P-coumaric acid biosensor and application thereof

Abstract

The invention discloses a p-coumaric acid biosensor and application thereof, and the p-coumaric acid biosensor is characterized in that a coding gene of a CarR mutant, a Plpp1.6 promoter, a coding gene of a green fluorescent protein and a Pcar promoter mutant for controlling the expression of the green fluorescent protein are located on an expression vector pTrc99a; the biosensor provided by the invention realizes real-time sensitive monitoring of the concentration of coumaric acid by a recombinant escherichia coli strain containing the coumaric acid biosensor, can be combined with a high-throughput mutagenesis and screening system, and is beneficial to rapid and efficient screening of high-yield engineering strains of coumaric acid; and a technical guarantee is provided for production of p-coumaric acid and derivatives by efficient fermentation of microorganisms. The p-coumaric acid biosensor based on transcriptional regulatory factors is constructed, and a new strategy is provided for high-throughput screening.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to a p-coumaric acid biosensor and applications thereof. Background Art

[0002] p-Coumaric acid is a phenolic acid compound with important biological and pharmacological effects. It is also a precursor of many phenylpropanoids such as polyphenols, flavonoids, and certain polyketides. The demand for p-coumaric acid derivative products is growing rapidly. In the past decade, the biosynthesis of p-coumaric acid has been achieved in a variety of microorganisms, including Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Pseudomonas sp. by rationally reconfiguring the microbial chassis metabolic network. Most prominently, Liu et al. achieved a p-coumaric acid production level of 12.5 g / L within 96 h by rewiring carbon metabolism and precisely adjusting the metabolic flux of the p-coumaric acid biosynthetic pathway in yeast. However, limited by a deep understanding of the intrinsic complexity of the cellular metabolic network and the relationship between enzyme structure and function, efficient biosynthesis of p-coumaric acid through traditional metabolic engineering strategies such as push-pull blocking and modularization remains difficult. In particular, the high cytotoxicity of p-coumaric acid and the low catalytic activity of tyrosine ammonia lyase severely limit the overproduction of p-coumaric acid. Although non-rational engineering strategies such as laboratory adaptive evolution, random mutagenesis, and directed evolution can avoid these cognitive limitations, the capacity of such non-rational mutation libraries is extremely large, and the lack of screening quantity makes it difficult to screen out mutants with desired traits. Therefore, it is imperative to develop high-throughput methods for isolating p-coumaric acid high-producing strains.

[0003] At present, some p-coumaric acid biosensors have been designed based on the p-coumaric acid-responsive transcriptional regulator PadR that exists in nature. Li et al. and Jiang et al. constructed p-coumaric acid biosensor systems using BsPadR of Bacillus subtilis and BaPadR of Bacillus amyloliquefaciens, respectively, and optimized their dynamic performance by changing the expression intensity of PadR, site-directed mutagenesis, and hybrid promoter construction. However, these biosensors still have problems such as large fluorescence leakage expression, small dynamic range, or the presence of hypothetical genes (yveF and yveG) on the effector promoter. In general, the rapid screening and isolation of p-coumaric acid high-producing strains are still greatly limited. Mining new transcription factors is crucial to expand the availability of these tool sets for biosensors. Summary of the invention

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

[0005] The second object of the present invention is to provide a p-coumaric acid biosensor.

[0006] The third object of the present invention is to provide a recombinant Escherichia coli strain containing the above-mentioned p-coumaric acid biosensor.

[0007] The fourth object of the present invention is to provide the use of the above-mentioned recombinant Escherichia coli strain to screen for high-coumaric acid production strains.

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

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

[0010] A p-coumaric acid biosensor is a gene encoding the CarR mutant, a P lpp1.6 Promoter, green fluorescent protein coding gene and P controlling green fluorescent protein expression car The promoter mutants were located on the expression vector pTrc99a (commercial product);

[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 that 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 The promoter and the P car The promoter mutants have the opposite orientation.

[0016] A recombinant Escherichia coli strain containing the above-mentioned p-coumaric acid biosensor.

[0017] The application of the above recombinant Escherichia coli strain to screen high-coumaric acid producing strains.

[0018] The above application comprises the following steps: mutagenizing the above recombinant Escherichia coli strain to obtain mutant strains, fermenting and culturing, sorting by flow cytometry, and selecting cells with increased green fluorescent protein signals, which are high-producing strains of p-coumaric acid.

