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

Abstract

The invention discloses a genetically engineered bacterium for producing tanshinol as well as a construction method and application of the genetically engineered bacterium, and the construction method comprises the following steps: (1) taking escherichia coli W3110 as an original strain, and reconstructing a tanshinol synthesis route in cytoplasm; (2) positioning flavin reductase from klebsiella pneumoniae and a 4-hydroxyphenylacetic acid-3 hydroxylase mutant in a periplasmic space by using periplasmic space positioning signal peptide sufI. The genetically engineered bacterium for producing the tanshinol disclosed by the invention has the advantages that the yield of the tanshinol reaches 2.8 g / L after being subjected to shake flask fermentation for 24 hours, and the yield of the tanshinol reaches 26.6 g / L after being fermented in a 5L fermentation tank for 48 hours.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and specifically relates to a genetic engineering bacterium for stably and efficiently producing danshensu, and a construction method and application thereof. Background Art

[0002] Salvianic acid A (SAA), chemically known as β-(3,4-dihydroxyphenyl) lactic acid, is a phenylpropanoid compound that is mainly found in the medicinal herb Salvia miltiorrhiza and is also an important precursor for the synthesis of plant phenolic acids such as rosmarinic acid.

[0003] Danshensu has powerful antioxidant and medicinal functions, such as preventing cardiovascular diseases (myocardial ischemia-reperfusion, atherosclerosis, hypertension), cerebrovascular diseases (such as cognitive decline and anxiety), improving inflammation, inhibiting the malignant biological behavior of cancer cells, inhibiting pulmonary fibrosis, protecting visual function and protecting the nervous system. Therefore, Danshensu has a broad application prospect in the future functional food and pharmaceutical industries.

[0004] At present, the method of obtaining danshensu mainly relies on direct extraction from danshensu roots, but this production mode depends on varieties and climate, is affected by seasonal changes, has unstable yields and cumbersome extraction steps. In recent years, the production of SAA by in vitro multi-enzyme cascades and whole-cell biotransformation methods has attracted considerable attention. These methods usually bypass the difficult hydroxylation reaction and directly use levodopa (DOPA) as a precursor, which greatly increases the cost of the substrate. In addition, it has been recently reported that the production of SAA by 4-hydroxyphenylacetate (4-HPA) 3-hydroxylase (EcHpaBC) and D-lactate dehydrogenase (D-LDH Y52A ) was introduced into the endogenous tyrosine synthesis pathway of Escherichia coli, and de novo synthesis of danshensu was achieved in Escherichia coli. After 70 hours of fermentation in a 5L fermenter, the yield of danshensu was 7.1g / L. Although the above research has made a major breakthrough in the synthesis of SAA, the problem of by-product accumulation caused by the promiscuous activity of hydroxylase has not been solved, and the yield of SAA is still low, making it difficult to achieve large-scale production. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a genetically engineered bacterium for producing danshensu.

[0006] The second object of the present invention is to provide a method for constructing a genetically engineered bacterium for producing tanshinone.

[0007] The third object of the present invention is to provide the use of the above-mentioned genetically engineered bacteria for producing danshensu by fermentation.

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

[0009] A method for constructing a genetically engineered bacterium for producing danshensu comprises the following steps:

[0010] (1) Using Escherichia coli W3110 as the starting strain, the tanshinone synthesis pathway was reconstructed in the cytoplasm, and the steps were as follows:

[0011] ① Knock out the lactose operon gene lacIZ and the transcriptional regulatory factor gene tyrR in Escherichia coli W3110;

[0012] ②Integration of 4-hydroxyphenylpyruvate biosynthesis pathway gene aroG S180F ,tyrA M53I / A354V , aroK and ydiB, and knocking out the prephenate dehydrogenase gene pheA;

[0013] ③Integrate the D-2-hydroxyacid dehydrogenase encoding gene BlfldH from Bifidobacterium longum;

[0014] ④Integrate the 4-hydroxyphenylacetate-3-hydroxylase mutant gene EchpaB E216G / E294I / Q434T and the flavin reductase gene KphpaC from Klebsiella pneumoniae , of which EchpaB was derived from Escherichia coli BL21(DE3);

[0015] ⑤ Integrate the L-amino acid deaminase gene PvlaaD from Proteus vulgaris; obtain the Escherichia coli engineered strain SAA-11; (2) use the periplasmic space localization signal peptide sufI to localize the flavin reductase and 4-hydroxyphenylacetic acid-3-hydroxylase mutant from Klebsiella pneumoniae in the periplasmic space, the steps are:

[0016] Construction of a nucleotide sequence-guided sufI containing genes KphpaC and EchpaB E216G / E294I / Q434T The fusion fragment is introduced into the Escherichia coli engineering strain SAA-11 to obtain the Escherichia coli engineering strain SAA-12. The nucleotide sequence of sufI is shown in SEQ ID NO.112.

