An engineered bacterium with high yield of observation blue and a construction method and application thereof

Abstract

The application relates to an engineering bacterium for high yield of observation blue and a construction method and application thereof, and belongs to the technical field of biological synthesis of natural dyes. The engineering bacterium for high yield of observation blue expresses icd, bpsA, glnA Y405F and gdhA; the engineering bacterium knocks out acnR, yggB, glsK, aceA and ldh. By knocking out the yggB and aceA genes, performing site-directed mutation (Y405F) on the glnA gene, replacing the glsK gene with the glnA Y405F gene, replacing the acnR gene with the icd gene, replacing the ldh gene with the gdhA gene, and integrating the bpsA gene, the application can block the formation of by-products such as lactate and succinic acid in the observation blue synthesis process, improve the flow direction of the citric acid->isocitric acid->alpha-ketoglutaric acid->glutamic acid->glutamine path, and then improve the intracellular glutamine concentration, so that the yield of observation blue is improved.

Description

Technical Field

[0001] This application relates to the field of biosynthetic natural dye technology, and in particular to an engineered bacterium that produces high-yield blue dyes, its construction method, and its application. Background Technology

[0002] Indigoidine is a non-toxic, natural blue pigment primarily used in the dye industry. Its synthesis in vivo mainly involves the enzyme 4-indigoidine synthase converting two molecules of glutamine into one molecule of indigoidine. Indigoidine is a water-insoluble blue pigment, but it is soluble in a few organic solvents, such as DMF, DMSO, tetrahydrofuran, pyridine, and N-methylpyrrolidone. It can also be reduced to its leuco form by sodium dithionite.

[0003] Currently, the highest known yield of microbial fermentation for producing bluewort is 15.48 g / L, with a conversion rate of 22.1%. The low yield, low conversion rate, and low fermentation stability of the inducible strains result in high costs, and large-scale industrial production applications are still some distance away.

[0004] To address the problems existing in the prior art, this invention discloses a genetically engineered strain and fermentation method for producing blue violet, which has the potential to reduce costs, improve efficiency, and achieve industrial application.

[0005] Previously, the applicant modified Corynebacterium glutamicum to enable it to synthesize blue violet (application number CN202410284862.5). However, during subsequent modifications, it was found that the blue violet yield of the strain decreased instead of increasing, and it was impossible to further improve the blue violet yield. Therefore, it is still necessary to develop an engineered strain that can stably and efficiently produce blue violet. Summary of the Invention

[0006] The purpose of this application is to overcome the shortcomings of the prior art and provide an engineered bacterium for the efficient production of blue-green plants, its construction method, and its application.

[0007] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0008] Firstly, this application provides a high-yield engineered bacterium for producing blue pigments, wherein the engineered bacterium expresses isocitrate dehydrogenase ICD, blue pigment synthase bpsA, and glutamine synthase glnA. Y405F and glutamate dehydrogenase GDHA;

[0009] The engineered bacteria knocked out aconitase inhibitor acnR, glutamate mechanosensitive channel yggB, glutaminase glsK, isocitrate lyase aceA, and lactate dehydrogenase ldh.

[0010] This application involves knocking out the yggB and aceA genes, performing a site-directed mutation (Y405F) on the glnA gene, and replacing the glsK gene with glnA. Y405F By replacing the acnR gene with the icd gene, the ldh gene with the gdhA gene, and integrating the bpsA gene, the formation of byproducts such as lactate and succinic acid during the synthesis of sage can be blocked. This enhances the flow of the citric acid → isocitrate → α-ketoglutarate → glutamate → glutamine pathway, thereby increasing the intracellular glutamine concentration and thus improving the yield of sage.

[0011] Aconitase inhibitor acnR can inhibit the conversion of citric acid to isocitrate, while isocitrate dehydrogenase icd can utilize isocitrate to synthesize α-ketoglutarate. Replacing acnR with icd can increase the flux of citric acid to isocitrate and isocitrate to α-ketoglutarate, thereby increasing the intracellular level of glutamate and thus increasing the yield of blue oxalis.

[0012] The glutamate mechanosensitive channel yggB can transport intracellular glutamate to the extracellular space. Knocking out yggB can prevent glutamate from being transported to the extracellular space, further increasing the level of intracellular glutamate and providing more precursors for glutamine.

[0013] As a preferred embodiment of the engineered bacteria described in this application, the isocitrate dehydrogenase ICD is derived from Corynebacterium glutamicum; the coding sequence of the isocitrate dehydrogenase ICD is shown in SEQ ID NO.1.

[0014] As a preferred embodiment of the engineered bacteria described in this application, the glutamine synthase glnA Y405F It is encoded by glutamine synthase glnA containing a mutation site, wherein glutamine synthase glnA Y405F The coding sequence is shown in SEQ NO.2. This application increases the copy number of glnA in engineered bacteria by performing a site-directed mutation at position 405 of glnA and then inserting it into the site where glsK has been knocked out. This further promotes the conversion of glutamate to glutamine and increases the concentration of glutamine.