[0019] Advantages of the present invention:

[0020] A p-coumaric acid biosensor of the present invention links the p-coumaric acid concentration with the intensity of the fluorescent signal, visualizes the p-coumaric acid concentration of the strain to be tested, and realizes real-time sensitive monitoring of the p-coumaric acid concentration of the recombinant Escherichia coli strain containing a p-coumaric acid biosensor. The biosensor of the present invention can be combined with a high-throughput mutagenesis and screening system, which is helpful for rapid and efficient screening of p-coumaric acid high-yield engineering strains, and provides technical support for efficient fermentation production of p-coumaric acid and its derivatives by microorganisms. The construction of a p-coumaric acid biosensor based on transcriptional regulatory factors provides a new strategy for realizing high-throughput screening (rapid identification of target mutants). BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is an example spectrum of the p-coumaric acid biosensor;

[0022] Figure 2 is the fluorescence signal response curve of p-coumaric acid biosensor;

[0023] Figure 3 This is a graph showing the p-coumaric acid production of each screened strain. DETAILED DESCRIPTION

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

[0025] Example 1

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

[0027] Example 2

[0028] A p-coumaric acid biosensor (control) is constructed, comprising the following steps:

[0029] The CarR coding gene was codon optimized according to the codon preference of Escherichia coli, and the gene synthesis was commissioned to Qingke Company to obtain the plasmid pUC57-CarR containing the CarR coding gene (SEQ ID No. 2).

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

[0031] The primers F2 (SEQ ID No. 9) and R2 (SEQ ID No. 10) were used to obtain the P sequence for controlling the expression of green fluorescent protein. car Nucleotide fragment of the promoter mutant (SEQ ID No. 5);

[0032] The 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). Quorum sensing-based dual-function switch and its application in solving two key metabolic engineering problems), primers F4 and R4 were used to amplify the green fluorescent protein coding gene egfp (SEQ ID No. 4) by ordinary PCR method;

[0034] Using the E. coli vector pTrc99a as a template (commercially available), using primers F5 (SEQ ID No.15) and R5 (SEQ IDNo.16), F6 (SEQ ID No.17) and R6 (SEQ ID No.18), the ordinary PCR method was used to amplify the pTrc99a-1 and pTrc99a-2 plasmid backbones in turn.

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

[0036] P lpp1.6 Promoter and P car The promoter mutants have the opposite orientation.

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

[0038] In order to ensure the correctness of the sequence in the amplification, the PCR reaction was carried out using the currently commercially available high-efficiency super-fidelity DNA polymerase. The PCR 50μL amplification system was: 1μL Phanta Max Super-Fidelity DNA polymerase, 25μL 2×PhantaMax Buffer, 4μL 2.5mM dNTP Mix, 2μL 10μM upstream primer, 2μL 10μM downstream primer, 1μL template, and ddH2O was used to make up to 50μL.

[0039] PCR amplification conditions were as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 60°C for 10 s, extension at 72°C for 2 min, 35 cycles; and extension at 72°C for 5 min.

[0040] Efficient and seamless cloning using commercially available recombinase-based Plasmid assembly was performed using the technology. The recombination reaction system was: 5 μL 2×2X MultiF Seamless Assembly Mix, 2 μL Express II, 0.03 pmol vector backbone, 0.06 pmol insert fragment, and ddH2O was added to 10 μL system. The recombination reaction conditions were: configure the recombination reaction system on ice, react at 50°C for 30 min, transfer the entire connected system to the DH5a competent medium, mix well, ice bath for 20 min, heat shock at 42°C for 60 s, and then immediately ice bath for 2-3 min, add 900 μL LB liquid culture medium, recover at 37°C for 1 h, centrifuge at 8000 rpm for 2 min, discard part of the supernatant, keep about 100 μL of the bacteria, resuspend and apply to a plate containing 100 mg / L ampicillin, and culture at 37°C overnight. After a single colony grows on the plate, it is identified and sequenced by colony PCR, and the positive transformants are selected. After transferring to a shake tube culture, it is extracted with a plasmid extraction kit to obtain pCA-Bio.

[0041] Example 3

[0042] A p-coumaric acid biosensor is constructed, comprising the following steps:

[0043] Using pCA-Bio obtained in Example 2 as a template, primers F7 (SEQ ID No. 19) and R7 (SEQ ID No. 20) were used to amplify the linear plasmid using high-fidelity Phanta Max Super-Fidelity DNA polymerase. Technology was used to assemble the plasmids obtained above, and after screening and verification, the biosensor plasmid pCA-Bio-R29 / I30G was finally obtained, which was then sequenced and confirmed by the company.