[0017] The above method constructs a genetically engineered bacterium for producing danshensu.

[0018] The application of the above-mentioned genetically engineered bacteria for producing danshensu by fermentation to produce danshensu.

[0019] Beneficial effects of the present invention:

[0020] The genetically engineered bacteria for producing danshensu of the present invention can produce 2.8 g / L danshensu after 24 h of shake flask fermentation and 26.6 g / L danshensu after 48 h of fermentation in a 5L fermenter. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 2 is a plasmid map, where a is pREDCas9 and b is pGRB plasmid.

[0022] Figure 2 The production and OD of Danshensu (SAA) of Escherichia coli engineered strain SAA-12 in a 5L fermenter 600 picture. DETAILED DESCRIPTION

[0023] Escherichia coli W3110 is commercially available.

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

[0025] 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.

[0026] Gene editing methods involved in the embodiments:

[0027] 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.

[0028] The specific steps of the CRISPR / Cas9 gene editing method are as follows:

[0029] 1. X-sgRNA plasmid construction

[0030] The pGRB plasmid (commercially available) was used as a template and primers pGRB-F (SEQ ID NO. 107) and pGRB-R (SEQ ID NO. 108) were used to obtain the pGRB plasmid vector by PCR.

[0031] 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.

[0032] 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.

[0033] 2. Preparation of recombinant DNA fragments

[0034] 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.

[0035] 3. Preparation of Electroporated Competent Cells

[0036] 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;

[0037] 2) Prepare competent cells. The competent cells preparation process is performed according to conventional standard operation.

[0038] 4. Electroporation

[0039] 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.

[0040] 5. Elimination of Plasmid

[0041] 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.

[0042] The genes involved in strain construction are shown in Table 1

[0043] Table 1 Gene accession numbers

[0044] Gene name accession number (GenBank or Uniprot) lacI BAE76127.1 lacZ BAE76126.1 tyrBAA14905.1 tehB BAA15059.1 aroG BAA35416.1 tyrA BAA16485.2 pheA BAA16484.1 aroK BAE77901.1 ydiB BAA15449.1 BlfldH WP_060620770.1 EchpaB Q57160 KphpaC A6THU1 PvlaaD A0A6M8R9V3

[0045] Example 1

[0046] Construction of the engineered Escherichia coli strain SAA-11

[0047] 1.1 Knockout of the lactose operon gene lacIZ

[0048] Using Escherichia coli W3110 (hereinafter referred to as W3110) as a template, primers lacIZ-UP-F (SEQ ID NO.1) and lacIZ-UP-R (SEQ ID NO.2) were used to obtain the upstream homology arm fragment of lacIZ knockout by PCR, primers lacIZ-DN-F (SEQID NO.3) and lacIZ-DN-R (SEQ ID NO.4) were used to obtain the downstream homology arm fragment of lacIZ knockout by PCR, and primers lacIZ-UP-F (SEQ ID NO.1) and lacIZ-DN-R (SEQ ID NO.4) were used to fuse the upstream and downstream homology arm fragments by PCR to obtain the fusion fragment of lacIZ knockout.

[0049] 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.

[0050] Prepare W3110 electroporation competent cells, and electroporate the pREDCas9 plasmid into the W3110 electroporation competent cells to obtain strain W3110-pREDCas9.

[0051] W3110-pREDCas9 electroporation competent medium was prepared, and the lacIZ knockout fusion fragment and 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). The correct strain was verified and the Escherichia coli engineered strain SAA-1, referred to as SAA-1, was obtained after plasmid elimination.

[0052] 1.2 Knockout of transcriptional regulator gene tyrR

[0053] 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.

[0054] 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.