[0015] In a preferred embodiment of the engineered bacteria described in this application, the glutamate dehydrogenase gdhA is derived from Corynebacterium glutamicum; the coding sequence of the glutamate dehydrogenase gdhA is shown in SEQ ID NO.3. This application increases the copy number of gdhA in the engineered bacteria by inserting gdhA into the ldh site, thereby improving the expression level of gdhA and preventing the restricted conversion of α-ketoglutarate to glutamate.

[0016] As a preferred embodiment of the engineered bacteria described in this application, the engineered bacteria overexpress glutamine synthase glnA and glutamate dehydrogenase gdhA.

[0017] As a preferred embodiment of the engineered bacteria described in this application, the blue pigment synthase bpsA is derived from Streptomyces lavendulae; the coding sequence of the blue pigment synthase bpsA is shown in SE Q ID NO.4.

[0018] Secondly, this application provides a method for constructing the aforementioned project, which includes the following steps:

[0019] A1. Knock out glutaminase glsK in Corynebacterium glutamicum and replenish glutamin synthase glnA. Y405F Engineered strain I was obtained by applying the glsK site;

[0020] A2. Knock out lactate dehydrogenase ldh in engineered bacteria I obtained in step A1, and add glutamate dehydrogenase gdhA to the ldh site to obtain engineered bacteria II.

[0021] A3. Knock out the aconitase inhibitor acnR in the engineered strain II obtained in step A2, and add isocitrate dehydrogenase icd to the acnR site to obtain engineered strain III;

[0022] A4. Knock out the glutamate mechanosensitive channel yggB in the engineered bacteria III obtained in step A3 to obtain engineered bacteria IV;

[0023] A5. Knock out the isocitrate lyase aceA encoding gene in engineered bacteria IV obtained in step A4 to obtain engineered bacteria V;

[0024] A6. Transfer the plasmid containing the encoding gene of blue pigment synthase bpsA into the engineered bacteria V obtained in step A5 to obtain engineered bacteria VI. The engineered bacteria VI is a high-yield blue pigment engineered bacteria.

[0025] In a preferred embodiment of the construction method described in this application, in step A6, the plasmid containing the encoding gene for the blue pigment synthase bpsA also contains the p1929 promoter, the sequence of which is shown in SEQ ID NO.5. The p1929 promoter originates from a high-transcriptional-level gene in Corynebacterium glutamicum. Combining the p1929 promoter with heterologous bpsA allows Corynebacterium glutamicum to better heterologously express bpsA, while activating bpsA transcription and translation without the need for additional reagents, resulting in higher stability and reduced processing steps in the production process.

[0026] In a preferred embodiment of the construction method described in this application, in step A6, the coding gene of the blue pigment synthase bpsA is integrated into engineered bacteria V after codon optimization.

[0027] Thirdly, this application provides the application of the aforementioned engineered bacteria in the production of bluebells.

[0028] Fourthly, this application provides a method for producing blue violet, which involves fermenting the seed culture of the aforementioned engineered bacteria with a fermentation culture medium in a bioreactor.

[0029] This application provides a method for producing *Gynostemma pentaphyllum*, which involves inducing high-yield engineered bacteria to produce *Gynostemma pentaphyllum* through a series of biochemical reactions using glucose as a substrate. The engineered bacteria used in the synthesis process can block the conversion of pyruvate to lactate, the cleavage of isocitrate to succinic acid, the hydrolysis of glutamine to glutamate, and the extracellular transport of glutamate in *Corynebacterium glutamicum*. This increases the flux of citric acid to isocitrate and isocitrate to α-ketoglutarate, reducing byproduct formation while preventing intracellular glutamate loss, maximizing the concentration of the *Gynostemma pentaphyllum* precursor glutamine, and thus increasing the yield of *Gynostemma pentaphyllum*. The shake-flask fermentation yield of *Gynostemma pentaphyllum* can reach 21.34 g / L.

[0030] In a preferred embodiment of the production method described in this application, the seed liquid is a secondary seed liquid. The secondary seed liquid described in this application can be prepared by various culture media suitable for the growth of Corynebacterium glutamicum. The culture media suitable for the growth of Corynebacterium glutamicum include, but are not limited to, BHISG medium. The formula of BHISG medium is: brain heart extract 37 g / L glucose 10 g / L.

[0031] As a preferred embodiment of the production method described in this application, the fermentation conditions are fermentation at 18-22℃ and 180-220rpm for 72-96h.

[0032] In a preferred embodiment of the production method described in this application, the fermentation medium comprises the following components: 68-72 g / L glucose, 18-22 g / L ammonium sulfate, 0.5-1.5 g / L potassium dihydrogen phosphate, 0.01-0.1 g / L ferric sulfate, 0.01-0.10 g / L manganese sulfate, 2-6 μg / L biotin, 10-30 μg / L thiamine, 12-17 mL / L soy sauce, and 45-55 g / L calcium carbonate.

