A genetically engineered Escherichia coli strain that produces emodin, its construction method, and its application.

Abstract

This application relates to the field of genetically engineered bacteria technology, specifically disclosing a genetically engineered *E. coli* strain for producing emodin, its construction method, and its applications. This application uses *E. coli* BAP1 as the substrate strain. This strain carries its own sfp in its genome, which can be used to activate polyketide synthase. Furthermore, *E. coli* itself grows rapidly, has a vigorous metabolism, and the culture medium is low-cost, facilitating high-density fermentation. Through gene editing, the uptake and utilization of glucose are improved, the flux of acetic acid and lactic acid diverted from pyruvate is reduced, and the ATP supply is enhanced to increase the flux of acetyl-CoA and malonyl-CoA within the bacteria. This provides sufficient substrate for the synthesis of emodin by polyketide synthase, thioesterase, and decarboxylase, thereby increasing the yield of emodin. The emodin yield achieved by this application through shake-flask fermentation reaches 576.4 mg / L, which is currently the highest level in the *E. coli* system.

Description

Technical Field

[0001] This application relates to the field of genetically engineered bacteria technology, and in particular to a genetically engineered Escherichia coli strain that produces emodin, its construction method, and its application. Background Technology

[0002] Emodin is a naturally occurring anthraquinone compound with the chemical formula C. 15 H 10 O5 is mainly found in the rhizomes of various medicinal plants, such as *Rheum palmatum*, *Rheum tanguticum*, and *Rheum palmatum*. Emodin is one of the main active and representative components of traditional Chinese purgatives. Emodin possesses multiple pharmacological effects, including anti-inflammatory, antibacterial, analgesic, antioxidant, hepatoprotective, and antitumor properties. Emodin itself is also a natural pigment that can be used in health products and daily chemical products, such as hair care and skin care.

[0003] Emodin is mainly produced through three methods: plant extraction, chemical synthesis, and microbial fermentation. Currently, plant extraction remains the primary method, but it faces challenges such as low emodin content in raw materials, long extraction cycles, and low separation and purification efficiency. Chemical synthesis of emodin suffers from low yields and complex routes. Microbial fermentation technology, due to its mild, environmentally friendly, and highly sustainable nature, is an important direction for green manufacturing; however, the technology is not yet fully mature and faces challenges related to catalytic efficiency and precursor supply. Currently, most microbial strains used for emodin synthesis are fungi, such as *Aspergillus nidus*, which can achieve a yield of 100.8 mg / L, and *Saccharomyces cerevisiae*, which can achieve a yield of 528.4 ± 62.7 mg / L. However, fungal fermentation has long fermentation cycles and low conversion efficiency. Summary of the Invention

[0004] The purpose of this application is to overcome the shortcomings of the prior art and provide a genetically engineered Escherichia coli strain for producing emodin, its construction method and application. The genetically engineered Escherichia coli strain provided by this application has advantages such as fast growth rate and short fermentation cycle.

[0005] To achieve the above objectives, the technical solution adopted in this application is as follows: This application provides a method for constructing a genetically engineered Escherichia coli strain that produces emodin, comprising the following steps: S1. Based on the chassis strain BAP1, the pta gene was knocked out sequentially, and J23119-ppk was introduced into the gene locus and named strain EM01. S2. Based on strain EM01, the poxB gene was knocked out, and J23119-accBC was introduced into this gene locus, and the strain was named EM02. S3. Based on strain EM02, the ldhA gene was knocked out, and J23119-accAD was introduced into the gene locus, and the strain was named EM03. S4. Based on strain EM03, the galR gene was knocked out, and the strain was named strain EM04. S5. The 95S expression plasmid containing the SlACAS gene, HyTE gene, and AfDC gene was transferred into strain EM04 to construct an Escherichia coli genetically engineered strain that produces emodin, and it was named strain EM05.