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

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

[0046] The correlation between the fluorescence intensity of the recombinant strains containing different biosensors and the p-coumaric acid concentration was plotted with the exogenous p-coumaric acid concentration as the horizontal axis and the relative fluorescence intensity as the vertical axis. Figure 2 As shown, by molecularly modifying the CarR transcriptional regulatory factor, a p-coumaric acid biosensor with better performance was obtained.

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

[0048] 1. Gene Editing Methods

[0049] The present invention adopts CRISPR / Cas9 gene editing method, and refers to 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 elimination system of pGRB, the Red recombination system of lambda phage and the Cas9 protein expression system, 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, 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] Use the CHOPCHOP website tool to find a suitable target sequence (PAM: 5'-NGG-3'), design and synthesize two reverse complementary primers, anneal the two primers to form a double-stranded DNA, and connect them to the pGRB plasmid vector using a seamless cloning enzyme. Transfer the entire connected system to the DH5a competent medium, mix well, ice bath for 20 minutes, heat shock at 42°C for 60 seconds, and then immediately ice bath for 2-3 minutes, add 900μL LB liquid culture medium, resuscitate at 37°C for 1 hour, centrifuge at 8000rpm for 2 minutes, discard part of the supernatant, keep about 100μL of the bacteria, resuspend and apply to a plate containing 100mg / L ampicillin, and culture at 37°C overnight. After a single colony grows on the plate, it is identified and sequenced by colony PCR, and the positive transformants are selected, transferred to shake tube culture, and extracted with a plasmid extraction kit.

[0053] 1.2 Preparation of recombinant DNA fragments

[0054] The recombinant fragment used for knockout or integration is amplified by high-fidelity enzyme PCR and then subjected to overlapping PCR. After gel running to verify the correct band, it is recovered using a DNA purification recovery kit.

[0055] 1.3 Preparation of electrocompetent cells

[0056] Pick a single clone from the plate or transfer 10uL of bacterial solution from the glycerol tube to an LB shake tube (add 100mg / L spectinomycin to the plasmid carrying pREDCas9), culture overnight and transfer to 2×YT medium and culture at 32℃ until OD 600 =0.1-0.2, add IPTG with a final concentration of 0.1 mM and continue culturing until OD 600 =0.6-0.7, and the competent state preparation was carried out. The competent state preparation process was carried out according to the conventional standard operation.

[0057] 1.4 Electroporation

[0058] The pGRB plasmid and the recombinant DNA fragment were electroporated into competent cells at the same time. The recovered cultured bacterial solution after electroporation was centrifuged and all of them were spread on LB plates containing ampicillin and spectinomycin. After overnight culture at 32°C, colony PCR was performed to verify the selection of positive transformants and preserve the bacteria.

[0059] 1.5 Elimination of plasmid

[0060] The positive transformants were transferred to LB shake tubes containing 0.2% arabinose and spectinomycin for overnight culture, and single clones were obtained by three-zone streaking. Single colonies were picked and spotted on LB plates with ampicillin and spectinomycin resistance, and no growth was found on the ampicillin plate. Single colonies that grew on the spectinomycin-resistant plate were preserved and transferred to LB shake tubes without resistance, and cultured at 42°C overnight. Single clones were obtained by three-zone streaking and spotted on LB plates with spectinomycin resistance and without resistance, and no growth was found on the spectinomycin-resistant plate. Single colonies that grew on the non-resistant plate were preserved.

[0061] 2. Genes involved in strain construction

[0062] Table 1 Gene accession numbers

[0063]

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

[0065] 3.1 Knockout of lacIZ gene

[0066] Using Escherichia coli W3110 as a template, primers lacIZ-UP-F (SEQ ID NO.21) and lacIZ-UP-R (SEQID NO.22) were used to obtain the upstream homology arm fragment of lacIZ knockout, primers lacIZ-DN-F (SEQ ID NO.23) and lacIZ-DN-R (SEQ ID NO.24) were used to obtain the downstream homology arm fragment of lacIZ knockout, and primers lacIZ-UP-F (SEQID NO.21) and lacIZ-DN-R (SEQ ID NO.24) were used to fuse the upstream and downstream homology arm fragments to obtain the fusion fragment of lacIZ knockout.