[0033] Compared with the prior art, the beneficial effects of this application are as follows:

[0034] (1) This application involves knocking out the yggB and aceA genes, performing a site-directed mutation (Y405F) on the glnA gene, and replacing the glsK gene with glnA. Y405FBy replacing the acnR gene with the icd gene, the ldh gene with the gdhA gene, and integrating the bpsA gene, the formation of byproducts such as lactate and succinic acid during the synthesis of sage can be blocked. This enhances the flow of the citric acid → isocitrate → α-ketoglutarate → glutamate → glutamine pathway, thereby increasing the intracellular glutamine concentration and thus improving the yield of sage.

[0035] (2) This application provides a method for producing *Gynostemma pentaphyllum*, which prepares *Gynostemma pentaphyllum* through a series of biochemical reactions using glucose as a substrate by inducing engineered bacteria that produce high-yield *Gynostemma pentaphyllum*. The engineered bacteria used in the synthesis process can block the conversion of pyruvate to lactate, the cleavage of isocitrate to succinic acid, the hydrolysis of glutamine to glutamate, and the extracellular transport of glutamate in *Corynebacterium glutamicum*. This increases the flux of citric acid to isocitrate and isocitrate to α-ketoglutarate, reduces the formation of byproducts, avoids the loss of intracellular glutamate, and maximizes the concentration of glutamine, the precursor of *Gynostemma pentaphyllum*, thereby increasing the yield of *Gynostemma pentaphyllum*. The shake-flask fermentation yield of the obtained *Gynostemma pentaphyllum* can reach 21.34 g / L. Attached Figure Description

[0036] Figure 1 This application describes the entire process of blue biosynthesis. Detailed Implementation

[0037] To better illustrate the purpose, technical solution, and advantages of this application, the following will provide further explanation of this application in conjunction with specific embodiments.

[0038] Unless otherwise specified, the materials and reagents used in the examples, comparative examples and experimental examples are commercially available.

[0039] Escherichia coli DH5α (hereinafter referred to as DH5α) can be used for vector construction and is available commercially.

[0040] Corynebacterium glutamicum ATCC 13032 (hereinafter referred to as ATCC 13032) was purchased from Shanghai Beinuo Biotechnology Co., Ltd.

[0041] Plasmids pTargetF and pJYS3-ΔcrtYf were purchased from BioWind.

[0042] The plasmid pTRCmob was constructed according to the following literature: Qian Liu; Shao-ping Ouyang; Jon ghyok Kim; Guo-Qiang Chen. (2007). The impact of PHB accumulation on l-glutamate production by recombinant Corynebacterium glutamicum., 132(3), 273-279. doi:10.1016 / j.jbiotec.2007.03.014.

[0043] High-fidelity DNA polymerase, restriction endonuclease, The Snap Assembly Master Mix was purchased from TAKARA.

[0044] Plasmid extraction kit, DNA purification kit, gel extraction kit, and bacterial genomic DNA extraction kit were purchased from OMEGA.

[0045] LB medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl, add water to make up to a total volume of 1 L, sterilize at 121℃ for 20 min; if preparing a solid medium, add 15-20 g / L agar powder.

[0046] BHISG medium: 37 g / L brain and heart extract, 10 g / L glucose, add water to make up to a total volume of 1 L, sterilize at 121℃ for 20 min; if preparing a solid medium, add 15-20 g / L agar powder; brain and heart extract was purchased from Nanjing You Nuo Jia Experimental Equipment Technology Co., Ltd.

[0047] GAP medium: 70 g / L glucose, 20 g / L ammonium sulfate, 1 g / L potassium dihydrogen phosphate, 0.01 g / L ferric sulfate, 0.01 g / L manganese sulfate, 4 μg / L biotin, 20 μg / L thiamine hydrochloride, 15 mL / L soy sauce, 50 g / L calcium carbonate, add water to make up to a total volume of 1 L, sterilize at 121℃ for 20 min. The soy sauce can be any commercially available soy sauce.

[0048] Unless otherwise specified, the plasmid transformation into E. coli DH5α in the following examples and comparative examples are all chemical transformations. The chemical transformation includes the following steps: the transformation system is added to DH5α competent cells that have been thawed on ice, incubated on ice for 30 min, then heat-shocked at 42°C for 1 min, incubated on ice again for 2 min, then 900 μL of pre-cooled LB medium is added, and incubated at 37°C and 220 rpm for 60 min; 100 μL of the incubated bacterial solution is added to LB medium containing streptomycin resistance and cultured overnight at 30°C / 37°C.

[0049] Electroporation transformation includes the following steps: Take 200-500 μg of the fragment to be transformed and add it to the prepared electroporation competent cells. After gently mixing, add it to the electroporation cuvette and incubate on ice for 5-10 min. After 2-3 electroporations at 1.8 KV, quickly add BHISG medium preheated to 46℃ and heat shock in a water bath at 46℃ for 6 minutes. Then, incubate the heat-shocked transformation solution in a shaker at 30℃ for 2 hours. After incubation, take 100 μL of the transformation solution and spread it on a BHISG plate. Incubate in a 30℃ incubator for 48 h.