[0006] The *E. coli* genetically engineered strain provided in this application was obtained through metabolic engineering. The chassis strain EM04 uses *E. coli* BAP1 as the chassis strain, knocking out the acetic acid production pathway and the lactate production pathway, reducing the pyruvate diversion pathway, and directing more acetyl-CoA to acetyl-CoA. Subsequently, the ACC complex is further enhanced to increase the synthesis of malonyl-CoA from acetyl-CoA. Furthermore, since the synthesis of malonyl-CoA from acetyl-CoA requires a large amount of ATP, and the synthesis product requires the diversion of a large amount of acetyl-CoA... A cannot enter the tricarboxylic acid cycle to provide a large amount of energy, so ppk is enhanced to provide sufficient ATP for substrate synthesis; finally, galR is knocked out to increase glucose uptake and provide sufficient substrate for the growth and metabolism of the strain. Then, the 95S expression plasmid containing the SlACAS gene, HyTE gene, and AfDC gene (expression vector 95S-SlACAS-HyTE-AfDC) is introduced into the chassis strain EM04 to construct the genetically engineered strain EM05. The constructed genetically engineered strain EM05 can increase the yield of emodin.

[0007] Containing the SlACAS gene, HyTE gene, and AfDC gene, these are key genes for heterologous expression of emodin synthesis within the Escherichia coli host.

[0008] As a preferred embodiment of the method for constructing the Escherichia coli genetically engineered bacteria for producing emodin as described in this application, the nucleotide sequence of J23119-ppk in step S1 is shown in SEQ ID NO: 53; the nucleotide sequence of J23119-accBC in step S2 is shown in SEQ ID NO: 54; and the nucleotide sequence of J23119-accAD in step S3 is shown in SEQ ID NO: 55.

[0009] As a preferred embodiment of the method for constructing the Escherichia coli genetically engineered bacteria for producing emodin as described in this application, the method for constructing the 95S expression plasmid containing the SlACAS gene, HyTE gene, and AfDC gene in step S5 includes the following steps: Using pLY2 plasmid as a template, the SlACAS fragment with homologous arms was amplified by PCR using primers 95S-SlACAS-F and TE-SlACAS-R; the HyTE fragment with homologous arms was amplified by PCR using primers S-HyTE-F and DC-HyTE-R; the AfDC fragment with homologous arms was amplified by PCR using primers TE-AfDC-F and 95S-AfDC-R; and the 95S plasmid backbone was amplified by PCR using primers 95S-bpsA and 95S-R. The SlACAS, HyTE, AfDC fragments, and the 95S plasmid backbone were assembled using Gibson DNA, and then transformed into DH5α to obtain a 95S expression plasmid containing the SlACAS, HyTE, and AfDC genes. The nucleotide sequence of 95S-SlACAS-F is shown in SEQ ID NO: 47, the nucleotide sequence of TE-SlACAS-R is shown in SEQ ID NO: 48, the nucleotide sequence of S-HyTE-F is shown in SEQ ID NO: 49, the nucleotide sequence of DC-HyTE-R is shown in SEQ ID NO: 50, the nucleotide sequence of TE-AfDC-F is shown in SEQ ID NO: 51, the nucleotide sequence of 95S-AfDC-R is shown in SEQ ID NO: 52, the nucleotide sequence of 95S-F is shown in SEQ ID NO: 53, and the nucleotide sequence of 95S-R is shown in SEQ ID NO: 54.

[0010] The nucleotide sequence of the SlACAS fragment is shown in SEQ ID NO: 56; the nucleotide sequence of the HyTE fragment is shown in SEQ ID NO: 57; and the nucleotide sequence of the AfDC fragment is shown in SEQ ID NO: 58.

[0011] This application also provides Escherichia coli genetically engineered bacteria prepared using the above-mentioned method for constructing genetically engineered Escherichia coli.

[0012] This application also provides the application of the above-mentioned genetically engineered Escherichia coli in the production of emodin.

[0013] In the technical solution of this application, the Escherichia coli genetically engineered strain uses BAP1 as the starting strain, knocking out the acetic acid and lactic acid production pathways of E. coli, reducing pyruvate shunting, strengthening ACC intensity, increasing malonyl-CoA flux, and simultaneously increasing glucose uptake rate. ATP supply provides sufficient substrate for the strain's growth and metabolism. The genetically engineered strain of this application utilizes glucose as a substrate to generate large amounts of acetyl-CoA and malonyl-CoA in vivo, efficiently synthesizing emodin.

