 electrocompetent states, ΔmbhA::P trc -tyrA M53I / A354V The fusion fragment and pGRB-mbhA plasmid were simultaneously electrotransferred into SAA-3-pREDCas9 electrocompetent cells, and strain SAA-4 was obtained according to the methods shown in 1.4 and 1.5.

[0081] 3.5 Knockout of the pheA gene

[0082] Using Escherichia coli W3110 as a template, the upstream homologous arm fragment of pheA knockout was obtained by PCR with primers pheA-UP-F (SEQ ID NO.55) and pheA-UP-R (SEQ ID NO.56), the downstream homologous arm fragment of pheA knockout was obtained by PCR with primers pheA-DN-F (SEQ ID NO.57) and pheA-DN-R (SEQ ID NO.58), and the fusion fragment of pheA knockout was obtained by PCR by fusing the upstream and downstream homologous arm fragments with primers pheA-UP-F (SEQ ID NO.55) and pheA-DN-R (SEQ ID NO.58).

[0083] The DNA double-stranded fragment was obtained by annealing with primers pheA-gRNA-F (SEQ ID NO.59) and pheA-gRNA-R (SEQ ID NO.60), and the fragment was ligated to the pGRB linear plasmid using the method shown in 1.1 to obtain the pGRB-pheA plasmid.

[0084] SAA-4-pREDCas9 electroporation competent cells were prepared by simultaneously electroporating the pheA knockout fusion fragment and the pGRB-pheA plasmid into the SAA-4-pREDCas9 electroporation competent cells, and strain SAA-5 was obtained according to the methods shown in 1.4 and 1.5.

[0085] 3.6 Integration of aroK at the rph site

[0086] Using *E. coli* W3110 as a template, the upstream homologous arm fragment of the knocked-out rph was obtained by PCR using primers rph-UP-F (SEQ ID NO. 61) and rph-UP-R (SEQ ID NO. 62). The downstream homologous arm fragment of the knocked-out rph was obtained by PCR using primers rph-DN-F (SEQ ID NO. 63) and rph-DN-R (SEQ ID NO. 64). The aroK fragment was obtained by PCR using primers aroK-F (SEQ ID NO. 67) and aroK-R (SEQ ID NO. 68). Finally, the above three fragments were fused by PCR using primers rph-UP-F (SEQ ID NO. 61) and rph-DN-R (SEQ ID NO. 64) to obtain Δrph::P. trc -aroK fusion fragment.

[0087] The DNA double-stranded fragment was obtained by annealing with primers rph-gRNA-F (SEQ ID NO.65) and rph-gRNA-R (SEQ ID NO.66), and the fragment was ligated to the pGRB linear plasmid using the method shown in 1.1 to obtain the pGRB-rph plasmid.

[0088] To prepare SAA-5-pREDCas9 electrocompetent states, Δrph::P trc The -aroK fusion fragment and pGRB-rph plasmid were simultaneously electrotransformed into SAA-5-pREDCas9 electrotransformation competent cells, and strain SAA-6 was obtained according to the methods shown in 1.4 and 1.5.

[0089] 3.7 Integration of ydiB at the yeep site

[0090] Using *E. coli* W3110 as a template, the upstream homologous arm fragment of yeep knockout was obtained by PCR using primers yeeP-UP-F (SEQ ID NO. 69) and yeeP-UP-R (SEQ ID NO. 70). The downstream homologous arm fragment of yeeP knockout was obtained by PCR using primers yeeP-DN-F (SEQ ID NO. 71) and yeeP-DN-R (SEQ ID NO. 72). The ydiB fragment was obtained by PCR using primers ydiB-F (SEQ ID NO. 75) and ydiB-R (SEQ ID NO. 76). Finally, the above three fragments were fused by PCR using primers yeeP-UP-F (SEQ ID NO. 69) and yeeP-DN-R (SEQ ID NO. 72) to obtain ΔyeeP::P. trc -ydiB fusion fragment.

[0091] The DNA double-stranded fragment was obtained by annealing with primers yeeP-gRNA-F (SEQ ID NO.73) and yeeP-gRNA-R (SEQ ID NO.74), and the fragment was ligated to the pGRB linear plasmid using the method shown in 1.1 to obtain the pGRB-yeeP plasmid.