[0050] The Gibson assembly technique (hereinafter referred to as Gibson) is operated with reference to the following literature:

[0051] Gibson,DG,et al.(2009).Enzymatic assembly of DNA molecules up toseveral hundred kilobases.Nature Methods,6(5),343-345.

[0052] Gibson,DG,et al.(2010).Creation of a bacterial cell controlled byachemically synthesized genome.Science,329(5987),52-56.

[0053] Example 1

[0054] This embodiment provides a high-yield engineered bacterium for producing blue-green foliage and its construction method, which includes the following steps:

[0055] I. Construction of engineered bacteria CG01

[0056] 1.1 Using plasmid pTargetF as a template, the pTarget-N24 plasmid backbone was obtained by PCR amplification using PT-N24R and PT-N24F primers;

[0057] Using pJYS3-ΔcrtYf as a template, the crRNA-crtYf fragment was amplified by PCR using primers 23119-crRNA-F and 23119-crRNA-R.

[0058] The pTarget-N24-crtYf fragment was obtained by assembling the pTarget-N24 plasmid backbone and the crRNA-crtYf fragment using Gibson.

[0059] Using pTarget-N24-crtYf as a template, PCR amplification was performed using pT-N24-glsK-F and pT-N24-glsK-R primers. The plasmid was then digested with DpnI enzyme and transformed into DH5α to obtain pTarget-N24-glsK plasmid.

[0060] The primer sequences for PT-N24R, PT-N24F, 23119-crRNA-F, 23119-crRNA-R, pT-N24-glsK-F, and pT-N24-glsK-R are shown in Table 1.

[0061] 1.2 Using ATCC 13032 as a template, the ptuf promoter was amplified by PCR using Ptuf-F and Ptuf-R primers;

[0062] Using 95S-rhtA plasmid as a template, the ptuf-S plasmid backbone was obtained by PCR amplification using 95S-F and 95S-Ptuf-R primers.

[0063] Using ATCC 13032 as a template, and utilizing glnA*-Ptuf-F and glnA Y405F -R primers were used for PCR amplification to obtain glnA Y405F Upstream segment, using glnA Y405F PCR amplification using primers -F and glnA*-Ptuf-R yielded glnA Y405F Downstream segment; glnA Y405F See SEQ ID NO.2 for the sequence;

[0064] The ptuf-S plasmid backbone, ptuf promoter, and glnA were assembled using Gibson. Y405F Upstream and downstream fragments are transferred into DH5α to obtain ptuf-S-glnA Y405F plasmids;

[0065] Ptuf-F, Ptuf-R, 95S-F, 95s-Ptuf-R, glnA*-Ptuf-F, glnA Y405F -R、glnA Y405F The primer sequences for -F and glnA*-Ptuf-R are shown in Table 1.

[0066] 1.3 Using ATCC 13032 as a template, the ΔglsK::glnAup fragment was amplified by PCR using primers glsK-up1200F and glsK::glnA*-up500R;

[0067] Using ATCC 13032 as a template, the ΔglsK::glnA down fragment was amplified by PCR using glsK::glnA*-down500F-T1 and glsK-down1200R primers;

[0068] The ptuf-S-glnA obtained in step 1.2 Y405F Using Ptuf-F and rrnB-R1-T1 primers, ptuf-glnA was amplified by PC R to obtain ptuf-glnA. Y405F Fragment;

[0069] With ΔglsK::glnAup, ptuf-glnA Y405F Using ΔglsK::glnAdown as a template, ΔglsK::glnA was amplified by overlapping extension PCR with primers S3-glsK-up1000F and S3-glsK-down1000R to obtain ΔglsK::glnA. Y405F donor-T1 fusion fragment;

[0070] The primer sequences of glsK-up1200F, glsK::glnA*-up500R, glsK::glnA*-down500F-T1, glsK-down1200R, rrnB-R1-T1, S3-glsK-up1000F and S3-glsK-down1000R are shown in Table 1.

[0071] 1.4 Using pTarget-N24-glsK obtained in step 1.1 as a template, the crRNA-glsK fragment was amplified by PCR using primers 23119-crRNA-F and 23119-crRNA-R;

[0072] Using pJYS3-ΔcrtYf as a template, the pJYS3 plasmid backbone was obtained by PCR amplification using pJYS3-vector-F and pJYS3-vector-R primers.

[0073] crRNA-glsK and ΔglsK::glnA obtained in step 1.3 were assembled using Gibson. Y405F The donor-T1 and pJYS3 plasmid backbones were transformed into DH5α to obtain pJYS3-ΔglsK::glnA Y405F -T1 plasmid;

[0074] The primer sequences for pJYS3-vector-F and pJYS3-vector-R are shown in Table 1.

[0075] 1.5 The pJYS3-ΔglsK::glnA obtained in step 1.4 is converted using an electrostatic conversion method. Y405FThe -T1 plasmid was transformed into AT CC13032, and ΔglsK::glnA was obtained by colony PCR screening. Y405F The target strain was obtained, and engineered strain CG01 was obtained.