[0014] This application also provides a method for shake-flask fermentation, including the following steps: The above-mentioned Escherichia coli genetically engineered bacteria were activated, inoculated into shake flask culture medium, and cultured in a shaker to obtain rhein shake flask culture medium.

[0015] The yield of emodin obtained by this application through shake-flask fermentation reached 576.4 mg / L, which is the highest level in the current E. coli system.

[0016] In a preferred embodiment of the shake-flask fermentation method described in this application, the shake-flask culture medium comprises components of a certain mass concentration: Tryptone 8~12g / L, yeast powder 4~6g / L, Na2HPO4 3~4g / L, KH2PO4 3~4g / L, NH4Cl 1.2~2.5g / L, Na2SO4 0.5~1.5g / L, MgSO4·7H2O 0.2~1g / L, 100× trace elements 10ml / L, defoamer 150μl / L~1ml / L, glycerol 2~4g / L, glucose 4~6g / L, biotin 50~100 μg / L, and polyphosphate 0.5~2g / L.

[0017] In a preferred embodiment of the shake-flask fermentation method described in this application, the 100× trace elements comprise the following components at the following mass concentrations: CuSO40.2~0.4g / L, CaCl2·2H2O 1.5~3.1g / L, MnCl2·4H2O 0.2~0.4g / L, ZnSO4·7H2O 5~6g / L, CoCl2·6H2O 0.3~0.5g / L, FeSO4·7H2O 2~3g / L, Na2MO4·2H2O 0.4~0.6g / L and 0.5M EDTA 80~100ml / L.

[0018] As a preferred embodiment of the shake flask fermentation method described in this application, the shaker culture includes the following conditions: shaker temperature of 28~32℃, rotation speed of 180~225rpm, and fermentation time of 60h.

[0019] In a preferred embodiment of the shake-flask fermentation method described in this application, the shake-flask culture medium is further supplemented with streptomycin at a final concentration of 50-100 μg / ml.

[0020] In some specific embodiments, the inoculum size of the genetically engineered Escherichia coli is 1%.

[0021] Compared with the prior art, this application has the following beneficial effects: This application provides a genetically engineered *E. coli* strain for producing emodin, its construction method, and its applications. This application uses *E. coli* BAP1 as the substrate strain. This strain carries its own sfp in its genome, which can be used to activate polyketide synthase. Furthermore, *E. coli* itself grows rapidly, has a vigorous metabolism, and the culture medium is low-cost and suitable for high-density fermentation. Through gene editing, the uptake and utilization of glucose are improved, the flux of acetic acid and lactic acid diverted from pyruvate is reduced, and the ATP supply is enhanced to increase the flux of acetyl-CoA and malonyl-CoA within the strain. This provides sufficient substrate for the synthesis of emodin by polyketide synthase, thioesterase, and decarboxylase, thereby increasing the yield of emodin. The emodin yield achieved by this application through shake-flask fermentation reaches 576.4 mg / L, which is currently the highest level in the *E. coli* system. The genetically engineered strain of this application can further promote the process of emodin biosynthesis. Attached Figure Description

[0022] Figure 1 High-performance liquid chromatogram of emodin standard; Figure 2 This is a high-performance liquid chromatogram of the fermentation broth in Example 6; Figure 3 Standard curve for the detection of emodin by high performance liquid chromatography. Detailed Implementation

[0023] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0024] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified, and the raw materials used in each parallel experiment are the same.

[0025] In the following embodiments: LB medium: 10 g / L sodium chloride, 10 g / L peptone and 5 g / L yeast extract.

[0026] The ZYBP fermentation medium formula is as follows: tryptone 10g / L, yeast extract 5g / L, Na2HPO4 3.55g / L, KH2PO4 3.4g / L, NH4Cl 2g / L, Na2SO4 0.71g / L, MgSO4·7H2O 0.5g / L, 100× trace elements 10ml / L, Guanhe sample 806 polyether defoamer 0.3ml / L, glycerol 2g / L, glucose 5g / L, biotin 50 μg / L and polyphosphate 1g / L.

