Genetically engineered bacterium for producing rosmarinic acid as well as construction method and application of genetically engineered bacterium

Abstract

The invention discloses a genetically engineered bacterium for producing rosmarinic acid as well as a construction method and application of the genetically engineered bacterium. The construction method comprises the following steps: knocking out genes lacIZ and tyrR of escherichia coli W3110; the method comprises the following steps: integrating genes aroGS180F, tyrAM53I / A354V, aroK and ydiB, and knocking out pheA; the method comprises the following steps: integrating genes BlfldH, EchpaBE216G / E294I / Q434T and KphpaC, and knocking out a ptsG gene and a crr gene; the RA-19 is obtained by integrating genes FjTAL, FbCarB, MoRAS, EchpaBE216G / E294I / Q434T and KphpaC and integrating genes RTSmCYP98A14 and RTSmCPR, the yield of the rosmarinic acid reaches 471.34 mg / L after the strain is subjected to shake flask fermentation, and the yield of the rosmarinic acid reaches 8.74 g / L after the strain is fermented in a fermentation tank.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to a genetic engineering bacterium for producing rosmarinic acid, a construction method and an application thereof. Background Art

[0002] Rosmarinic acid (RA) is a water-soluble natural phenolic acid compound isolated from rosemary, a plant of the Lamiaceae family. There are two hydroxyl groups on each of the two aromatic rings in the structure, which enables it to scavenge superoxide anion free radicals and hydroxyl free radicals. Its antioxidant activity is higher than that of vitamin E, caffeic acid, chlorogenic acid, folic acid, etc. At the same time, rosmarinic acid also has anti-inflammatory, antibacterial, anti-allergic and anti-tumor activities, and has shown important application value in the fields of pharmaceuticals, food, health products and cosmetics.

[0003] In 2014, Sarah E. Bloch et al. analyzed the natural metabolic pathway of rosmarinic acid in plants, identified multiple enzyme functions, and constructed a bacterial-plant chimeric rosmarinic acid synthesis pathway in Escherichia coli. Subsequently, the biosynthesis of rosmarinic acid was studied one after another. In 2023, Zhou Jingwen's team constructed an engineered Escherichia coli through modular caffeic acid and tanshinone synthesis pathways, key enzyme mining, knockout competition pathways, cofactor engineering and other means. After fermentation in a 5L fermenter for 68 hours, the rosmarinic acid production reached 5780.6 mg / L. After early efforts, although the use of synthetic biology to produce rosmarinic acid has achieved certain results, the low yield is still difficult to meet the needs of industrialization, and the construction of efficient rosmarinic acid production strains is still a very arduous challenge. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a genetically engineered bacterium for producing rosmarinic acid.

[0005] The second object of the present invention is to provide a method for constructing a genetically engineered bacterium for producing rosmarinic acid.

[0006] The third object of the present invention is to provide the use of the above-mentioned genetically engineered bacteria for producing rosmarinic acid by fermentation.

[0007] The technical solution of the present invention is summarized as follows:

[0008] A method for constructing a genetically engineered bacterium for producing rosmarinic acid comprises the following steps:

[0009] Knock out the lactose operon gene lacIZ and transcriptional regulatory factor gene tyrR of the starting strain Escherichia coli W3110; integrate the 4-hydroxyphenylpyruvate synthesis pathway gene aroG S180F ,tyrA M53I / A354V, aroK and ydiB; (Escherichia coli)

[0010] and knocking out the pheA gene;

[0011] Integration of codon-optimized D-2-hydroxyacid dehydrogenase encoding gene BlfldH and 4-hydroxyphenylacetate-3-hydroxylase mutant gene EchpaB E216G / E294I / Q434T and the codon-optimized flavin reductase gene KphpaC,

[0012] The EchpaB is derived from Escherichia coli BL21 (DE3);

[0013] Knockout of the ptsG gene, component IIBC of the glucose-specific PTS enzyme, and the crr gene, component IIA of the glucose-specific PTS enzyme;

[0014] Integration of the codon-optimized tyrosine ammonia lyase gene FjTAL;

[0015] Integration of the codon-optimized caffeoyl-CoA synthase gene FbCarB;

[0016] Integration of the codon-optimized rosmarinic acid synthase gene MoRAS;

[0017] Integration of 4-hydroxyphenylacetate-3-hydroxylase mutant gene EchpaB E216G / E294I / Q434T and the codon-optimized flavin reductase gene KphpaC,

[0018] A genetically engineered bacterium RA-19 producing rosmarinic acid was obtained by integrating a codon-optimized, truncated P450 enzyme gene RTSmCYP98A14 and a codon-optimized, truncated P450 enzyme reductase gene RTSmCPR.

[0019] The above construction method is preferably:

[0020] The nucleotide sequence of the codon-optimized D-2-hydroxyacid dehydrogenase encoding gene BlfldH is shown in SEQ ID NO.153;

[0021] The nucleotide sequence of the codon-optimized flavin reductase gene KphpaC is shown in SEQ ID NO.154;

[0022] The nucleotide sequence of the codon-optimized tyrosine ammonia lyase gene FjTAL is shown in SEQ ID NO.155;

[0023] The nucleotide sequence of the codon-optimized caffeoyl-CoA synthase gene FbCarB is shown in SEQ ID NO.156;

[0024] The nucleotide sequence of the codon-optimized rosmarinic acid synthase gene MoRAS is shown in SEQ ID NO.157;

[0025] The nucleotide sequence of the codon-optimized, truncated P450 enzyme gene RTSmCYP98A14 is shown in SEQ ID NO.158;

[0026] The nucleotide sequence of the codon-optimized, truncated P450 enzyme reductase gene RTSmCPR is shown in SEQ ID NO.159;

[0027] The above construction method constructs a genetically engineered bacterium for producing rosmarinic acid.

[0028] The application of the above-mentioned genetically engineered bacteria for producing rosmarinic acid by fermentation to produce rosmarinic acid.

[0029] Beneficial effects of the present invention:

[0030] The genetically engineered bacteria for producing rosmarinic acid of the present invention can produce 471.34 mg / L of rosmarinic acid after fermentation in a shake flask for 24 hours, and can produce 8.74 g / L of rosmarinic acid after fermentation in a 5L fermenter for 48 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Figure 2 is a plasmid map, where a is pREDCas9 and b is pGRB plasmid.

[0032] Figure 2 The rosmarinic acid (RA) production and OD of the engineered Escherichia coli strain RA-19 in a 5L fermenter 600 picture. DETAILED DESCRIPTION

[0033] Sources of various genes of the present invention: D-2-hydroxyacid dehydrogenase encoding gene BlfldH is derived from Bifidobacterium longum;

[0034] The flavin reductase gene KphpaC is derived from Klebsiella pneumoniae;

[0035] The tyrosine ammonia lyase gene FjTAL is from Flavobacterium johnsoniae;

[0036] The caffeoyl-CoA synthase gene FbCarB is derived from Firmicutes bacterium

[0037] The rosmarinic acid synthase gene MoRAS is derived from Melissa officinalis;

[0038] P450 enzyme gene SmCYP98A14 from Salvia miltiorrhiza

[0039] The P450 enzyme reductase gene SmCPR is derived from Salvia miltiorrhiza.