[0067] The double-stranded DNA 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 connected with the pGRB linear plasmid using the method shown in 1.1 to obtain the pGRB-lacI plasmid.

[0068] The pREDCas9 plasmid was electroporated into the W3110 electroporation competent medium to obtain the strain W3110-pREDCas9. The W3110-pREDCas9 electroporation competent medium was prepared, and the fusion fragment with lacIZ knockout and the pGRB-lacI plasmid were simultaneously electroporated into the W3110-pREDCas9 electroporation competent medium, and the strain SAA-1 was obtained according to the methods shown in 1.4 and 1.5.

[0069] 3.2 Knockout of tyrR gene

[0070] Using Escherichia coli W3110 as a template, primers tyrR-UP-F (SEQ ID NO.27) and tyrR-UP-R (SEQ ID NO.28) were used to obtain the upstream homology arm fragment of tyrR knockout by PCR, primers tyrR-DN-F (SEQ ID NO.29) and tyrR-DN-R (SEQ ID NO.30) were used to obtain the downstream homology arm fragment of tyrR knockout by PCR, and primers tyrR-UP-F (SEQ ID NO.27) and tyrR-DN-R (SEQ ID NO.30) were used to fuse the upstream and downstream homology arm fragments by PCR to obtain the fusion fragment of tyrR knockout.

[0071] The double-stranded DNA 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 connected with the pGRB linear plasmid using the method shown in 1.1 to obtain the pGRB-tyrR plasmid.

[0072] Prepare SAA-1-pREDCas9 electroporation competent medium, electroporate the fusion fragment of tyrR knockout and pGRB-tyrR plasmid into SAA-1-pREDCas9 electroporation competent medium at the same time, and obtain strain SAA-2 according to the methods shown in 1.4 and 1.5.

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

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

[0075] The double-stranded DNA 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 connected with the pGRB linear plasmid using the method shown in 1.1 to obtain the pGRB-tehB plasmid.

[0076] Prepare SAA-2-pREDCas9 electroporation competent cells and transfer △tehB::P trc -aroG S180F The fusion fragment and pGRB-tehB plasmid were simultaneously electroporated into the SAA-2-pREDCas9 electroporation competent cell, and the 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 locus M53I / A354V

[0078] Using Escherichia coli W3110 as a template, primers mbhA-UP-F (SEQ ID NO.43) and mbhA-UP-R (SEQ ID NO.44) were used to obtain the upstream homology arm fragment of mbhA knockout, primers mbhA-DN-F (SEQ ID NO.45) and mbhA-DN-R (SEQ ID NO.46) were used to obtain the downstream homology arm fragment of mbhA knockout, and primers tyrA-F (SEQ ID NO.49) and tyrA-M53I-R (SEQ ID NO.50) were used to obtain the tyrA knockout fragment. M53I / A354V The first fragment of tyrA 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 of tyrA was obtained by PCR using primers tyrA-A354V-F (SEQ ID NO.53) and tyrA-R (SEQ ID NO.54). M53I / A354V The 5 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 Fusion fragments.

[0079] The double-stranded DNA 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 connected with the pGRB linear plasmid using the method shown in 1.1 to obtain the pGRB-mbhA plasmid.

[0080] Prepare SAA-3-pREDCas9 electroporation competent cells and transfer △mbhA::P trc -tyrA M53I / A354V The fusion fragment and pGRB-mbhA plasmid were simultaneously electroporated into the SAA-3-pREDCas9 electroporation competent cell, and the strain SAA-4 was obtained according to the methods shown in 1.4 and 1.5.

[0081] 3.5 Knockout of pheA gene

[0082] Using Escherichia coli W3110 as a template, primers pheA-UP-F (SEQ ID NO.55) and pheA-UP-R (SEQ ID NO.56) were used to obtain the upstream homology arm fragment of pheA knockout by PCR, primers pheA-DN-F (SEQ ID NO.57) and pheA-DN-R (SEQ ID NO.58) were used to obtain the downstream homology arm fragment of pheA knockout by PCR, and primers pheA-UP-F (SEQ ID NO.55) and pheA-DN-R (SEQ ID NO.58) were used to fuse the upstream and downstream homology arm fragments by PCR to obtain the fusion fragment of pheA knockout.