[0055] Prepare SAA-1 electroporation competent cells, and electroporate the pREDCas9 plasmid into the SAA-1 electroporation competent cells to obtain the strain SAA-1-pREDCas9.

[0056] The SAA-1-pREDCas9 electroporation competent medium was prepared, and the fusion fragment with tyrR knockout and the tyrR-sgRNA plasmid were simultaneously electroporated into the SAA-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). The correct strain was verified and the Escherichia coli engineered strain SAA-2 (SAA-2 for short) was obtained after plasmid elimination.

[0057] 1.3 Integration of 4-hydroxyphenylpyruvate biosynthesis pathway gene aroG at the tehB locus S180F

[0058] Using W3110 as a template, primers tehB-UP-F (SEQ ID NO.13) and tehB-UP-R (SEQ ID NO.14) were used to obtain the upstream homology arm fragment of tehB knockout, primers tehB-DN-F (SEQ ID NO.15) and tehB-DN-R (SEQ ID NO.16) were used to obtain the downstream homology arm fragment of tehB knockout, and primers aroG-F (SEQ ID NO.19) and aroG-S180F-R (SEQ ID NO.20) were used to obtain the aroG S180F The upstream fragment of 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 was amplified, and 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 the △tehB::Ptrc-aroGS180F fusion fragment.

[0059] 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 to obtain the tehB-sgRNA plasmid.

[0060] Prepare SAA-2 electroporation competent cells, and electroporate the pREDCas9 plasmid into the SAA-2 electroporation competent cells to obtain the strain SAA-2-pREDCas9.

[0061] Prepare SAA-2-pREDCas9 electroporation competent cells and transfer △tehB::P trc -aroG S180FThe fusion fragment and tehB-sgRNA plasmid were simultaneously electroporated into the SAA-2-pREDCas9 electroporation competent medium. 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). The correct strain was verified and the plasmid was eliminated to obtain the Escherichia coli engineered strain SAA-3 (abbreviated as SAA-3).

[0062] 1.4 Integration of tyrA at the mbhA locus M53I / A354V Gene

[0063] Using W3110 as a template, primers mbhA-UP-F (SEQ ID NO.23) and mbhA-UP-R (SEQ ID NO.24) were used to obtain the upstream homology arm fragment of mbhA knockout, primers mbhA-DN-F (SEQ ID NO.25) and mbhA-DN-R (SEQ ID NO.26) were used to obtain the downstream homology arm fragment of mbhA knockout, and primers tyrA-F (SEQ ID NO.29) and tyrA-M53I-R (SEQ ID NO.30) 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.31) and tyrA-A354V-R (SEQ ID NO.32). M53I / A354V The second fragment of tyrA was obtained by PCR using primers tyrA-A354V-F (SEQ ID NO.33) and tyrA-R (SEQ ID NO.34). M53I / A354V The 5 fragments were fused by PCR using primers mbhA-UP-F (SEQ ID NO.23) and mbhA-DN-R (SEQ ID NO.26) to obtain △mbhA::P trc -tyrA M53I / A354V Fusion fragments.

[0064] 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.

[0065] Prepare SAA-3 electroporation competent cells, and electroporate the pREDCas9 plasmid into the SAA-3 electroporation competent cells to obtain the strain SAA-3-pREDCas9.

[0066] Prepare SAA-3-pREDCas9 electroporation competent cells and transfer △mbhA::P trc -tyrA M53I / A354V The fusion fragment and mbhA-sgRNA plasmid were simultaneously electroporated into the SAA-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). The correct strain was verified and the plasmid was eliminated to obtain the Escherichia coli engineered strain SAA-4 (abbreviated as SAA-4).

[0067] 1.5 Integration of aroK gene at the rph locus

[0068] 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.

[0069] 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.

[0070] Prepare SAA-4 electroporation competent cells, and electroporate the pREDCas9 plasmid into the SAA-4 electroporation competent cells to obtain the strain SAA-4-pREDCas9.

[0071] Prepare SAA-4-pREDCas9 electroporation competent cells and transform △rph::P trcThe -aroK fusion fragment and rph-sgRNA plasmid were simultaneously electroporated into the SAA-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). The correct strain was verified and the plasmid was eliminated to obtain the Escherichia coli engineered strain SAA-5 (abbreviated as SAA-5).

[0072] 1.6 Integration of the ydiB gene at the yeep locus

[0073] 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.