[0040] The nucleotide sequence of the codon-optimized D-2-hydroxyacid dehydrogenase encoding gene BlfldH is shown in SEQ ID NO.153;

[0041] The nucleotide sequence of the codon-optimized flavin reductase gene KphpaC is shown in SEQ ID NO.154;

[0042] The nucleotide sequence of the codon-optimized tyrosine ammonia lyase gene FjTAL is shown in SEQ ID NO.155;

[0043] The nucleotide sequence of the codon-optimized caffeoyl-CoA synthase gene FbCarB is shown in SEQ ID NO.156;

[0044] The nucleotide sequence of the codon-optimized rosmarinic acid synthase gene MoRAS is shown in SEQ ID NO.157;

[0045] The nucleotide sequence of the codon-optimized, truncated P450 enzyme gene RTSmCYP98A14 is shown in SEQ ID NO.158;

[0046] The nucleotide sequence of the codon-optimized, truncated P450 enzyme reductase gene RTSmCPR is shown in SEQ ID NO.159;

[0047] Escherichia coli W3110 is commercially available.

[0048] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0049] Unless otherwise specified, the primer synthesis and sequencing services in the present invention are provided by Ascent, gene synthesis and DH5a competence are provided by Qingke, enzymes and reagents used in molecular operations are from Novozymes, and plasmid extraction and DNA purification and recovery kits are provided by Takara. The reaction system, PCR program, annealing system, reaction program, seamless cloning reaction system, plasmid extraction method and DNA purification and recovery method of PCR amplification are all referred to the relevant instructions.

[0050] Gene editing methods involved in the embodiments:

[0051] The present invention adopts the CRISPR / Cas9 gene editing method, which is carried out according 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 two plasmid maps used in this method are shown in Figure 1 The construction of pREDCas9 plasmid also refers to (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.), pREDCas9 plasmid carries pGRB elimination system, Red recombination system of λ phage and Cas9 protein expression system, spectinomycin resistance (working concentration: 100 mg / L), cultured at 32°C; pGRB plasmid is used to express gRNA-Cas9 binding region sequence and terminator sequence, ampicillin resistance (working concentration: 100 mg / L), cultured at 37°C.

[0052] The specific steps of the CRISPR / Cas9 gene editing method are as follows:

[0053] 1. X-sgRNA plasmid construction

[0054] Using pGRB plasmid (commercially available) as a template, PCR was performed using primers pGRB-F (SEQ ID NO. 151) and primer pGRB-R (SEQ ID NO. 152) to obtain the pGRB plasmid vector.

[0055] The CHOPCHOP website tool was used to find a suitable target sequence (PAM: 5'-NGG-3'), and two reverse complementary primers were designed and synthesized. The two primers were annealed to form an X-DNA double strand.

[0056] The X-DNA double strand obtained above was connected to the pGRB plasmid vector using seamless cloning enzyme. The connected system was transferred to DH5a competent medium, mixed and ice-bathed for 20 minutes, heat-shocked at 42°C for 60 seconds, and then immediately ice-bathed for 2-3 minutes, and 900 μL of LLB liquid culture medium was added. After resuscitation at 37°C for 1 hour, centrifuged at 8000 rpm for 2 minutes, part of the supernatant was discarded, and about 100 μL of bacterial cells were resuspended and coated on a plate containing 100 mg / L ampicillin, and cultured at 37°C overnight. After a single colony grows on the plate, it is identified and sequenced by colony PCR, and positive transformants are selected. After transferring to shake tube culture, it is extracted with a plasmid extraction kit to construct the X-sgRNA plasmid.

[0057] 2. Preparation of recombinant DNA fragments

[0058] 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.

[0059] 3. Preparation of Electroporated Competent Cells

[0060] 1) 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 (known) 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;

[0061] 2) Prepare competent cells. The competent cells preparation process is performed according to conventional standard operation.

[0062] 4. Electroporation

[0063] The X-sgRNA plasmid and the recombinant DNA fragment were electroporated into the electroporation competent cells at the same time. After electroporation, the revived cultured bacterial solution was centrifuged and all coated on LB resistance plates containing 100 mg / L ampicillin and 100 mg / L spectinomycin. After overnight culture at 32°C, colony PCR was performed to verify the screening of positive transformants and preserve the bacteria.

[0064] 5. Elimination of Plasmid

[0065] The positive transformants were transferred to LB shake tubes containing 0.2% arabinose and 100 mg / L spectinomycin for overnight culture, and single clones were obtained by three-zone streaking. Single colonies were picked and spotted on LB plates containing 100 mg / L ampicillin and 100 mg / L spectinomycin, respectively. No growth was found on the LB plate containing ampicillin. Single colonies grown on the LB plate containing spectinomycin were preserved and transferred to LB shake tubes without antibiotics, cultured at 42°C overnight, single clones were obtained by three-zone streaking and spotted on LB plates containing spectinomycin and LB plates without antibiotics, no growth was found on the LB plate containing spectinomycin, and single colonies grown on the LB plate without antibiotics were preserved.

[0066] The genes involved in strain construction are shown in Table 1

[0067] Table 1 Gene accession numbers

[0068]

[0069] Example 1: A method for constructing a genetically engineered bacterium (RA-19) for producing rosmarinic acid, comprising the following steps:

[0070] 1.1 Knockout of the lactose operon gene lacIZ

[0071] Using Escherichia coli W3110 (hereinafter referred to as W3110) as a template,

[0072] The upstream homology arm fragment of lacIZ knockout was obtained by PCR using primers lacIZ-UP-F (SEQ ID NO.1) and lacIZ-UP-R (SEQ ID NO.2).

[0073] The downstream homology arm fragment of lacIZ knockout was obtained by PCR using primers lacIZ-DN-F (SEQ ID NO.3) and lacIZ-DN-R (SEQ ID NO.4).

[0074] The upstream and downstream homology arm fragments were fused by PCR using primers lacIZ-UP-F (SEQ ID NO.1) and lacIZ-DN-R (SEQ ID NO.4) to obtain a fusion fragment with lacIZ knocked out.

[0075] The primers lacI-gRNA-F (SEQ ID NO.5) and lacI-gRNA-R (SEQ ID NO.6) were annealed to obtain a lacI-DNA double-stranded fragment, and the fragment was connected to the pGRB plasmid vector to obtain a lacI-sgRNA plasmid.

[0076] Prepare W3110 electroporation competent cells, and electroporate the pREDCas9 plasmid into the W3110 electroporation competent cells to obtain strain W3110-pREDCas9.