[0083] The double-stranded DNA 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 connected with the pGRB linear plasmid using the method shown in 1.1 to obtain the pGRB-pheA plasmid.

[0084] Prepare SAA-4-pREDCas9 electroporation competent medium, electroporate the pheA knockout fusion fragment and pGRB-pheA plasmid into SAA-4-pREDCas9 electroporation competent medium at the same time, and obtain strain SAA-5 according to the methods shown in 1.4 and 1.5.

[0085] 3.6 Integration of aroK at the rph site

[0086] Using Escherichia coli W3110 as a template, primers rph-UP-F (SEQ ID NO.61) and rph-UP-R (SEQ ID NO.62) were used to obtain the upstream homology arm fragment of rph knockout by PCR, primers rph-DN-F (SEQ ID NO.63) and rph-DN-R (SEQ ID NO.64) were used to obtain the downstream homology arm fragment of rph knockout by PCR, primers aroK-F (SEQ ID NO.67) and aroK-R (SEQ ID NO.68) were used to obtain the aroK fragment by PCR, and primers rph-UP-F (SEQ ID NO.61) and rph-DN-R (SEQ ID NO.64) were used to fuse the above three fragments by PCR to obtain △rph::P trc -aroK fusion fragment.

[0087] The double-stranded DNA 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 connected with the pGRB linear plasmid using the method shown in 1.1 to obtain the pGRB-rph plasmid.

[0088] Prepare SAA-5-pREDCas9 electroporation competent cells and transform △rph::P trc The -aroK fusion fragment and the pGRB-rph plasmid were simultaneously electroporated into the SAA-5-pREDCas9 electroporation competent cell, and the 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 locus

[0090] Using Escherichia coli W3110 as a template, primers yeeP-UP-F (SEQ ID NO.69) and yeeP-UP-R (SEQ ID NO.70) were used to obtain the upstream homology arm fragment of yeep knockout by PCR, primers yeeP-DN-F (SEQ ID NO.71) and yeeP-DN-R (SEQ ID NO.72) were used to obtain the downstream homology arm fragment of yeeP knockout by PCR, primers ydiB-F (SEQ ID NO.75) and ydiB-R (SEQ ID NO.76) were used to obtain the ydiB fragment by PCR, and primers yeeP-UP-F (SEQ ID NO.69) and yeeP-DN-R (SEQ ID NO.72) were used to fuse the above three fragments by PCR to obtain △yeeP::P trc -ydiB fusion fragment.

[0091] The double-stranded DNA 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 connected with the pGRB linear plasmid using the method shown in 1.1 to obtain the pGRB-yeeP plasmid.

[0092] Prepare SAA-6-pREDCas9 electroporation competent cells and transfer △yeeP::P trc The -ydiB fusion fragment and the pGRB-yeeP plasmid were simultaneously electroporated into the SAA-6-pREDCas9 electroporation competent cell, and the 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 locus

[0094] Using Escherichia coli W3110 as a template, primers yghE-UP-F (SEQ ID NO.77) and yghE-UP-R (SEQ ID NO.78) were used to obtain the upstream homology arm fragment of yghE knockout by PCR, and primers yghE-DN-F (SEQ ID NO.79) and yghE-DN-R (SEQ ID NO.80) were used to obtain the downstream homology arm fragment of yghE knockout by PCR. Using the synthetic sequence FjTAL (SEQ ID NO.81) as a template, primers FjTAL-F (SEQ ID NO.84) and FjTAL-R (SEQ ID NO.85) were used to obtain the FjTAL fragment by PCR, and primers yghE-UP-F (SEQ ID NO.77) and yghE-DN-R (SEQ ID NO.80) were used to fuse the upstream and downstream homology arm fragments of yghE with the FjTAL fragment to obtain △yghE::P trc -FjTAL fusion fragment.

[0095] The double-stranded DNA 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 connected with the pGRB linear plasmid using the method shown in 1.1 to obtain the pGRB-yghE plasmid.

[0096] Prepare SAA-10-pREDCas9 electroporation competent cells and transfer △yghE::P trc The -FjTAL fusion fragment and pGRB-yghE plasmid were simultaneously electroporated into the SAA-10-pREDCas9 electroporation competent cell, and the 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 p-coumaric acid biosensor

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

[0099] Example 5 Application of recombinant Escherichia coli strains to screen for high-coumaric acid production strains

[0100] As a small molecule compound, p-coumaric acid is difficult to detect inside cells. Based on a p-coumaric acid biosensor, it can sensitively sense the changes in p-coumaric acid concentration inside cells and output as a fluorescent signal. By coupling with high-throughput screening equipment such as flow cytometry, it can be quickly sorted at the fluorescence level, which will greatly improve the speed of high-yield strain selection and the potential for efficient screening of target strains from a diverse mutant library.