[0076] Table 1 Primers used in the construction of engineered bacteria CG01

[0077]

[0078]

[0079] II. Construction of engineered bacteria CG08

[0080] 2.1 Using pTarget-N24-crtYf obtained in step 1.1 as a template, PCR amplification was performed using primers pT-N24-ldh-F and pT-N24-ldh-R. The template was digested with DpnI enzyme and transformed into DH5α to obtain the pTarget-N24-ldh plasmid. The primer sequences of pT-N24-ldh-F and pT-N24-ldh-R are shown in Table 2.

[0081] 2.2 Using ATCC 13032 as a template, the gdhA fragment was amplified by PCR using pTuf-NcoⅠ-gdhA-F and pTuf-EcoRⅠ-gdh AR primers. The gdhA coding sequence is shown in SEQ ID NO.3.

[0082] The ptuf-S-glnA obtained in step 1.2 Y405F Using Ptuf-Vector-F and Ptuf-Vector-R primers as templates, the ptuf-S plasmid backbone was obtained by PCR amplification.

[0083] The ptuf-S plasmid backbone and gdhA were assembled using Gibson and then transformed into DH5α to obtain the ptuf-S-gdhA plasmid.

[0084] The primer sequences for pT-N24-ldh-F, pT-N24-ldh-R, pTuf-NcoⅠ-gdhA-F, pTuf-EcoRⅠ-gdhA-R, Ptuf-Vector-F, and Ptuf-Vector-R are shown in Table 2.

[0085] 2.3 Using ATCC 13032 as a template, the Δldh::gdhA up fragment was amplified by PCR using primers ldh-up1200F and ldh::gdhA-up500R;

[0086] Using ATCC 13032 as a template, the Δldh::gdhA down-T2 fragment was amplified by PCR using primers ldh::gdhA-down500F-T2 and ldh-down1200R.

[0087] Using the ptuf-S-gdhA obtained in step 2.2 as a template, the ptuf-gdhA fragment was amplified by PCR using primers Ptuf-F and rrnB-R1-T2;

[0088] Using Δldh::gdhA up, ptuf-gdhA and Δldh::gdhA down-T2 as templates, the Δldh::gdhA donor-T2 fusion fragment was amplified by overlapping extension PCR using S3-ldh-up1000F and S3-ldh-down1000R primers.

[0089] The primer sequences for ldh-up1200F, ldh::gdhA-up500R, ldh::gdhA-down500F-T2, ldh-down1200R, S3-ldh-up1000F, S3-ldh-down1000R, and rrnB-R1-T2 are shown in Table 2, and the primer sequences for Ptuf-F are shown in Table 1.

[0090] 2.4 Using pTarget-N24-ldh obtained in step 2.1 as a template, the crRNA-ldh fragment was amplified by PCR using primers 23119-crRNA-F and 23119-crRNA-R;

[0091] Using pJYS3-ΔcrtYf as a template, the pJYS3 plasmid backbone was obtained by PCR amplification using pJYS3-vector-F and pJYS3-vector-R primers.

[0092] The crRNA-ldh, the Δldh::gdhA donor-T2 obtained in step 2.3, and the pJYS3 plasmid backbone were assembled using Gibson and then transformed into DH5α to obtain the pJYS3-Δldh::gdhA-T2 plasmid.

[0093] The primer sequences for 23119-crRNA-F, 23119-crRNA-R, pJYS3-vector-F, and pJYS3-vector-R are shown in Table 1.

[0094] 2.5 The pJYS3-Δldh::gdhA-T2 plasmid obtained in step 2.4 was transformed into the engineered bacteria CG01 obtained in step 1.5 using electroporation. The target strain Δldh::gdhA was obtained by colony PCR screening, resulting in engineered bacteria CG08.

[0095] Table 2 Primers used in the construction of engineered bacteria CG08

[0096]

[0097]

[0098] III. Construction of engineered bacteria CG09

[0099] 3.1 Using pTarget-N24-crtYf obtained in step 1.1 as a template, PCR amplification was performed using pT-N24-acnR-F and pT-N24-acnR-R primers. The template was digested with DpnI enzyme and transformed into DH5α to obtain the pTarget-N24-acnR plasmid.

[0100] The primer sequences for pT-N24-acnR-F and pT-N24-acnR-R are shown in Table 3.

[0101] 3.2 Using ATCC 13032 as a template, the icd fragment was amplified by PCR using pTuf-NcoⅠ-icd-F and pTuf-EcoRⅠ-icd-R primers. The coding gene sequence of the icd fragment is shown in SEQ ID NO.1.

[0102] 3.3 Using the ptuf-S-glnA obtained in step 1.2 Y405F Using Ptuf-Vector-F and Ptuf-Vector-R primers as templates, the ptuf-S plasmid backbone was obtained by PCR amplification.