[0077] A W3110-pREDCas9 electroporation competent medium was prepared, and the lacIZ knockout fusion fragment and the lacI-sgRNA plasmid were simultaneously electroporated into the W3110-pREDCas9 electroporation competent medium. The transformed single clones were verified by colony PCR using primers lacIZ-UP-F (SEQ ID NO.1) and lacIZ-DN-R (SEQ ID NO.4). After the correct strain was verified, a genetically engineered bacterium RA-1 (abbreviated as RA-1) producing rosmarinic acid was obtained after plasmid elimination.

[0078] 1.2 Knockout of transcriptional regulator gene tyrR

[0079] Using W3110 as a template, primers tyrR-UP-F (SEQ ID NO.7) and tyrR-UP-R (SEQ ID NO.8) were used to obtain the upstream homology arm fragment of tyrR knockout, primers tyrR-DN-F (SEQ ID NO.9) and tyrR-DN-R (SEQ ID NO.10) were used to obtain the downstream homology arm fragment of tyrR knockout, and primers tyrR-UP-F (SEQ ID NO.7) and tyrR-DN-R (SEQ ID NO.10) were used to fuse the upstream and downstream homology arm fragments to obtain the fusion fragment of tyrR knockout.

[0080] The tyrR-gRNA-F (SEQ ID NO.11) and tyrR-gRNA-R (SEQ ID NO.12) primers were annealed to obtain a tyrR-DNA double-stranded fragment, and the fragment was connected to the pGRB plasmid vector to obtain the tyrR-sgRNA plasmid.

[0081] Prepare RA-1 electroporation competent cells, and electroporate the pREDCas9 plasmid into the RA-1 electroporation competent cells to obtain strain RA-1-pREDCas9.

[0082] An RA-1-pREDCas9 electroporation competent medium was prepared, and the fusion fragment with tyrR knockout and the tyrR-sgRNA plasmid were simultaneously electroporated into the RA-1-pREDCas9 electroporation competent medium. The single clones grown after transformation were verified by colony PCR using primers tyrR-UP-F (SEQ ID NO.7) and tyrR-DN-R (SEQ ID NO.10). After the correct strain was verified, a genetically engineered bacterium RA-2 (abbreviated as RA-2) producing rosmarinic acid was obtained after elimination of the plasmid.

[0083] 1.3 Integration of 4-hydroxyphenylpyruvate biosynthesis pathway gene aroG at the tehB locus S180F

[0084] Using W3110 as a template, PCR was performed with primers tehB-UP-F (SEQ ID NO.13) and tehB-UP-R (SEQ ID NO.14) to obtain the upstream homology arm fragment of tehB knockout.

[0085] The downstream homology arm fragment of tehB knockout was obtained by PCR using primers tehB-DN-F (SEQ ID NO.15) and tehB-DN-R (SEQ ID NO.16).

[0086] AroG was obtained by PCR using primers aroG-F (SEQ ID NO. 19) and aroG-S180F-R (SEQ ID NO. 20). S180F The upstream fragment of

[0087] AroG was obtained by PCR using primers aroG-S180F-F (SEQ ID NO. 21) and aroG-R (SEQ ID NO. 22). S180F The downstream fragment of

[0088] The above four fragments were fused by PCR using primers tehB-UP-F (SEQ ID NO.13) and tehB-DN-R (SEQ ID NO.16) to obtain △tehB::P trc -aroG S180F Fusion fragments.

[0089] The tehB-DNA double-stranded fragment was obtained by annealing with primers tehB-gRNA-F (SEQ ID NO.17) and tehB-gRNA-R (SEQ ID NO.18), and the fragment was connected to the pGRB plasmid vector (described above) to obtain the tehB-sgRNA plasmid.

[0090] Prepare RA-2 electroporation competent cells, and electroporate the pREDCas9 plasmid into the RA-2 electroporation competent cells to obtain strain RA-2-pREDCas9.

[0091] Prepare RA-2-pREDCas9 electroporation competent cells and transfer △tehB::P trc -aroG S180F The fusion fragment and tehB-sgRNA plasmid were simultaneously electroporated into the RA-2-pREDCas9 electroporation competent cell. The single clones grown after transformation were verified by colony PCR using primers tehB-UP-F (SEQ ID NO.13) and tehB-DN-R (SEQ ID NO.16). After the correct strain was verified, a genetically engineered bacterium RA-3 (abbreviated as RA-3) producing rosmarinic acid was obtained by eliminating the plasmid.

[0092] 1.4 Integration of tyrA at the mbhA locus M53I / A354V Gene

[0093] Using W3110 as a template, PCR was performed using primers mbhA-UP-F (SEQ ID NO.23) and mbhA-UP-R (SEQ ID NO.24) to obtain the upstream homology arm fragment of mbhA knockout.

[0094] The downstream homology arm fragment of mbhA knockout was obtained by PCR using primers mbhA-DN-F (SEQ ID NO.25) and mbhA-DN-R (SEQ ID NO.26).

[0095] TyrA was obtained by PCR using primers tyrA-F (SEQ ID NO. 29) and tyrA-M53I-R (SEQ ID NO. 30). M53I / A354V The first segment of

[0096] TyrA was obtained by PCR using primers tyrA-M53I-F (SEQ ID NO. 31) and tyrA-A354V-R (SEQ ID NO. 32). M53I / A354V The second segment of

[0097] TyrA was obtained by PCR using primers tyrA-A354V-F (SEQ ID NO. 33) and tyrA-R (SEQ ID NO. 34). M53I / A354V The third segment of

[0098] The above five fragments were fused by PCR using primers mbhA-UP-F (SEQ ID NO.23) and mbhA-DN-R (SEQ ID NO.26) to obtain ΔmbhA::P trc -tyrA M53I / A354V Fusion fragments.

[0099] The mbhA-DNA double-stranded fragment was obtained by annealing with primers mbhA-gRNA-F (SEQ ID NO.27) and mbhA-gRNA-R (SEQ ID NO.28), and the fragment was connected to the pGRB plasmid vector to obtain the mbhA-sgRNA plasmid.

[0100] Prepare RA-3 electroporation competent cells, and electroporate the pREDCas9 plasmid into the RA-3 electroporation competent cells to obtain strain RA-3-pREDCas9.

[0101] Prepare RA-3-pREDCas9 electroporation competent cells and transfer △mbhA::Ptrc -tyrA M53I / A354V The fusion fragment and mbhA-sgRNA plasmid were simultaneously electroporated into the RA-3-pREDCas9 electroporation competent cell. The transformed single clones were verified by colony PCR using primers mbhA-UP-F (SEQ ID NO.23) and mbhA-DN-R (SEQ ID NO.26). After the correct strain was verified, a genetically engineered bacterium RA-4 (abbreviated as RA-4) producing rosmarinic acid was obtained by eliminating the plasmid.