[0027] The formula for 100× trace elements is: CuSO4 0.32g / L, CaCl2·2H2O 2g / L, MnCl2·4H2O 0.32g / L, ZnSO4·7H2O 5.75g / L, CoCl2·6H2O 0.47g / L, FeSO4·7H2O 2.8g / L, Na2MO4·2H2O 0.48g / L, and 0.5M EDTA 80ml / L.

[0028] This application provides a method for constructing a genetically engineered Escherichia coli strain that produces emodin, comprising the following steps: S1. Based on the chassis strain BAP1, the pta gene was knocked out sequentially, and J23119-ppk was introduced into the gene locus and named strain EM01. S2. Based on strain EM01, the poxB gene was knocked out, and J23119-accBC was introduced into this gene locus, and the strain was named EM02. S3. Based on strain EM02, the ldhA gene was knocked out, and J23119-accAD was introduced into the gene locus, and the strain was named EM03. S4. Based on strain EM03, the galR gene was knocked out, and the strain was named strain EM04. S5. The 95S expression plasmid containing the SlACAS gene, HyTE gene, and AfDC gene was transferred into strain EM04 to construct an Escherichia coli genetically engineered strain that produces emodin, and it was named strain EM05.

[0029] The primers used to construct the recombinant plasmid in Example 1 are shown in Table 1.

[0030] Table 1 The primers used to construct the recombinant plasmid in Example 2 are shown in Table 2.

[0031] Table 2 The primers used to construct the recombinant plasmid in Example 3 are shown in Table 3.

[0032] Table 3 The primers used to construct the recombinant plasmid in Example 4 are shown in Table 4.

[0033] Table 4 The primers used to construct the recombinant plasmid in Example 5 are shown in Table 5.

[0034] Table 5 Example 1 The construction method of chassis strain EM01, utilizing the CRISPR-Cas9 technology principle, involves the following steps: (1) Construction of pTarget-pta: Using pTargetF as a template, PCR amplification was performed using pTarget-pta-F and pTarget-pta-R primers. The template was digested with DpnI and transformed into DH5α to obtain pTarget-pta.

[0035] (2) Construction of 95S-ppk: Using Escherichia coli BAP1 as a template, the linearized fragment of 95S-ppk was obtained by PCR amplification using primers 95S-ppk-F and 95S-ppk-R; using 95S-bpsA plasmid as a template, the 95S plasmid backbone was obtained by PCR amplification using primers 95S-F and 95S-R; the 95S plasmid backbone and the linearized fragment of 95S-ppk were assembled by Gibson, and then transformed into DH5α to obtain plasmid 95S-ppk.

[0036] (3) Construction of the Δpta::ppk-T1 donor fusion fragment: Using E. coli BAP1 as a template, the Δpta::ppk up fragment was obtained by PCR amplification using primers pta-up800F and pta::ppk-up500R; using E. coli BAP1 as a template, the Δpta::ppk-T1 down fragment was obtained by PCR amplification using primers pta::ppk-down500F-T1 and pta-down800R; using 95S-ppk as a template, the J23119-ppk-T1 fragment was obtained by PCR amplification using primers T5 / tac / 119-F1 and rrnB-R1-T1; using the Δpta::ppk up fragment, Δpta::ppk-T1 down fragment, and J23119-ppk-T1 fragment as templates, the Δpta::ppk-T1 fragment was obtained by PCR amplification using primers pta-up500F and pta-down500R overlap extension. donor fusion fragment.

[0037] (4) The pTarget-pta and Δpta::ppk-T1 donor fusion fragments were integrated into the chassis strain IN05 by electroporation. Single colonies were picked from the plate after 24 hours of incubation at 30°C and PCR verification was performed using pta-up800F and pta-down800R primers. Strains that underwent correct homologous recombination were screened to obtain the recombinant Escherichia coli gene-edited strain, which was named EM01.

[0038] The specific process of the above transformation is as follows: Take the system from the above steps and add it to the DH5α-competent cells that have been thawed on ice. After incubating on ice for 30 min, heat shock at 42℃ for 1 min, and then incubate on ice again for 2 min. Then add 900 uL of pre-cooled LB medium and incubate at 37℃ and 220 rpm for 60 min. Take 100 μL of the incubated bacterial solution and add it to LB medium containing streptomycin resistance and incubate overnight at 37℃.