[0103] The ptuf-S plasmid backbone and the icd fragment obtained in step 3.2 were assembled using Gibson and then transformed into DH5α to obtain the ptuf-S-icd plasmid.

[0104] The primer sequences for pTuf-NcoⅠ-icd-F and pTuf-EcoRⅠ-icd-R are shown in Table 3, and the primer sequences for Ptuf-Vector-F and Ptuf-Vector-R are shown in Table 2.

[0105] 3.4 Using ATCC 13032 as a template, the ΔacnR::icd up fragment was amplified by PCR using primers acnR-up1200F and acnR::icd-up500R;

[0106] Using ATCC 13032 as a template, the ΔacnR::icd down-T3 fragment was amplified by PCR using primers acnR::icd-down500F-T3 and acnR-down1200R;

[0107] Using the ptuf-S-icd obtained in step 3.3 as a template, the ptuf-icd fragment was amplified by PCR using Ptuf-F and rrnB-R1-T3 primers;

[0108] Using ΔacnR::icd up, ptuf-icd and ΔacnR::icd down-T3 as templates, the ΔacnR::icd donor-T3 fusion fragment was obtained by PCR amplification with overlapping extension primers S3-acnR-up1000F and S3-acnR-down1000R.

[0109] The primer sequences for acnR::icd-down500F-T3, acnR-down1200R, and rrnB-R1-T3 are shown in Table 3, and the primer sequences for Ptuf-F are shown in Table 1.

[0110] 3.5 Using pTarget-N24-acnR obtained in step 3.1 as a template, the crRNA-acnR fragment was amplified by PCR using primers 23119-crRNA-F and 23119-crRNA-R;

[0111] Using pJYS3-ΔcrtYf as a template, the pJYS3 plasmid backbone was obtained by PCR amplification using pJYS3-vector-F and pJYS3-vector-R primers.

[0112] The crRNA-acnR, the ΔacnR::icd donor-T3 obtained in step 3.4, and the pJYS3 plasmid backbone were assembled using Gibson and then transformed into DH5α to obtain the pJYS3-ΔacnR::icd-T3 plasmid.

[0113] The primer sequences for 23119-crRNA-F, 23119-crRNA-R, pJYS3-vector-F, and pJYS3-vector-R are shown in Table 1.

[0114] 3.6 The pJYS3-ΔacnR::icd-T3 plasmid obtained in step 3.5 was transformed into CG08 obtained in step 2.5 using electroporation transformation. The target strain ΔacnR::icd was obtained by colony PCR screening, and the engineered strain CG09 was obtained.

[0115] Table 3 Primers used in the construction of engineered bacteria CG09

[0116]

[0117]

[0118] IV. Construction of engineered bacteria CG10

[0119] 4.1 Using pTarget-N24-crtYf obtained in step 1.1 as a template, PCR amplification was performed using primers pT-N24-yggB-F and pT-N24-yggB-R. The template was digested with DpnI enzyme and transformed into DH5α to obtain the pTarget-N24-yggB plasmid. The primer sequences of pT-N24-yggB-F and pT-N24-yggB-R are shown in Table 4.

[0120] 4.2 Using ATCC 13032 as a template, the ΔyggB up-T4 fragment was amplified by PCR using primers yggB-up1200F and yggB-up500R-T4;

[0121] Using ATCC 13032 as a template, the ΔyggB down-T4 fragment was amplified by PCR using yggB-down500F-T4 and yggB-down1200R primers.

[0122] Using ΔyggB up-T4 and ΔyggB down-T4 as templates, the ΔyggB donor-T4 fusion fragment was obtained by PCR amplification with overlapping primers S3-yggB-up1000F and S3-yggB-down1000R.

[0123] The primer sequences of yggB-up1200F, yggB-up500R-T4, yggB-down500F-T4 and yggB-down1200R are shown in Table 4.

[0124] 4.3 Using pTarget-N24-yggB obtained in step 4.1 as a template, the crRNA-yggB fragment was amplified by PCR using primers 23119-crRNA-F and 23119-crRNA-R.

[0125] Using pJYS3-ΔcrtYf as a template, the pJYS3 plasmid backbone was obtained by PCR amplification using pJYS3-vector-F and pJYS3-vector-R primers.

[0126] The crRNA-yggB, the ΔyggB donor-T4 obtained in step 4.2, and the pJYS3 plasmid backbone were assembled using Gibson and then transformed into DH5α to obtain the pJYS3-ΔyggB-T4 plasmid.

[0127] The primer sequences for 23119-crRNA-F, 23119-crRNA-R, pJYS3-vector-F, and pJYS3-vector-R are shown in Table 1.

[0128] 4.4 The pJYS3-ΔyggB-T4 plasmid obtained in step 4.3 was transformed into the engineered bacteria CG09 obtained in step 3.6 using electroporation transformation. The target strain ΔyggB was obtained by colony PCR screening, and the engineered bacteria CG10 was obtained.