[0102] 1.5 Integration of aroK gene at the rph locus

[0103] Using W3110 as a template, primers rph-UP-F (SEQ ID NO.41) and rph-UP-R (SEQ ID NO.42) were used to obtain the upstream homology arm fragment of rph knockout by PCR, primers rph-DN-F (SEQ ID NO.43) and rph-DN-R (SEQ ID NO.44) were used to obtain the downstream homology arm fragment of rph knockout by PCR, primers aroK-F (SEQ ID NO.47) and aroK-R (SEQ ID NO.48) were used to obtain the aroK fragment by PCR, and primers rph-UP-F (SEQ ID NO.41) and rph-DN-R (SEQ ID NO.44) were used to fuse the above three fragments by PCR to obtain △rph::P trc -aroK fusion fragment.

[0104] The rph-DNA double-stranded fragment was obtained by annealing with primers rph-gRNA-F (SEQ ID NO.45) and rph-gRNA-R (SEQ ID NO.46), and the fragment was connected to the pGRB plasmid vector to obtain the rph-sgRNA plasmid.

[0105] Prepare RA-4 electroporation competent cells, and electroporate the pREDCas9 plasmid into the RA-4 electroporation competent cells to obtain strain RA-4-pREDCas9.

[0106] Prepare RA-4-pREDCas9 electroporation competent cells and transform △rph::P trc The -aroK fusion fragment and rph-sgRNA plasmid were simultaneously electroporated into the RA-4-pREDCas9 electroporation competent cell. The transformed single clones were verified by colony PCR using primers rph-UP-F (SEQ ID NO.41) and rph-DN-R (SEQ ID NO.44). After the correct strain was verified, a genetically engineered bacterium RA-5 (abbreviated as RA-5) producing rosmarinic acid was obtained by eliminating the plasmid.

[0107] 1.6 Integration of the ydiB gene at the yeep locus

[0108] Using W3110 as a template, primers yeeP-UP-F (SEQ ID NO.49) and yeeP-UP-R (SEQ ID NO.50) were used to obtain the upstream homology arm fragment of yeep knockout, primers yeeP-DN-F (SEQ ID NO.51) and yeeP-DN-R (SEQ ID NO.52) were used to obtain the downstream homology arm fragment of yeeP knockout, primers ydiB-F (SEQ ID NO.55) and ydiB-R (SEQ ID NO.56) were used to obtain the ydiB fragment, and primers yeeP-UP-F (SEQ ID NO.49) and yeeP-DN-R (SEQ ID NO.52) were used to fuse the above three fragments to obtain △yeeP::P trc -ydiB fusion fragment.

[0109] The yeeP-DNA double-stranded fragment was obtained by annealing with primers yeeP-gRNA-F (SEQ ID NO.53) and yeeP-gRNA-R (SEQ ID NO.54), and the fragment was connected to the pGRB plasmid vector to obtain the yeeP-sgRNA plasmid.

[0110] Prepare RA-5 electroporation competent cells, and electroporate the pREDCas9 plasmid into the RA-5 electroporation competent cells to obtain strain RA-5-pREDCas9.

[0111] Prepare RA-5-pREDCas9 electroporation competent cells and transfer △yeeP::P trc The -ydiB fusion fragment and yeeP-sgRNA plasmid were simultaneously electroporated into the RA-5-pREDCas9 electroporation competent cell. The transformed single clones were verified by colony PCR using primers yeeP-UP-F (SEQ ID NO.49) and yeeP-DN-R (SEQ ID NO.52). After the correct strain was verified, a genetically engineered bacterium RA-6 (abbreviated as RA-6) producing rosmarinic acid was obtained by eliminating the plasmid.

[0112] 1.7 Knockout of pheA gene

[0113] Using W3110 as a template, primers pheA-UP-F (SEQ ID NO.35) and pheA-UP-R (SEQ ID NO.36) were used to obtain the upstream homology arm fragment of pheA knockout by PCR, primers pheA-DN-F (SEQ ID NO.37) and pheA-DN-R (SEQID NO.38) were used to obtain the downstream homology arm fragment of pheA knockout by PCR, and primers pheA-UP-F (SEQ ID NO.35) and pheA-DN-R (SEQ ID NO.38) were used to fuse the upstream and downstream homology arm fragments by PCR to obtain the fusion fragment of pheA knockout.

[0114] The pheA-gRNA-F (SEQ ID NO. 39) and pheA-gRNA-R (SEQ ID NO. 40) primers were annealed to obtain a pheA-DNA double-stranded fragment, and the fragment was connected to the pGRB plasmid vector to obtain the pheA-sgRNA plasmid.

[0115] Prepare RA-6 electroporation competent cells, and electroporate the pREDCas9 plasmid into the RA-6 electroporation competent cells to obtain the strain RA-6-pREDCas9.

[0116] The RA-6-pREDCas9 electroporation competent medium was prepared, and the pheA knockout fusion fragment and the pheA-sgRNA plasmid were simultaneously electroporated into the RA-6-pREDCas9 electroporation competent medium. The transformed single clones were verified by colony PCR using primers pheA-UP-F (SEQ ID NO.35) and pheA-DN-R (SEQ ID NO.38). After the correct strain was verified, a genetically engineered bacterium RA-7 (abbreviated as RA-7) producing rosmarinic acid was obtained after plasmid elimination.

[0117] 1.8 Integration of the BlfldH gene at the yjgX locus

[0118] Using W3110 as a template, PCR was performed with primers yjgX-UP-F (SEQ ID NO.57) and yjgX-UP-R (SEQ ID NO.58) to obtain the upstream homology arm fragment of yjgX knockout, and PCR was performed with primers yjgX-DN-F (SEQ ID NO.59) and yjgX-DN-R (SEQID NO.60) to obtain the downstream homology arm fragment of yjgX knockout;

[0119] The synthetic sequence BlfldH (SEQ ID NO.153) was used as a template, and primers BlfldH-F (SEQ ID NO.63) and BlfldH-R (SEQ ID NO.64) were used to obtain the BlfldH fragment by PCR, and the above three fragments were fused by PCR using primers yjgX-UP-F (SEQ ID NO.57) and yjgX-DN-R (SEQ ID NO.60) to obtain △yjgX::P trc -BlfldH fusion fragment.

[0120] The yjgX-DNA double-stranded fragment was obtained by annealing with primers yjgX-gRNA-F (SEQ ID NO.61) and yjgX-gRNA-R (SEQ ID NO.62), and the fragment was connected to the pGRB plasmid vector to obtain the yjgX-sgRNA plasmid.

[0121] Prepare RA-7 electroporation competent cells, and electroporate the pREDCas9 plasmid into the RA-7 electroporation competent cells to obtain the strain RA-7-pREDCas9.

[0122] Prepare RA-7-pREDCas9 electroporation competent cells and transfer △yjgX::P trc The -BlfldH fusion fragment and yjgX-sgRNA plasmid were simultaneously electroporated into the RA-7-pREDCas9 electroporation competent medium. The transformed single clones were verified by colony PCR using primers yjgX-UP-F (SEQ ID NO.57) and yjgX-DN-R (SEQ ID NO.60). After the correct strain was verified, a genetically engineered bacterium RA-8 (abbreviated as RA-8) producing rosmarinic acid was obtained by eliminating the plasmid.