[0101] The screening process steps are as follows: the recombinant E. coli p-CA3-BIO constructed in Example 4 was inoculated into 5 mL LB liquid culture medium, and after shaking culture at 37°C and 200 rpm for 10 h, the bacterial cells were collected by centrifugation, and the cells were washed with 10% glycerol and resuspended to a cell concentration of OD 600 =1.0. Take 10 μL of bacterial cells and use the atmospheric pressure room temperature plasma ARTP mutagenesis breeding instrument for random mutagenesis, where the instrument parameters are set to 120W power, 10SLM gas volume, and 35s treatment time. Suspend the mutagenized bacterial cells in 1mL LB fresh liquid culture medium and shake at 37℃ and 200rpm for 1h for recovery. After recovery, calculate the initial concentration OD 600 =0.1 was inoculated into fresh p-coumaric acid fermentation medium and fermented at 37°C and 220 rpm for 12 h. After the fermentation was completed, the cells were washed with pH 7.4 PBS buffer and resuspended to OD 600 =0.1, the bacterial cells were screened by flow cytometry, and the cells with fluorescence intensity in the top 0.5% were selected, directly collected and plated on LB solid culture medium. After static culture at 37°C for 24 hours, after obvious single colonies appeared on the LB plate, the colonies were picked up and rescreened in 96 deep-well plates containing p-coumaric acid fermentation medium, and fermented and cultured at 30°C and 220rpm for 24 hours. The bacteria were collected by centrifugation, resuspended with an equal volume of 0.9% NaCl aqueous solution, and the relative fluorescence intensity was determined by Varioskan LUX multifunctional microplate reader, where the excitation wavelength was set to 488nm and the emission wavelength was set to 520nm. At the same time, the bacterial OD was measured 600 Values, fluorescence intensity and OD 600The ratio of was defined as the value of relative fluorescence intensity, and the mutant strains with higher relative fluorescence intensity than the starting strain p-CA3-BIO were selected and subcultured to remove the biosensor plasmid pCA-Bio-R29 / I30G, and then the subsequent shake flask fermentation test was carried out. After 24 hours of fermentation, samples were taken to determine the content of p-coumaric acid.

[0102] The components of the p-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, ferrous sulfate heptahydrate 30mg / L, manganese sulfate monohydrate 10mg / L, trace element mixed solution 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, glucose 20g / L.

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

[0104] Through shake flask fermentation test, ten mutant strains with higher p-coumaric acid fermentation yield than the starting strain p-CA3-BIO were finally obtained, such as Figure 3As shown, they are named M1, M2, M3, M4, M5, M6, M7, M8, M9 and M10 respectively. The p-coumaric acid production of the M5 mutant is about 1.59 times that of the starting strain p-CA3. The above embodiments show that a p-coumaric acid biosensor of the present invention can sensitively respond to the characteristics of p-coumaric acid, and a screening platform for p-coumaric acid high-yield strains can be efficiently developed and established by coupling screening methods such as flow cytometry. Based on this screening platform, p-coumaric acid high-yield strains can be quickly and efficiently screened.

Claims

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

2. A p-coumaric acid biosensor, characterized in that The coding gene of the CarR mutant according to claim 1, P lpp1.6 Promoter, green fluorescent protein coding gene and P controlling green fluorescent protein expression car The promoter mutants were located on the expression vector pTrc99a; 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 that controls the expression of green fluorescent protein car The nucleotide sequence of the promoter mutant is shown in SEQ ID No. 5; Among them, the P lpp1.6 The promoter and the P car The promoter mutants have the opposite orientation.

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

4. Use of the recombinant Escherichia coli strain of claim 3 to screen for p-coumaric acid high-producing strains.

5. The use according to claim 4, characterized in that The method comprises the following steps: mutagenizing the recombinant Escherichia coli strain of claim 3 to obtain mutant strains, fermenting and culturing, sorting by flow cytometry, and selecting cells with increased green fluorescent protein signals, which are p-coumaric acid high-yield strains.

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