[0092] To prepare SAA-6-pREDCas9 electrocompetent states, ΔyeeP::P trc The -ydiB fusion fragment and the pGRB-yeeP plasmid were simultaneously electrotransferred into SAA-6-pREDCas9 electrocompetent cells, and strain SAA-7 was obtained according to the methods shown in 1.4 and 1.5.

[0093] 3.8 Integration of FjTAL at the yghE site

[0094] Using *E. coli* W3110 as a template, the upstream homologous arm fragment of yghE knockout was obtained by PCR using primers yghE-UP-F (SEQ ID NO.77) and yghE-UP-R (SEQ ID NO.78). The downstream homologous arm fragment of yghE knockout was obtained by PCR using primers yghE-DN-F (SEQ ID NO.79) and yghE-DN-R (SEQ ID NO.80). Using the synthesized sequence FjTAL (SEQ ID NO.81) as a template, the FjTAL fragment was obtained by PCR using primers FjTAL-F (SEQ ID NO.84) and FjTAL-R (SEQ ID NO.85). The upstream and downstream homologous arm fragments of yghE and the FjTAL fragment were then fused by PCR using primers yghE-UP-F (SEQ ID NO.77) and yghE-DN-R (SEQ ID NO.80) to obtain ΔyghE::P. trc -FjTAL fusion fragment.

[0095] The DNA double-stranded fragment was obtained by annealing with primers yghE-gRNA-F (SEQ ID NO.82) and yghE-gRNA-R (SEQ ID NO.83), and the fragment was ligated to the pGRB linear plasmid using the method shown in 1.1 to obtain the pGRB-yghE plasmid.

[0096] To prepare SAA-10-pREDCas9 electrocompetent states, ΔyghE::P trc The -FjTAL fusion fragment and the pGRB-yghE plasmid were simultaneously electrotransformed into SAA-10-pREDCas9 electrocompetent cells, and strain p-CA3 was obtained according to the methods shown in 1.4 and 1.5.

[0097] 4. Construction of a recombinant Escherichia coli strain for a coumaric acid biosensor

[0098] The p-coumaric acid biosensor pCA-Bio-R29 / I30G was introduced into strain p-CA3 via electroporation to obtain recombinant Escherichia coli p-CA3-BIO.

[0099] Example 5: Application of Recombinant Escherichia coli Strain Screening to High-Yield Coumaric Acid Strains

[0100] Coumaric acid, being a small molecule compound, is difficult to detect intracellularly. A coumaric acid biosensor can sensitively detect changes in intracellular coumaric acid concentration and output a fluorescence signal. By coupling with high-throughput screening equipment such as flow cytometry, rapid sorting at the fluorescence level can be achieved, significantly improving the speed of breeding high-yield strains and the potential for efficient screening of target strains from diverse mutant libraries.

[0101] The screening procedure is as follows: The recombinant Escherichia coli p-CA3-BIO constructed in Example 4 was inoculated into 5 mL of LB liquid medium and cultured at 37°C with shaking at 200 rpm for 10 h. After centrifugation, the bacterial cells were collected, washed with 10% glycerol, and resuspended to a cell concentration of OD. 600 =1.0. Take 10 μL of bacterial cells and perform random mutagenesis using an ambient pressure room temperature plasma ARTP mutagenesis breeding instrument. The instrument parameters are set as follows: power 120W, gas flow rate 10 SLM, and treatment time 35s. The mutagenized bacterial cells are suspended in 1 mL of LB fresh liquid culture medium and incubated at 37℃ with shaking at 200 rpm for 1 h for recovery. After recovery, the cells are incubated at the initial concentration OD. 600 =0.1 g of the bacterial culture medium was inoculated into fresh p-coumaric acid fermentation medium and fermented at 37°C and 220 rpm for 12 h. After fermentation was completed, the bacterial cells were washed with pH 7.4 PBS buffer and resuspended at OD200. 600 =0.1, the bacterial cells were screened using flow cytometry, and cells with fluorescence intensity in the top 0.5% were directly collected and spotted onto LB agar plates. After static incubation at 37°C for 24 hours, when clear single colonies appeared on the LB plates, the colonies were picked and transferred to 96-well plates containing p-coumaric acid fermentation medium for rescreening. Fermentation was carried out at 30°C and 220 rpm for 24 hours. The cells were collected by centrifugation, resuspended in an equal volume of 0.9% NaCl aqueous solution, and their relative fluorescence intensity was measured using a Varioskan LUX multi-functional microplate reader, with the excitation wavelength set at 488 nm and the emission wavelength at 520 nm. Simultaneously, the bacterial OD was measured. 600 Value, combining fluorescence intensity and OD 600The ratio was defined as the relative fluorescence intensity. Mutant strains with higher relative fluorescence intensity than the starting strain p-CA3-BIO were selected and passaged to remove the biosensor plasmid pCA-Bio-R29 / I30G before subsequent shake-flask fermentation. After 24 hours of fermentation, samples were taken to determine the coumaric acid content.