[0123] 1.9 Integration of EchpaB at the yciQ site E216G / E294I / Q434T Gene

[0124] Using W3110 as a template, PCR was performed with primers yciQ-UP-F (SEQ ID NO.65) and yciQ-UP-R (SEQ ID NO.66) to obtain the upstream homology arm fragment of yciQ knockout, and PCR was performed with primers yciQ-DN-F (SEQ ID NO.67) and yciQ-DN-R (SEQID NO.68) to obtain the downstream homology arm fragment of yciQ knockout;

[0125] Using the genome of Escherichia coli BL21 (DE3) (commercially available) as a template, PCR was performed using primers EchpaB-F (SEQ ID NO. 71) and EchpaB (E216G) -R (SEQ ID NO. 72) to obtain EchpaB E216G / E294I / Q434TThe first fragment was obtained by PCR using primers EchpaB(E216G)-F (SEQ ID NO.73) and EchpaB(E294I)-R (SEQ ID NO.74). E216G / E294I / Q434T The second fragment was obtained by PCR using primers EchpaB(E294I)-F (SEQ ID NO.75) and EchpaB(Q434T)-R (SEQ ID NO.76). E216G / E294I / Q434T The third fragment was obtained by PCR using primers EchpaB(Q434T)-F (SEQ ID NO.77) and EchpaB-R (SEQ ID NO.78). E216G / E294I / Q434T The fourth fragment was obtained by PCR using primers EchpaB-F (SEQ ID NO.71) and EchpaB-R (SEQ ID NO.78) to fuse the above four fragments. trc -EchpaB E216G / E294I / Q434T fragment; then use primers yciQ-UP-F (SEQ ID NO.65) and yciQ-DN-R (SEQ ID NO.68) to knock out the upstream and downstream homology arm fragments of yciQ and P trc -EchpaB E216G / E294I / Q434T Fragment fusion PCR to obtain △yciQ::P trc -EchpaB E216G / E294I / Q434T Fusion fragments.

[0126] The yciQ-DNA double-stranded fragment was obtained by annealing with primers yciQ-gRNA-F (SEQ ID NO.69) and yciQ-gRNA-R (SEQ ID NO.70), and the fragment was connected to the pGRB plasmid vector to obtain the yciQ-sgRNA plasmid.

[0127] Prepare RA-8 electroporation competent cells, and electroporate the pREDCas9 plasmid into the RA-8 electroporation competent cells to obtain the strain RA-8-pREDCas9.

[0128] Prepare RA-8-pREDCas9 electroporation competent cells and transfer △yciQ::P trc -EchpaB E216G / E294I / Q434T The fusion fragment and yciQ-sgRNA plasmid were simultaneously electroporated into the RA-8-pREDCas9 electroporation competent cell. The transformed single clones were verified by colony PCR using primers yciQ-UP-F (SEQ ID NO.65) and yciQ-DN-R (SEQ ID NO.68). After the correct strain was verified, a genetically engineered bacterium RA-9 (abbreviated as RA-9) producing rosmarinic acid was obtained by eliminating the plasmid.

[0129] 1.10 Integration of KphpaC gene at the yjiV locus

[0130] Using W3110 as a template, primers yjiV-UP-F (SEQ ID NO.79) and yjiV-UP-R (SEQ ID NO.80) were used to obtain the upstream homology arm fragment of yjiV knockout, and primers yjiV-DN-F (SEQ ID NO.81) and yjiV-DN-R (SEQ ID NO.82) were used to obtain the downstream homology arm fragment of yjiV knockout; using the synthetic sequence KphpaC (SEQ ID NO.154) as a template, primers KphpaC-R (SEQ ID NO.85) and KphpaC-F (SEQ ID NO.86) were used to obtain the KphpaC fragment, and primers yjiV-UP-F (SEQ ID NO.79) and yjiV-DN-R (SEQ ID NO.82) were used to fuse the above three fragments to obtain △yjiV::P trc -KphpaC fusion fragment.

[0131] The yjiV-DNA double-stranded fragment was obtained by annealing with primers yjiV-gRNA-F (SEQ ID NO.83) and yjiV-gRNA-R (SEQ ID NO.84), and the fragment was connected to the pGRB plasmid vector to obtain the yjiV-sgRNA plasmid.

[0132] Prepare RA-9 electroporation competent cells, and electroporate the pREDCas9 plasmid into the RA-9 electroporation competent cells to obtain the strain RA-9-pREDCas9.

[0133] Prepare RA-9-pREDCas9 electroporation competent cells and transfer △yjiV::P trc -KphpaC fusion fragment and yjiV-sgRNA plasmid were simultaneously electroporated into RA-9-pREDCas9 electroporation competent medium, and the transformed single clones were verified by colony PCR using primers yjiV-UP-F (SEQ ID NO.79) and yjiV-DN-R (SEQ ID NO.82). After the correct strain was verified, a genetically engineered bacterium RA-10 (abbreviated as RA-10) producing rosmarinic acid was obtained by eliminating the plasmid.

[0134] 1.11 Knockout of ptsG gene

[0135] Using W3110 as a template, primers ptsG-UP-F (SEQ ID NO.87) and ptsG-UP-R (SEQ ID NO.88) were used to obtain the upstream homology arm fragment of ptsG knockout by PCR, and primers ptsG-DN-F (SEQ ID NO.89) and ptsG-DN-R (SEQID NO.90) were used to obtain the downstream homology arm fragment of ptsG knockout by PCR. Primers ptsG-UP-F (SEQ ID NO.87) and ptsG-DN-R (SEQ ID NO.90) were used to fuse the upstream and downstream homology arm fragments by PCR to obtain the fusion fragment of ptsG knockout.

[0136] The ptsG-gRNA double-stranded fragment was obtained by annealing with primers ptsG-gRNA-F (SEQ ID NO.91) and ptsG-gRNA-R (SEQ ID NO.92), and the fragment was connected to the pGRB plasmid vector to obtain the ptsG-sgRNA plasmid.

[0137] Prepare RA-10 electroporation competent cells, and electroporate the pREDCas9 plasmid into the RA-10 electroporation competent cells to obtain the strain RA-10-pREDCas9.

[0138] The RA-10-pREDCas9 electroporation competent medium was prepared, and the ptsG knockout fusion fragment and the ptsG-sgRNA plasmid were simultaneously electroporated into the RA-10-pREDCas9 electroporation competent medium. The single clones grown after transformation were verified by colony PCR using primers ptsG-UP-F (SEQ ID NO.87) and ptsG-DN-R (SEQ ID NO.90). After the correct strain was verified, a genetically engineered bacterium RA-11 (abbreviated as RA-11) producing rosmarinic acid was obtained after plasmid elimination.