[0129] Table 4 Primers used in the construction of engineered bacteria CG10

[0130]

[0131] V. Construction of engineered bacteria CG11

[0132] 5.1 Using pTarget-N24-crtYf as a template, PCR amplification was performed using pT-N24-aceA-F and pT-N24-aceA-R primers. The template was digested with DpnI enzyme and transformed into DH5α to obtain the pTarget-N24-aceA plasmid.

[0133] The primer sequences for pT-N24-aceA-F and pT-N24-aceA-R are shown in Table 5.

[0134] 5.2 Using ATCC 13032 as a template, the ΔaceA up-T5 fragment was amplified by PCR using primers aceA-up1200F and aceA-up500R-T5;

[0135] Using ATCC 13032 as a template, the ΔaceA down-T5 fragment was amplified by PCR using primers aceA-down500F-T5 and aceA-down1200R;

[0136] Using ΔaceA up-T5 and ΔaceA down-T5 as templates, the ΔaceA donor-T5 fusion fragment was amplified by overlapping extension PCR using S3-aceA-up1000F and S3-aceA-down1000R primers.

[0137] The primer sequences of aceA-up1200F, aceA-up500R, aceA-down500F, aceA-down1200R, S3-aceA-up1000F and S3-aceA-down1000R are shown in Table 5.

[0138] 5.3 Using pTarget-N24-aceA obtained in step 5.1 as a template, the crRNA-aceA fragment was amplified by PCR using primers 23119-crRNA-F and 23119-crRNA-R;

[0139] Using pJYS3-ΔcrtYf as a template, the pJYS3 plasmid backbone was obtained by PCR amplification using pJYS3-vector-F and pJYS3-vector-R primers.

[0140] The crRNA-aceA, the ΔaceA donor-T5 obtained in step 5.2, and the pJYS3 plasmid backbone were assembled using Gibson and then transformed into DH5α to obtain the pJYS3-ΔaceA-T5 plasmid.

[0141] The primer sequences for 23119-crRNA-F, 23119-crRNA-R, pJYS3-vector-F, and pJYS3-vector-R are shown in Table 1.

[0142] 5.4 The pJYS3-ΔaceA plasmid obtained in step 5.3 was transformed into the engineered bacteria CG10 obtained in step 4.4 using electroporation transformation. The target strain of ΔaceA was obtained by colony PCR screening, and the engineered bacteria CG11 was obtained.

[0143] Table 5 Primers used in the construction of engineered bacteria CG11

[0144]

[0145] VI. Construction of engineered bacteria CG12

[0146] 6.1 The BpsA gene sequence of blue pigment synthase from Streptomyces lavendulae was codon optimized to synthesize the bpsA gene fragment. The codon-optimized bpsA sequence is shown in SEQ ID NO.4.

[0147] 6.2 Using the bpsA gene fragment obtained in step 5.1 as a template, the bpsA fragment with homologous arms was amplified by PCR using primers R5bpsA-p1929-F and bpsA-p1929-R.

[0148] Using ATCC 13032 as a template, the p1929 promoter fragment was obtained by PCR amplification using p1929-F and p1929-R5-R primers. The p1929 promoter sequence is shown in SEQ ID NO.5.

[0149] Using pTRCmob plasmid as a template, the p1929 plasmid backbone with the p1929 promoter was obtained by PCR amplification using p1929-Vector-F and p1929-Vector-R primers.

[0150] The bpsA, p1929 promoter and p1929 plasmid backbone were assembled by Gibson and then transformed into DH5α to obtain the p1929-R5bpsA plasmid.

[0151] The primer sequences R5bpsA-p1929-F, bpsA-p1929-R, R5bpsA-p1929-F, bpsA-p1929-R, p1929-F, p1929-R5-R, p1929-Vector-F, and p1929-Vector-R are shown in Table 6.

[0152] 6.3 The p1929-R5bpsA plasmid obtained in step 6.2 was transformed into the engineered bacteria CG11 obtained in step 5.5 using electroporation transformation. The target strain with R5bpsA was obtained by colony PCR screening, and the engineered bacteria CG12 was obtained.

[0153] Table 6 Primers used in the construction of engineered bacteria CG12

[0154]

[0155] Example 2

[0156] This embodiment provides a method for producing orchids, which includes the following steps:

[0157] (1) The engineered bacteria CG12 of Example 1 was cultured in BHISG solid medium at 30°C for 48h. A single colony was picked and inoculated into 5mL BHISG medium and cultured at 30°C and 220rpm for 12-16h to obtain CG07 primary seed liquid.

[0158] (2) Take 350 μL of the CG12 primary seed solution obtained in step (1) and inoculate it into 35 mL of BHISG medium. Incubate at 30 °C and 220 rpm for 16 h to obtain the CG12 secondary seed solution.

[0159] (3) Centrifuge the CG12 secondary seed culture obtained in step (2) at 4℃ and 4500rpm for 10min, retain the cells, transfer them to a 250mL baffle shake flask containing 25mL GAP medium, and incubate at 30℃ and 200rpm for 72h to obtain Guanlan.