[0039] The specific process of the above electroporation transformation is as follows: Take 200 ng of pTarget-pta and 400 ng of Δpta::ppk donor fusion fragment and add them to the prepared IN05 electroporation competent cells. After gently mixing, add them to the electroporation cuvette and incubate on ice for 5-10 min. After electroporation at 2.5 KV, quickly add pre-cooled LB medium. Then, incubate the transformation solution in a shaker at 30℃ for 2 hours. After incubation, take 100 μL of transformation solution and spread it on an LB plate. Incubate in a 30℃ incubator for 24 h.

[0040] Example 2 The construction method of chassis strain EM02, utilizing the CRISPR-Cas9 technology principle, involves the following steps: (1) Construction of pTarget-poxB: Using pTarget-pta as a template, PCR amplification was performed using pTarget-poxB-F and pTarget-poxB-R primers. The template was digested with DpnI and transformed into DH5α to obtain pTarget-poxB.

[0041] (2) Construction of 95S-accBC: Using Escherichia coli BAP1 as a template, the linearized fragment of 95S-accBC was obtained by PCR amplification using primers 95S-accB-F and 95S-accC-R; using 95S-bpsA plasmid as a template, the 95S plasmid backbone was obtained by PCR amplification using primers 95S-F and 95S-R; the 95S plasmid backbone and the linearized fragment of 95S-accBC were assembled by Gibson, and then transformed into DH5α to obtain plasmid 95S-accBC.

[0042] (3) Construction of the ΔpoxB::accBC-T2 donor fusion fragment: Using E. coli BAP1 as a template, the ΔpoxB::accBC up fragment was obtained by PCR amplification using primers poxB-up800F and poxB::accBC-up500R; using E. coli BAP1 as a template, the ΔpoxB::accBC-T2 down fragment was obtained by PCR amplification using primers poxB::accBC-down500F-T2 and poxB-down800R; using 95S-accBC as a template, the J23119-accBC-T2 fragment was obtained by PCR amplification using primers T5 / tac / 119-F1 and rrnB-R1-T2; using the ΔpoxB::accBC up fragment and ΔpoxB::accBC-T2... Using the down fragment and the J23119-accBC-T2 fragment as templates, the ΔpoxB::accBC-T2 donor fusion fragment was obtained by PCR amplification using overlapping primers of poxB-up500F and poxB-down500R.

[0043] (4) The pTarget-poxB and ΔpoxB::accBC-T2 donor fusion fragments were integrated into the chassis strain EM01 by electroporation. Single colonies were picked from the plate after 24 hours of incubation at 30°C and PCR verification was performed using poxB-up800F and poxB-down800R primers. Strains that underwent correct homologous recombination were screened to obtain the recombinant Escherichia coli gene-edited strain, which was named EM02.

[0044] Example 3 The construction method of chassis strain EM03, utilizing the CRISPR-Cas9 technology principle, involves the following steps: (1) Construction of pTarget-ldhA: Using pTarget-pta as a template, PCR amplification was performed using pTarget-ldhA-F and pTarget-ldhA-R primers. The template was digested with DpnI and transformed into DH5α to obtain pTarget-ldhA.

[0045] (2) Construction of 95S-accAD: Using Escherichia coli BAP1 as a template, the linearized fragment of 95S-accA was obtained by PCR amplification using primers 95S-accA-F and accAD-R; using Escherichia coli BAP1 as a template, the linearized fragment of 95S-accD was obtained by PCR amplification using primers accAD-F and 95S-accD-R; using 95S-bpsA plasmid as a template, the 95S plasmid backbone was obtained by PCR amplification using primers 95S-F and 95S-R; the 95S plasmid backbone, 95S-accA, and 95S-accD linearized fragments were assembled by Gibson and transformed into DH5α to obtain plasmid 95S-accAD.