[0139] 1.12 Knockout of crr gene

[0140] Using W3110 as a template, primers crr-UP-F (SEQ ID NO.93) and crr-UP-R (SEQ ID NO.94) were used to PCR to obtain the upstream homology arm fragment of crr knockout, primers crr-DN-F (SEQ ID NO.95) and crr-DN-R (SEQ ID NO.96) were used to PCR to obtain the downstream homology arm fragment of crr knockout, and primers crr-UP-F (SEQ ID NO.93) and crr-DN-R (SEQ ID NO.96) were used to fuse the upstream and downstream homology arm fragments to obtain the fusion fragment of crr knockout.

[0141] The crr-DNA double-stranded fragment was obtained by annealing with primers crr-gRNA-F (SEQ ID NO.97) and crr-gRNA-R (SEQ ID NO.98), and the fragment was connected to the pGRB plasmid vector to obtain the crr-sgRNA plasmid.

[0142] Prepare RA-11 electroporation competent cells, and electroporate the pREDCas9 plasmid into the RA-11 electroporation competent cells to obtain the strain RA-11-pREDCas9.

[0143] RA-11-pREDCas9 electroporation competent cells were prepared, and the crr knockout fusion fragment and crr-sgRNA plasmid were simultaneously electroporated into the RA-11-pREDCas9 electroporation competent cells. The transformed single clones were verified by colony PCR using primers crr-UP-F (SEQ ID NO.93) and crr-DN-R (SEQ ID NO.96). After the correct strain was verified, a genetically engineered bacterium RA-12 (abbreviated as RA-12) producing rosmarinic acid was obtained by eliminating the plasmid.

[0144] 1.13 Integration of FjTAL gene at yghE locus

[0145] Using W3110 as a template, primers yghE-UP-F (SEQ ID NO.99) and yghE-UP-R (SEQ ID NO.100) were used to obtain the upstream homology arm fragment of yghE knockout, and primers yghE-DN-F (SEQ ID NO.101) and yghE-DN-R (SEQID NO.102) were used to obtain the downstream homology arm fragment of yghE knockout; using the synthetic sequence FjTAL (SEQ ID NO.155) as a template, primers FjTAL-F (SEQ ID NO.105) and FjTAL-R (SEQ ID NO.106) were used to obtain the FjTAL fragment, and primers yghE-UP-F (SEQ ID NO.99) and yghE-DN-R (SEQ ID NO.102) were used to fuse the above three fragments to obtain △yghE::P trc -FjTAL fusion fragment.

[0146] The yghE-DNA double-stranded fragment was obtained by annealing with primers yghE-gRNA-F (SEQ ID NO.103) and yghE-gRNA-R (SEQ ID NO.104), and the fragment was connected to the pGRB plasmid vector to obtain the yghE-sgRNA plasmid.

[0147] Prepare RA-12 electroporation competent cells, and electroporate the pREDCas9 plasmid into the RA-12 electroporation competent cells to obtain the strain RA-12-pREDCas9.

[0148] Prepare RA-12-pREDCas9 electroporation competent cells and transfer △yghE::P trc The -FjTAL fusion fragment and yghE-sgRNA plasmid were simultaneously electroporated into the RA-12-pREDCas9 electroporation competent cell. The transformed single clones were verified by colony PCR using primers yghE-UP-F (SEQ ID NO.99) and yghE-DN-R (SEQ ID NO.102). After the correct strain was verified, a genetically engineered bacterium RA-13 (abbreviated as RA-13) producing rosmarinic acid was obtained by eliminating the plasmid.

[0149] 1.14 Integration of FbCarB gene at yeeL locus

[0150] Using W3110 as a template, primers yeeL-UP-F (SEQ ID NO.107) and yeeL-UP-R (SEQ ID NO.108) were used to obtain the upstream homology arm fragment of yeeL knockout, and primers yeeL-DN-F (SEQ ID NO.109) and yeeL-DN-R (SEQ ID NO.110) were used to obtain the downstream homology arm fragment of yeeL knockout; using the synthetic sequence FbCarB (SEQ ID NO.156) as a template, primers FbCarB-F (SEQ ID NO.113) and FbCarB-R (SEQ ID NO.114) were used to obtain the FbCarB fragment, and primers yeeL-UP-F (SEQ ID NO.107) and yeeL-DN-R (SEQ ID NO.110) were used to fuse the above three fragments to obtain △yeeL::P trc -FbCarB fusion fragment.

[0151] The yeeL-DNA double-stranded fragment was obtained by annealing with primers yeeL-gRNA-F (SEQ ID NO.111) and yeeL-gRNA-R (SEQ ID NO.112), and the fragment was connected to the pGRB plasmid vector to obtain the yeeL-sgRNA plasmid.

[0152] Prepare RA-13 electroporation competent cells, and electroporate the pREDCas9 plasmid into the RA-13 electroporation competent cells to obtain the strain RA-13-pREDCas9.

[0153] Prepare RA-13-pREDCas9 electroporation competent cells and transfer △yeeL::P trc-FbCarB fusion fragment and yeeL-sgRNA plasmid were simultaneously electroporated into RA-13-pREDCas9 electroporation competent cells. The transformed single clones were verified by colony PCR using primers yeeL-UP-F (SEQ ID NO.107) and yeeL-DN-R (SEQ ID NO.110). After the correct strain was verified, a genetically engineered bacterium RA-14 (abbreviated as RA-14) producing rosmarinic acid was obtained by eliminating the plasmid.

[0154] 1.15 Integration of MoRAS gene at ycgH locus

[0155] Using W3110 as a template, primers ycgH-UP-F (SEQ ID NO.115) and ycgH-UP-R (SEQ ID NO.116) were used to obtain the upstream homology arm fragment of ycgH knockout, and primers ycgH-DN-F (SEQ ID NO.117) and ycgH-DN-R (SEQ ID NO.118) were used to obtain the downstream homology arm fragment of ycgH knockout; using the synthetic sequence MoRAS (SEQ ID NO.157) as a template, primers MoRAS-F (SEQ ID NO.121) and MoRAS-R (SEQ ID NO.122) were used to obtain the MoRAS fragment, and primers ycgH-UP-F (SEQ ID NO.115) and ycgH-DN-R (SEQ ID NO.118) were used to fuse the above three fragments to obtain △ycgH::P trc -MoRAS fusion fragment.

[0156] The ycgH-DNA double-stranded fragment was obtained by annealing with primers ycgH-gRNA-F (SEQ ID NO.119) and ycgH-gRNA-R (SEQ ID NO.120), and the fragment was connected to the pGRB plasmid vector to obtain the ycgH-sgRNA plasmid.

[0157] Prepare RA-14 electroporation competent cells, and electroporate the pREDCas9 plasmid into the RA-14 electroporation competent cells to obtain the strain RA-14-pREDCas9.