[0074] 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.

[0075] Prepare SAA-5 electroporation competent cells, and electroporate the pREDCas9 plasmid into the SAA-5 electroporation competent cells to obtain the strain SAA-5-pREDCas9.

[0076] Prepare SAA-5-pREDCas9 electroporation competent cells and transfer △yeeP::P trc The -ydiB fusion fragment and yeeP-sgRNA plasmid were simultaneously electroporated into the SAA-5-pREDCas9 electroporation competent medium. 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). The correct strain was verified and the Escherichia coli engineered strain SAA-6 (abbreviated as SAA-6) was obtained after plasmid elimination.

[0077] 1.7 Knockout of pheA gene

[0078] 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.

[0079] 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.

[0080] Prepare SAA-6 electroporation competent cells, and electroporate the pREDCas9 plasmid into the SAA-6 electroporation competent cells to obtain the strain SAA-6-pREDCas9.

[0081] The SAA-6-pREDCas9 electroporation competent medium was prepared, and the pheA knockout fusion fragment and pheA-sgRNA plasmid were simultaneously electroporated into the SAA-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). The correct strain was verified and the Escherichia coli engineered strain SAA-7 (SAA-7 for short) was obtained after plasmid elimination.

[0082] 1.8 Integration of the D-2-hydroxyacid dehydrogenase encoding gene BlfldH from Bifidobacterium longum at the yjgX locus

[0083] 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;

[0084] The synthetic sequence BlfldH (SEQ ID NO.109) 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.

[0085] 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.

[0086] Prepare SAA-7 electroporation competent cells, and electroporate the pREDCas9 plasmid into the SAA-7 electroporation competent cells to obtain the strain SAA-7-pREDCas9.

[0087] Prepare SAA-7-pREDCas9 electroporation competent cells and transfer △yjgX::P trc The -BlfldH fusion fragment and yjgX-sgRNA plasmid were simultaneously electroporated into the SAA-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). The correct strain was verified and the Escherichia coli engineered strain SAA-8 (abbreviated as SAA-8) was obtained after plasmid elimination.

[0088] 1.9 Integration of 4-hydroxyphenylacetate-3-hydroxylase mutant gene EchpaB at the yciQ locus E216G / E294I / Q434T

[0089] 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;

[0090] 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 EchpaBE216G / E294I / Q434T The 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.

[0091] 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.

[0092] Prepare SAA-8 electroporation competent cells, and electroporate the pREDCas9 plasmid into the SAA-8 electroporation competent cells to obtain the strain SAA-8-pREDCas9.

[0093] Prepare SAA-8-pREDCas9 electroporation competent cells and transfer △yciQ::P trc -EchpaB E216G / E294I / Q434TThe fusion fragment and yciQ-sgRNA plasmid were simultaneously electroporated into the SAA-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). The correct strain was verified and the plasmid was eliminated to obtain the Escherichia coli engineered strain SAA-9 (abbreviated as SAA-9).

[0094] 1.10 Integration of the flavin reductase gene KphpaC from Klebsiella pneumoniae into the yjiV locus

[0095] 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.110) 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.

[0096] 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.

[0097] Prepare SAA-9 electroporation competent cells, and electroporate the pREDCas9 plasmid into the SAA-9 electroporation competent cells to obtain the strain SAA-9-pREDCas9.

[0098] Prepare SAA-9-pREDCas9 electroporation competent cells and transfer △yjiV::P trc-KphpaC fusion fragment and yjiV-sgRNA plasmid were simultaneously electroporated into SAA-9-pREDCas9 electroporation competent medium. 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). The correct strain was verified and the Escherichia coli engineered strain SAA-10 (SAA-10 for short) was obtained after plasmid elimination.

[0099] 1.11 Integration of the L-amino acid deaminase gene PvlaaD from Proteus vulgaris at the yeeL locus

[0100] Using W3110 as a template, PCR was performed with primers yeeL-UP-F (SEQ ID NO.99) and yeeL-UP-R (SEQ ID NO.100) to obtain the upstream homology arm fragment of yjiV knockout, and PCR was performed with primers yeeL-DN-F (SEQ ID NO.101) and yeeL-DN-R (SEQID NO.102) to obtain the downstream homology arm fragment of yeeL knockout;

[0101] The synthesized sequence PvlaaD (SEQ ID NO.111) was used as a template, and primers PvlaaD-F (SEQ ID NO.105) and PvlaaD-R (SEQ ID NO.106) were used to obtain the PvlaaD fragment by PCR, and the above three fragments were fused by PCR using primers yeeL-UP-F (SEQ ID NO.99) and yeeL-DN-R (SEQ ID NO.102) to obtain △yeeL::P trc -PvlaaD fusion fragment.