[0046] (3) Construction of ΔldhA::accAD-T3 donor fusion fragment: Using E. coli BAP1 as a template, the ΔldhA::accAD up fragment was obtained by PCR amplification using primers ldhA-up800F and ldhA::accAD-up500R; using E. coli BAP1 as a template, the ΔldhA::accAD-T3 down fragment was obtained by PCR amplification using primers ldhA::accAD-down500F-T3 and ldhA-down800R; using 95S-accAD as a template, the J23119-accAD-T3 fragment was obtained by PCR amplification using primers T5 / tac / 119-F1 and rrnB-R1-T3; using the ΔldhA::accAD up fragment and ΔldhA::accAD-T3 donor fusion fragment... Using the down fragment and the J23119-accAD-T3 fragment as templates, the ΔldhA::accAD-T3 donor fusion fragment was obtained by PCR amplification with overlapping extension primers ldhA-up500F and ldhA-down500R.

[0047] (4) The pTarget-ldhA and ΔldhA::accAD-T3 donor fusion fragments were integrated into the chassis strain EM02 by electroporation. Single colonies were picked from the plates after 24 hours of incubation at 30°C. PCR verification was performed using ldhA-up800F and ldhA-down800R primers. Strains that underwent correct homologous recombination were screened to obtain the recombinant Escherichia coli gene-edited strain, which was named EM03.

[0048] Example 4 The construction method of chassis strain EM04, utilizing the CRISPR-Cas9 technology principle, involves the following steps: (1) Construction of pTarget-galR: Using pTarget-pta as a template, PCR amplification was performed using pTarget-galR-F and pTarget-galR-R primers. The template was digested with DpnI and transformed into DH5α to obtain pTarget-galR.

[0049] (2) Construction of ΔgalR-T4 donor fusion fragment: Using Escherichia coli BAP1 as a template, the ΔgalR-T4 up fragment was obtained by PCR amplification using galR-up800F and galR-up500R-T4 primers; using Escherichia coli BAP1 as a template, the ΔgalR::accAD-T3 down fragment was obtained by PCR amplification using galR-down500F-T4 and galR-down800R primers; using the ΔgalR-T4 up fragment and the ΔgalR-T4 down fragment as templates, the ΔgalR-T4 donor fusion fragment was obtained by PCR amplification using galR-up500F and galR-down500R primers with overlap extension.

[0050] (3) The pTarget-galR and ΔgalR-T4 donor fusion fragments were integrated into the chassis strain EM03 by electroporation. Single colonies were picked from the plate after 24 hours of incubation at 30°C. PCR verification was performed using galR-up800F and galR-down800R primers. Strains that underwent correct homologous recombination were screened to obtain the recombinant Escherichia coli gene-edited strain, which was named EM04.

[0051] Example 5 The construction method of the genetically engineered strain EM05 is as follows: (1) Construction of 95S-SlACAS-HyTE-AfDC: Using pLY2 plasmid as a template, the SlACAS fragment with homologous arms was amplified by PCR using 95S-SlACAS-F and TE-SlACAS-R primers; the HyTE fragment with homologous arms was amplified by PCR using pLY2 plasmid as a template using S-HyTE-F and DC-HyTE-R primers; the AfDC fragment with homologous arms was amplified by PCR using pRS425-AfDC plasmid as a template using TE-AfDC-F and 95S-AfDC-R primers; the 95S plasmid backbone was amplified by PCR using 95S-bpsA plasmid as a template using 95S-F and 95S-R primers; the SlACAS fragment, HyTE fragment, AfDC fragment and 95S plasmid backbone were assembled by Gibson and transformed into DH5α to obtain 95S-SlACAS-HyTE-AfDC.

[0052] (2) 95S-SlACAS-HyTE-AfDC was integrated into the gene-edited strain EM04 of Escherichia coli through chemical transformation to obtain the genetically engineered Escherichia coli that produces emodin, which was named EM05.

[0053] Example 6 This embodiment provides a method for shake-flask fermentation, including the following steps: The above-mentioned Escherichia coli genetically engineered strain EM05 was activated and inoculated into 50 ml shake flasks at a 1% inoculum. The shake flask medium was ZYBP medium, with streptomycin added to the medium at a final concentration of 50 μg / ml. The shaker temperature was 30℃ and the rotation speed was 225 rpm. 5 g / L glucose and 0.25 g / L polyphosphate were added at 16 h, 32 h, and 48 h, respectively. Fermentation was completed after 60 h to obtain the emodin fermentation broth. The emodin was dissolved in DMSO and detected by high performance liquid chromatography. The final emodin yield was calculated using a standard curve, reaching 576.4 mg / L.