[0158] Prepare RA-14-pREDCas9 electroporation competent cells and transfer △ycgH::P trc-MoRAS fusion fragment and ycgH-sgRNA plasmid were simultaneously electroporated into RA-14-pREDCas9 electroporation competent cells. The transformed single clones were verified by colony PCR using primers ycgH-UP-F (SEQ ID NO.115) and ycgH-DN-R (SEQ ID NO.118). After the correct strain was verified, a genetically engineered bacterium RA-15 producing rosmarinic acid was obtained by eliminating the plasmid, referred to as RA-15.

[0159] 1.16 Integration of EchpaB at the ylbE locus E216G / E294I / Q434T Gene

[0160] Using W3110 as a template, primers ylbE-UP-F (SEQ ID NO.123) and ylbE-UP-R (SEQ ID NO.124) were used to obtain the upstream homology arm fragment of knocking out ylbE by PCR, and primers ylbE-DN-F (SEQ ID NO.125) and ylbE-DN-R (SEQ ID NO.126) were used to obtain the downstream homology arm fragment of knocking out ylbE by PCR; primers ylbE-UP-F (SEQ ID NO.123) and ylbE-DN-R (SEQ ID NO.126) were used to cross-link the upstream and downstream homology arm fragments of ylbE with the P obtained in 1.9 trc -EchpaB E216G / E294I / Q434T Fragment fusion PCR to obtain △ylbE::P trc -EchpaB E216G / E294I / Q434T Fusion fragments.

[0161] The ylbE-gRNA double-stranded fragment was obtained by annealing with primers ylbE-gRNA-F (SEQ ID NO.127) and ylbE-gRNA-R (SEQ ID NO.128), and the fragment was connected to the pGRB plasmid vector to obtain the ylbE-sgRNA plasmid.

[0162] Prepare RA-15 electroporation competent cells, and electroporate the pREDCas9 plasmid into the RA-15 electroporation competent cells to obtain the strain RA-15-pREDCas9.

[0163] Prepare RA-15-pREDCas9 electroporation competent cells and transfer △ylbE::P trc -EchpaB E216G / E294I / Q434TThe fusion fragment and ylbE-sgRNA plasmid were simultaneously electroporated into the RA-15-pREDCas9 electroporation competent cell. The transformed single clones were verified by colony PCR using primers ylbE-UP-F (SEQ ID NO.123) and ylbE-DN-R (SEQ ID NO.126). After the correct strain was verified, a genetically engineered bacterium RA-16 (abbreviated as RA-16) producing rosmarinic acid was obtained by eliminating the plasmid.

[0164] 1.17 Integration of KphpaC gene at yjiT site

[0165] Using W3110 as a template, primers yjiT-UP-F (SEQ ID NO.129) and yjiT-UP-R (SEQ ID NO.130) were used to obtain the upstream homology arm fragment of yjiT knockout by PCR, and primers yjiT-DN-F (SEQ ID NO.131) and yjiT-DN-R (SEQ ID NO.132) were used to obtain the downstream homology arm fragment of yjiT knockout by PCR; primers yjiT-UP-F (SEQ ID NO.129) and yjiT-DN-R (SEQ ID NO.132) were used to fuse the upstream and downstream homology arm fragments of yjiT with the KphpaC fragment obtained in 1.10 by PCR to obtain △yjiT::P trc -KphpaC fusion fragment.

[0166] The yjiT-DNA double-stranded fragment was obtained by annealing with primers yjiT-gRNA-F (SEQ ID NO.133) and yjiT-gRNA-R (SEQ ID NO.134), and the fragment was connected to the pGRB plasmid vector to obtain the yjiT-sgRNA plasmid.

[0167] Prepare RA-16 electroporation competent cells, and electroporate the pREDCas9 plasmid into the RA-16 electroporation competent cells to obtain the strain RA-16-pREDCas9.

[0168] Prepare RA-16-pREDCas9 electroporation competent cells and transfer △yjiT::P trc -KphpaC fusion fragment and yjiT-sgRNA plasmid were simultaneously electroporated into RA-16-pREDCas9 electroporation competent cells. The transformed single clones were verified by colony PCR using primers yjiT-UP-F (SEQ ID NO.129) and yjiT-DN-R (SEQ ID NO.132). After the correct strain was verified, a genetically engineered bacterium RA-17 (abbreviated as RA-17) producing rosmarinic acid was obtained by eliminating the plasmid.

[0169] 1.18 Integration of the RTSmCYP98A14 gene at the ykgH-betA position

[0170] Using W3110 as a template, primers ykgH-betA-UF (SEQ ID NO.135) and ykgH-betA-UR (SEQ ID NO.136) were used to obtain the upstream homology arm fragment of knocking out ykgH-betA, and primers ykgH-betA-DF (SEQ ID NO.137) and ykgH-betA-DR (SEQ ID NO.138) were used to obtain the downstream homology arm fragment of knocking out ykgH-betA; using the synthetic sequence RTSmCYP98A14 (SEQ ID NO.158) as a template, primers RT-SmCYP98A14-F (SEQ ID NO.141) and SmCYP98A14-R (SEQ ID NO.142) were used to obtain the RTSmCYP98A14 fragment; primers ykgH-betA-UF (SEQ ID NO.135) and ykgH-betA-DR (SEQ ID NO.137) were used to obtain the downstream homology arm fragment of knocking out ykgH-betA. NO.138) The upstream and downstream homology arm fragments of ykgH-betA were fused with the RTSmCYP98A14 fragment by PCR to obtain △ykgH-betA::P trc -RTSmCYP98A14 fusion fragment.

[0171] The ykgH-betA-gRNA-F (SEQ ID NO.139) and ykgH-betA-gRNA-R (SEQ ID NO.140) primers were annealed to obtain a ykgH-betA-DNA double-stranded fragment, and the fragment was connected to the pGRB plasmid vector to obtain the ykgH-betA-sgRNA plasmid.

[0172] Prepare RA-17 electroporation competent cells, and electroporate the pREDCas9 plasmid into the RA-17 electroporation competent cells to obtain the strain RA-17-pREDCas9.

[0173] Prepare RA-17-pREDCas9 electroporation competent cells and transfer △ykgH-betA::P trc The -RTSmCYP98A14 fusion fragment and the ykgH-betA-sgRNA plasmid were simultaneously electroporated into the RA-17-pREDCas9 electroporation competent cell. The transformed single clones were verified by colony PCR using primers ykgH-betA-UF (SEQ ID NO.135) and ykgH-betA-DR (SEQ ID NO.138). After the correct strain was verified, a genetically engineered bacterium RA-18 (abbreviated as RA-18) producing rosmarinic acid was obtained by eliminating the plasmid.