[0102] The yeeL-DNA double-stranded fragment was obtained by annealing with primers yeeL-gRNA-F (SEQ ID NO.103) and yeeL-gRNA-R (SEQ ID NO.104), and the fragment was connected to the pGRB plasmid vector to obtain the yeeL-sgRNA plasmid.

[0103] Prepare SAA-10 electroporation competent cells, and electroporate the pREDCas9 plasmid into the SAA-10 electroporation competent cells to obtain the strain SAA-10-pREDCas9.

[0104] Prepare SAA-10-pREDCas9 electroporation competent cells and transfer △yeeL::P trc-PvlaaD fusion fragment and yeeL-sgRNA plasmid were simultaneously electroporated into SAA-10-pREDCas9 electroporation competent cells. The transformed single clones were verified by colony PCR using primers yeeL-UP-F (SEQ ID NO.99) and yeeL-DN-R (SEQ ID NO.102). The correct strain was verified and the plasmid was eliminated to obtain the E. coli engineered strain SAA-11. (Abbreviated as SAA-11)

[0105] Example 2

[0106] Construction of the engineered Escherichia coli strain SAA-12

[0107] In order to localize the flavin reductase and 4-hydroxyphenylacetate-3-hydroxylase mutants from Klebsiella pneumoniae to the periplasmic space of Escherichia coli for expression, the sufI signal peptide was used as a guide in this example.

[0108] Construction of a nucleotide sequence guided by sufI containing genes KphpaC and EchpaB E216G / E294I / Q434T The two genes were connected by Linker (GSTSSGSSG), and the fusion fragment was introduced into the Escherichia coli engineering strain SAA-11 to obtain the Escherichia coli engineering strain SAA-12. The nucleotide sequence of sufI is shown in SEQ ID NO.112.

[0109] Using W3110 as a template, PCR was performed with primers yghE-UP-F (SEQ ID NO.87) and yghE-UP-R (SEQ ID NO.88) to obtain the upstream homology arm fragment of yghE knockout, and PCR was performed with primers yghE-DN-F (SEQ ID NO.89) and yghE-DN-R (SEQID NO.90) to obtain the downstream homology arm fragment of yghE knockout;

[0110] Using W3110 as template, PCR was performed with primers sufI-2F-F (SEQ ID NO.93) and sufI-R2 (SEQ ID NO.94) to obtain the signal peptide sufI fragment;

[0111] Using the synthesized sequence KphpaC (SEQ ID NO.110) as a template, PCR was performed using primers FL-KphpaC-R (SEQ ID NO.95) and FL-EchpaB-F (SEQ ID NO.96) to obtain the FL-KphpaC fragment;

[0112] The P obtained from Example 1 at 1.9 trc -EchpaB E216G / E294I / Q434TThe fragment was used as a template, and primers EchpaB-KpHpaC-F (SEQ ID NO.97) and EchpaB-KpHpaC-R (SEQ ID NO.98) were used to obtain FL-EchpaB E216G / E294I / Q434T The nucleotide sequence of linker: GSTSGSSG was designed in FL-KphpaC-R (SEQ ID NO.95) and EchpaB-KpHpaC-F (SEQ ID NO.97). Finally, the upstream and downstream homology arm fragments of yghE were knocked out and sufI, FL-KphpaC, FL-EchpaB were linked with primers yghE-UP-F (SEQ ID NO.87) and yghE-DN-R (SEQ ID NO.90). E216G / E294I / Q434T Fragment fusion PCR to obtain △yghE::P trc -sufI-KphpaC-GSTSSGSSG-EchpaB E216G / E294I / Q434T Fusion fragments.

[0113] The yghE-DNA double-stranded fragment was obtained by annealing with primers yghE-gRNA-F (SEQ ID NO.91) and yghE-gRNA-R (SEQ ID NO.92), and the fragment was connected to the pGRB plasmid vector to obtain the yghE-sgRNA plasmid.