[0102] The components of the coumaric acid fermentation medium described in this embodiment are: yeast powder 3g / L, ammonium sulfate 1g / L, potassium dihydrogen phosphate 4g / L, sodium sulfate heptahydrate 1g / L, ammonium citrate 0.5g / L, ferric sulfate heptahydrate 30mg / L, manganese sulfate monohydrate 10mg / L, trace element mixture 1mL / L (Na2MoO4·2H2O 2.5g / L, AlCl3·6H2O 2.5g / L, NiSO4·6H2O 2.5g / L, CoCl2·6H2O 1.75g / L, CaCl2·2H2O 10g / L, ZnSO4·7H2O 0.5g / L, CuCl2·2H2O 0.25g / L, H3BO3 0.125g / L), vitamin B1 0.5mg / L, vitamin H 0.5mg / L, and glucose 20g / L.

[0103] The method for determining the coumaric acid content described in this embodiment uses an Agilent 1260 high-performance liquid chromatography system equipped with a reversed-phase C18 column (4.6 × 250 mm, Thermo, USA) and a flow rate of 1.0 mL / min. -1 The temperature was 40℃ (see Wang, L et al. (2023). Enhancing caffeic acid production in Escherichia coliby engineering the biosynthesis pathway and transporter. Bioresour Technol. 368, 128320.). The content of p-coumaric acid in the fermentation sample was calculated based on the standard curve corresponding to p-coumaric acid and absorbance.

[0104] Through shake-flask fermentation tests, ten mutant strains with higher coumaric acid fermentation yields than the starting strain p-CA3-BIO were finally obtained, such as... Figure 3As shown, they were named M1, M2, M3, M4, M5, M6, M7, M8, M9, and M10, respectively. The M5 mutant produced approximately 1.59 times the p-coumaric acid yield of the starting strain p-CA3. The above examples demonstrate that the p-coumaric acid biosensor of this invention can sensitively respond to the characteristics of p-coumaric acid, and can be coupled with flow cytometry and other screening methods to efficiently develop and establish a screening platform for high-coumaric acid-producing strains. Based on this screening platform, high-coumaric acid-producing strains can be rapidly and efficiently screened.

Claims

1. A CarR mutant, wherein CarR is a transcriptional regulatory factor derived from *Acetobacterium woodii*, and the amino acid sequence of CarR is shown in SEQ ID NO.1, characterized in that... The CarR mutant is an amino acid sequence shown in SEQ ID NO.1 with arginine missing at position 29 and isoleucine at position 30 mutated to glycine.

2. A coumaric acid biosensor, characterized in that... The coding gene of the CarR mutant of claim 1, P lpp1.6 Promoter, gene encoding green fluorescent protein, and P that controls green fluorescent protein expression car The promoter mutant is located on the expression vector pTrc99a; where P lpp1.6 The promoter controls the expression of the coding gene in the CarR mutant; The nucleotide sequence of the gene encoding the CarR mutant is shown in SEQ ID No. 3; P lpp1.6 The nucleotide sequence of the promoter is shown in SEQ ID No. 6; The nucleotide sequence of the gene encoding green fluorescent protein is shown in SEQ ID No. 4; P controls the expression of green fluorescent protein car The nucleotide sequence of the promoter mutant is shown in SEQ ID No. 5; Wherein, P lpp1.6 promoter and the P car The promoter mutants are in the opposite direction.

3. A recombinant Escherichia coli strain containing a p-coumaric acid biosensor according to claim 2.

4. The application of the recombinant Escherichia coli strain screening method of claim 3 to high-coumaric acid-producing strains.

5. The application according to claim 4, characterized in that... The process includes the following steps: mutagenesis of the recombinant Escherichia coli strain of claim 3 to obtain a mutant strain, fermentation culture, sorting by flow cytometry, and selecting cells with rising green fluorescent protein signals, which are the high-yield paracoumaric acid strains.

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