[0174] 1.19 Integration of the RTSmCPR gene at the ypjC-ileY locus

[0175] Using W3110 as a template, primers ypjC-ileY-UF (SEQ ID NO.143) and ypjC-ileY-UR (SEQ ID NO.144) were used to obtain the upstream homology arm fragment of knocking out ypjC-ileY, and primers ypjC-ileY-DF (SEQ ID NO.145) and ypjC-ileY-DR (SEQ ID NO.146) were used to obtain the downstream homology arm fragment of knocking out ypjC-ileY; using the synthetic sequence RTSmCPR (SEQ ID NO.159) as a template, primers RT-SmCPR-F (SEQ ID NO.149) and SmCPR-R (SEQID NO.150) were used to obtain the RTSmCPR fragment; primers ypjC-ileY-UF (SEQ ID NO.143) and ypjC-ileY-DR (SEQ ID NO.146) were used to obtain the downstream homology arm fragment of knocking out ypjC-ileY. NO.146) The upstream and downstream homology arm fragments of ypjC-ileY were fused with the RTSmCPR fragment by PCR to obtain △ypjC-ileY::P trc -RTSmCPR fusion fragment.

[0176] The ypjC-ileY-gRNA double-stranded fragment was obtained by annealing with primers ypjC-ileY-gRNA-F (SEQ ID NO.147) and ypjC-ileY-gRNA-R (SEQ ID NO.148), and the fragment was connected to the pGRB plasmid vector to obtain the ypjC-ileY-sgRNA plasmid.

[0177] Prepare RA-18 electroporation competent cells, and electroporate the pREDCas9 plasmid into the RA-18 electroporation competent cells to obtain the strain RA-18-pREDCas9.

[0178] Prepare RA-18-pREDCas9 electroporation competent cells and transfer △ypjC-ileY::P trc The -RTSmCPR fusion fragment and ypjC-ileY-sgRNA plasmid were simultaneously electroporated into the RA-18-pREDCas9 electroporation competent cell. The transformed single clones were verified by colony PCR using primers ypjC-ileY-UF (SEQ ID NO.143) and ypjC-ileY-DR (SEQ ID NO.146). After the correct strain was verified, a genetically engineered bacterium RA-19 (abbreviated as RA-19) producing rosmarinic acid was obtained by eliminating the plasmid.

[0179] Embodiment 2:

[0180] Application of a genetically engineered bacterium RA-19 (abbreviated as RA-19) for producing rosmarinic acid by fermentation

[0181] (1) Shake flask fermentation:

[0182] A single RA-19 colony was picked from the plate and transferred to 5 mL LB medium, and cultured at 37°C in a shaker at 220 rpm for 12 h. Then it was transferred to a 30 mL LB shake flask and fermented at 37°C in a shaker at 220 rpm for 10 h to obtain seed liquid. 1 mL of seed liquid was transferred to 26 mL fermentation medium and fermented at 37°C in a shaker at 220 rpm for 24 h. The rosmarinic acid yield reached 471.34 mg / L.

[0183] (2) Fermentation in 5L fermenter

[0184] Slant activation culture: scrape a loop of RA-19 bacteria from the -80℃ freezer tube, spread it evenly on the LB solid slant medium, and culture at 37℃ for 12h;

[0185] Seed culture: Then transfer all into a 5L fermenter containing 3L fermentation medium. Culture OD 600 After reaching 10, 15% of the seed liquid was transferred to a 5L fermentation tank containing 2L of fresh fermentation medium for fermentation and culture: during the fermentation process, the pH was controlled to be stable at around 7.0, the temperature was maintained at 35°C, and the dissolved oxygen was around 15%; when the glucose in the culture medium was consumed, 80% (m / v) glucose solution was added.

[0186] Fermentation medium formula:

[0187] 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, glucose 20g / L.

[0188] Embodiment 3:

[0189] The concentration of rosmarinic acid in the fermentation broth was detected by HPLC, as follows:

[0190] Take 1 mL of fermentation broth, centrifuge at 12000 rpm for 5 min, and then take the supernatant and pass it through a membrane for HPLC detection.

[0191] The detection conditions are as follows: Thermo Fisher C18 (250 mm×4.6 mm) chromatographic column; PDA detector; detection wavelength 281 nm; flow rate 1 mL / min. The mobile phase is: 20% methanol, 79.9% water and 0.1% formic acid.

[0192] like Figure 2 As shown, RA-19 was cultured in a 5L fermenter for 48h, and the rosmarinic acid production reached 8.74g / L, which is better than that reported in the prior art.

[0193] The above-described embodiments are only preferred embodiments of the present invention and are not intended to limit the scope of the present invention. All simple changes and modifications made according to the claims and the contents of the specification of the present invention are within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A method for constructing a genetically engineered bacterium for producing rosmarinic acid, characterized in that The following steps are included: Knock out the lactose operon gene lacIZ and transcriptional regulatory factor gene tyrR of the starting strain Escherichia coli W3110; integrate the 4-hydroxyphenylpyruvate synthesis pathway gene aroG S180F ,tyrA M53I / A354V , aroK and ydiB; and knocking out the pheA gene; Integration of codon-optimized D-2-hydroxyacid dehydrogenase encoding gene BlfldH and 4-hydroxyphenylacetate-3-hydroxylase mutant gene EchpaB E216G / E294I / Q434T and the codon-optimized flavin reductase gene KphpaC, The EchpaB is derived from Escherichia coli BL21 (DE3); Knockout of the ptsG gene, component IIBC of the glucose-specific PTS enzyme, and the crr gene, component IIA of the glucose-specific PTS enzyme; Integration of the codon-optimized tyrosine ammonia lyase gene FjTAL; Integration of the codon-optimized caffeoyl-CoA synthase gene FbCarB; Integration of the codon-optimized rosmarinic acid synthase gene MoRAS; Integration of 4-hydroxyphenylacetate-3-hydroxylase mutant gene EchpaB E216G / E294I / Q434T and the codon-optimized flavin reductase gene KphpaC, A genetically engineered bacterium RA-19 producing rosmarinic acid was obtained by integrating a codon-optimized, truncated P450 enzyme gene RTSmCYP98A14 and a codon-optimized, truncated P450 enzyme reductase gene RTSmCPR.

2. The construction method according to claim 1, characterized in that Said The nucleotide sequence of the codon-optimized D-2-hydroxyacid dehydrogenase encoding gene BlfldH is shown in SEQ ID NO.153; The nucleotide sequence of the codon-optimized flavin reductase gene KphpaC is shown in SEQ ID NO.154; The nucleotide sequence of the codon-optimized tyrosine ammonia lyase gene FjTAL is shown in SEQ ID NO.155; The nucleotide sequence of the codon-optimized caffeoyl-CoA synthase gene FbCarB is shown in SEQ ID NO.156; The nucleotide sequence of the codon-optimized rosmarinic acid synthase gene MoRAS is shown in SEQ ID NO.157; The nucleotide sequence of the codon-optimized, truncated P450 enzyme gene RTSmCYP98A14 is shown in SEQ ID NO.158; the nucleotide sequence of the codon-optimized, truncated P450 enzyme reductase gene RTSmCPR is shown in SEQ ID NO.

159.

3. A genetically engineered bacterium for producing rosmarinic acid constructed by the construction method of claim 1.

4. Use of a rosmarinic acid-producing genetically engineered bacterium according to claim 3 in fermenting rosmarinic acid.

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