[0054] Comparative Example 1 Comparative Example 1 used BAP1 as the chassis strain; the expression vector 95S-SlACAS-HyTE-AfDC from Example 5 was introduced into BAP1 through chemical transformation to obtain control strain 1. Following the steps and methods of Example 6, the final emodin yield during shake-flask fermentation was 78.3 mg / L.

[0055] Comparative Example 2 Comparative Example 2 uses strain EM01 obtained in Example 1 as the chassis strain; the expression vector 95S-SlACAS-HyTE-AfDC in Example 5 above is introduced into EM01 by chemical transformation to obtain control strain 2. Following the steps and methods of Example 6, the final emodin yield of shake-flask fermentation is 214.8 mg / L.

[0056] Comparative Example 3 Comparative Example 3 uses strain EM02 obtained in Example 2 as the chassis strain; the expression vector 95S-SlACAS-HyTE-AfDC in Example 5 above is introduced into EM02 by chemical transformation to obtain control strain 3. Following the steps and methods of Example 6, the final emodin yield of shake-flask fermentation is 394.7 mg / L.

[0057] Comparative Example 4 Comparative Example 4 uses strain EM03 obtained in Example 3 as the chassis strain; the expression vector 95S-SlACAS-HyTE-AfDC in Example 5 above is introduced into EM03 by chemical transformation to obtain control strain 4. Following the steps and methods of Example 6, the final emodin yield of shake-flask fermentation is 478.1 mg / L.

[0058] The yield results of shake-flask tests for different strains are shown in Table 6.

[0059] Table 6 Example 7 This embodiment provides a high-performance liquid chromatography (HPLC) method for the detection of emodin, including the following steps: The Escherichia coli genetically engineered strain from Example 5 was used in the method of Example 6 to produce emodin, which was dissolved in dimethyl sulfoxide (DMSO). A Welchrom C18 4.6x250mm 5-Micron column was used with a mobile phase of methanol:0.1% phosphoric acid water = 80:20, isocratic elution, a flow rate of 1.0 mL / min, a column temperature of 35℃, a detection wavelength of 440 nm, an injection volume of 10 μL, and a run time of 20 minutes.

[0060] The standard curve for emodin content was determined using high-performance liquid chromatography (HPLC). A stock solution of 1.135 mg / mL emodin standard was prepared and then serially diluted to solutions of different concentrations: 0.1135 mg / mL, 0.227 mg / mL, 0.3405 mg / mL, 0.454 mg / mL, 0.5675 mg / mL, 0.681 mg / mL, and 0.7945 mg / mL. HPLC analysis was then performed on these solutions. The standard curve was plotted with the prepared sample concentration as the ordinate and the corresponding peak area as the abscissa. The obtained standard curve result for emodin was y = 1.2501 × 10⁻⁶. -7 R² = 0.9999. The high-performance liquid chromatogram of the emodin standard is shown below. Figure 1 As shown; the high-performance liquid chromatogram of the fermentation broth in Example 6 is shown below. Figure 2 As shown; the standard curve for the detection of emodin by high performance liquid chromatography is as follows. Figure 3 As shown.

[0061] The above results demonstrate that this application has successfully constructed a genetically engineered Escherichia coli strain EM05 for the production of emodin through genetic engineering. This genetically engineered strain, through gene editing, increases the flux of acetyl-CoA and malonyl-CoA, providing sufficient substrate for emodin synthesis. Simultaneously, it enhances sugar uptake and ATP supply, providing sufficient energy for strain growth and product synthesis, ultimately achieving a yield of 576.4 mg / L. This effectively shortens the fermentation cycle for emodin production and accelerates the development of emodin biosynthesis.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A method for constructing a genetically engineered *Escherichia coli* strain that produces emodin, characterized in that, Includes the following steps: S1. Based on the chassis strain BAP1, the pta gene was knocked out sequentially, and J23119-ppk was introduced into the gene locus and named strain EM01. S2. Based on strain EM01, the poxB gene was knocked out, and J23119-accBC was introduced into this gene locus, and the strain was named strain EM02. S3. Based on strain EM02, the ldhA gene was knocked out, and J23119-accAD was introduced into the gene locus, and the strain was named EM03. S4. Based on strain EM03, the galR gene was knocked out, and the strain was named strain EM04. S5. The 95S expression plasmid containing the SlACAS gene, HyTE gene, and AfDC gene was transferred into strain EM04 to construct an Escherichia coli genetically engineered strain that produces emodin, and it was named strain EM05.