[0114] Prepare SAA-11 electroporation competent cells, and electroporate the pREDCas9 plasmid into the SAA-11 electroporation competent cells to obtain the strain SAA-11-pREDCas9.

[0115] Prepare SAA-11-pREDCas9 electroporation competent cells.

[0116] △yghE::P trc -sufI-KphpaC-GSTSSGSSG-EchpaB E216G / E294I / Q434T The fusion fragment and yghE-sgRNA plasmid were simultaneously electroporated into the SAA-11-pREDCas9 electroporation competent medium. The transformed single clones were verified by colony PCR using primers yghE-UP-F (SEQ ID NO.87) and yghE-DN-R (SEQ ID NO.90). The correct strain was eliminated after plasmid elimination to obtain the E. coli engineered strain SAA-12. (Abbreviated as SAA-12)

[0117] Example 3

[0118] The method for producing danshensu by fermentation of Escherichia coli engineered strain SAA-12 is as follows:

[0119] 3.1 Shake flask fermentation:

[0120] A single colony of the engineered E. coli strain SAA-12 constructed in Example 1 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 a seed solution. 1 mL of the seed solution was transferred to 30 mL of fermentation medium, and fermented at 37°C in a shaker at 220 rpm for 24 h.

[0121] 3.2 Fermentation in 5L fermenter

[0122] Slant activation culture: scrape a loop of bacteria from the -80℃ freezer tube, spread evenly on LB solid slant medium, and culture at 37℃ for 12h;

[0123] Seed culture: Then transfer all into a 5L fermenter containing 3L fermentation medium. Culture OD 600 After reaching 10, 15% of the seed solution was transferred to a 5 L fermentor containing 2 L of fresh fermentation medium.

[0124] Fermentation culture: During the fermentation process, the pH is controlled to be stable at about 7.0, the temperature is maintained at 35°C, and the dissolved oxygen is about 15%; when the glucose in the culture medium is consumed, 80% (m / v) glucose solution is added.

[0125] Fermentation medium formula:

[0126] 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.

[0127] Example 4

[0128] The concentration of danshensu in the fermentation broth was detected by HPLC, as follows:

[0129] 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.

[0130] 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.

[0131] like Figure 2 As shown, the Escherichia coli engineered strain SAA-12 was cultured in a 5L fermenter for 48 hours, and the tanshinone production reached 26.6 g / L, which is better than the existing technical reports and has good prospects for industrial application.

[0132] 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 danshensu, characterized in that: (1) Using Escherichia coli W3110 as the starting strain, the tanshinone synthesis pathway was reconstructed in the cytoplasm, and the steps were as follows: ① Knock out the lactose operon gene lacIZ and the transcriptional regulatory factor gene tyrR in Escherichia coli W3110; ②Integration of 4-hydroxyphenylpyruvate biosynthesis pathway gene aroG S180F ,tyrA M53I / A354V , aroK and ydiB, and knocking out the prephenate dehydrogenase gene pheA; ③Integrate the D-2-hydroxyacid dehydrogenase encoding gene BlfldH from Bifidobacterium longum; ④Integrate the 4-hydroxyphenylacetate-3-hydroxylase mutant gene EchpaB E216G / E294I / Q434T and the flavin reductase gene KphpaC from Klebsiella pneumoniae , of which EchpaB was derived from Escherichia coli BL21(DE3); ⑤ Integrate the L-amino acid deaminase gene PvlaaD from Proteus vulgaris; obtain the Escherichia coli engineered strain SAA-11; (2) use the periplasmic space localization signal peptide sufI to localize the flavin reductase and 4-hydroxyphenylacetic acid-3-hydroxylase mutant from Klebsiella pneumoniae in the periplasmic space, the steps are: Construction of a nucleotide sequence-guided sufI containing genes KphpaC and EchpaB E216G / E294I / Q434T The fusion fragment is introduced into the Escherichia coli engineering strain SAA-11 to obtain the Escherichia coli engineering strain SAA-12. The nucleotide sequence of sufI is shown in SEQ ID NO.

112.

2. A genetically engineered bacterium for producing danshensu constructed by the method of claim 1.

3. The use of a genetically engineered bacterium for producing danshensu according to claim 2 to produce danshensu by fermentation.

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