2. The method for constructing genetically engineered Escherichia coli as described in claim 1, characterized in that, The nucleotide sequence of J23119-ppk in step S1 is shown in SEQ ID NO: 53; the nucleotide sequence of J23119-accBC in step S2 is shown in SEQ ID NO: 54; and the nucleotide sequence of J23119-accAD in step S3 is shown in SEQ ID NO:

55.

3. The method for constructing genetically engineered Escherichia coli as described in claim 1, characterized in that, The method for constructing the 95S expression plasmid containing the SlACAS gene, HyTE gene, and AfDC gene in step S5 includes the following steps: Using pLY2 plasmid as a template, the SlACAS fragment with homologous arms was amplified by PCR using primers 95S-SlACAS-F and TE-SlACAS-R; the HyTE fragment with homologous arms was amplified by PCR using primers S-HyTE-F and DC-HyTE-R; the AfDC fragment with homologous arms was amplified by PCR using primers TE-AfDC-F and 95S-AfDC-R; and the 95S plasmid backbone was amplified by PCR using primers 95S-bpsA and 95S-R. The SlACAS, HyTE, AfDC fragments, and the 95S plasmid backbone were assembled using Gibson DNA, and then transformed into DH5α to obtain a 95S expression plasmid containing the SlACAS, HyTE, and AfDC genes. The nucleotide sequences of 95S-SlACAS-F are shown in SEQ ID NO: 47, TE-SlACAS-R are shown in SEQ ID NO: 48, S-HyTE-F are shown in SEQ ID NO: 49, DC-HyTE-R are shown in SEQ ID NO: 50, TE-AfDC-F are shown in SEQ ID NO: 51, 95S-AfDC-R are shown in SEQ ID NO: 52, 95S-F are shown in SEQ ID NO: 53, and 95S-R are shown in SEQ ID NO:

54.

4. The Escherichia coli genetically engineered bacteria prepared by the method for constructing Escherichia coli according to any one of claims 1 to 3.

5. The application of the genetically engineered Escherichia coli as described in claim 4 in the production of emodin.

6. A method for shake-flask fermentation, characterized in that, Includes the following steps: The genetically engineered Escherichia coli strain as described in claim 1 is activated, inoculated into a shake flask culture medium, and cultured on a shaker to obtain a rhein shake flask culture medium.

7. The shake-flask fermentation method as described in claim 6, characterized in that, The shake flask culture medium comprises components of varying mass concentrations: Tryptone 8~12g / L, yeast powder 4~6g / L, Na2HPO4 3~4g / L, KH2PO4 3~4g / L, NH4Cl 1.2~2.5g / L, Na2SO4 0.5~1.5g / L, MgSO4·7H2O 0.2~1g / L, 100× trace elements 10ml / L, defoamer 150μl / L~1ml / L, glycerol 2~4g / L, glucose 4~6g / L, biotin 50~100 μg / L, and polyphosphate 0.5~2g / L.

8. The shake-flask fermentation method as described in claim 7, characterized in that, The 100× trace elements include the following components at the following mass concentrations: CuSO40.2~0.4g / L, CaCl2·2H2O 1.5~3.1g / L, MnCl2·4H2O 0.2~0.4g / L, ZnSO4·7H2O 5~6g / L, CoCl2·6H2O 0.3~0.5g / L, FeSO4·7H2O 2~3g / L, Na2MO4·2H2O 0.4~0.6g / L and 0.5MEDTA 80~100ml / L.

9. The shake-flask fermentation method as described in claim 6, characterized in that, The shaker culture includes the following conditions: shaker temperature of 28~32℃, rotation speed of 180~225rpm, and fermentation time of 60h.

10. The shake-flask fermentation method as described in claim 6, characterized in that, The shake flask culture medium is also supplemented with streptomycin at a final concentration of 50-100 μg / ml.

Images