[0160] The route for synthesizing blue pigment using the engineered bacteria CG12 in this application is shown below. Figure 1 .

[0161] like Figure 1As shown, the engineered bacterium CG12 of this application uses glucose as a substrate to produce pyruvate through glycolysis. Pyruvate is converted to acetyl-CoA by pyruvate dehydrogenase. Acetyl-CoA enters the tricarboxylic acid cycle and is converted to citrate by citrate synthase. Citrate is converted to isocitrate by cis-aconitase. Isocitrate is converted to α-ketoglutarate and succinate by isocitrate dehydrogenase. α-ketoglutarate is converted to glutamate by glutamate dehydrogenase. Glutamate is converted to glutamine by glutamine synthase. Glutamine is converted to blue pigment by activated blue pigment synthase.

[0162] Comparative Example 1

[0163] This comparative example provides an engineered bacterium that produces high levels of blue-green foliage and its construction method. The construction method includes the following steps:

[0164] The p1929-R5bpsA plasmid was constructed according to steps 6.1-6.3 in Example 1. The p1929-R5bpsA plasmid was then transformed into ATCC 13032 by electroporation to obtain engineered bacteria CG13.

[0165] Comparative Example 2

[0166] This comparative example provides an engineered bacterium that produces high levels of blue-green foliage and its construction method. The construction method includes the following steps:

[0167] The p1929-R5bpsA plasmid was constructed according to steps 6.1-6.3 in Example 1. The p1929-R5bpsA plasmid was then transferred into the engineered bacteria CG01 obtained in step 1.5 of Example 1 by electroporation to obtain engineered bacteria CG14.

[0168] Comparative Example 3

[0169] This comparative example provides an engineered bacterium that produces high levels of blue-green foliage and its construction method. The construction method includes the following steps:

[0170] The p1929-R5bpsA plasmid was constructed according to steps 6.1-6.3 in Example 1. The p1929-R5bpsA plasmid was then transferred into the engineered bacteria CG08 obtained in step 2.5 of Example 1 by electroporation to obtain engineered bacteria CG15.

[0171] Comparative Example 4

[0172] This comparative example provides an engineered bacterium that produces high levels of blue-green foliage and its construction method. The construction method includes the following steps:

[0173] The p1929-R5bpsA plasmid was constructed according to steps 6.1-6.3 in Example 1. The p1929-R5bpsA plasmid was then transferred into the engineered bacteria CG09 obtained in step 3.6 of Example 1 by electroporation to obtain engineered bacteria CG16.

[0174] Comparative Example 5

[0175] This comparative example provides a high-yield blue-brightening engineered bacteria and its construction method, which is similar to that of Comparative Example 1 and includes the following steps:

[0176] The p1929-R5bpsA plasmid was constructed according to steps 6.1-6.3 in Example 1. The p1929-R5bpsA plasmid was then transferred into the engineered bacteria CG10 obtained in step 4.4 of Example 1 by electroporation to obtain engineered bacteria CG17.

[0177] Comparative Example 6

[0178] This comparative example provides a high-yield blue-bearing engineered bacterium, strain CG07, which was constructed according to the method in Example 1 of application number CN202410284862.5.

[0179] Example of effect

[0180] The engineered bacteria CG12 from Example 1, and the engineered bacteria CG13-CG17 and CG07 from Comparative Examples 1-6 were synthesized into Guanlan according to the method in Example 2.

[0181] The OD values ​​of 1-18 μg / mL blue standard solutions were measured at a wavelength of 612 nm. A standard curve was plotted with blue concentration on the x-axis and OD612 on the y-axis. The obtained standard curve was Y = 0.0694X + 0.0087, R0 2 =0.999.

[0182] The OD values ​​of different samples at 612 nm were measured using a UV spectrophotometer. The yield of Guanlan was calculated based on the standard curve. The results are shown in Table 7. The synthetic route is shown in [reference needed]. Figure 1 .

[0183] Table 7. Yield of *Gynostemma pentaphyllum* by shake-flask fermentation (g / L)

[0184]

[0185]

[0186] As shown in Table 7, the engineered strain CG12 of this application can successfully synthesize lanthanum through shake-flask fermentation, and the yield of lanthanum is 21.34 g / L. Compared with CG07, the yield is increased by 37.9%, and the glucose material cost is reduced by 27%.

[0187] During the synthesis of succinate, byproducts such as succinate and lactate are produced. Knocking out the encoding genes for lactate dehydrogenase and isocitrate lyase can reduce the formation of these byproducts, thereby increasing the throughput of the succinate synthesis pathway. Simultaneously, replacing the encoding gene for aconitase inhibitor with the encoding gene for isocitrate dehydrogenase, which can utilize isocitrate to synthesize α-ketoglutarate, can increase the intensity of the tricarboxylic acid cycle, leading to the accumulation of more α-ketoglutarate and thus increasing glutamate levels. Furthermore, to retain glutamate within the cell, knocking out the encoding gene for the glutamate mechanosensitive channel prevents intracellular glutamate from being transported extracellularly, thereby increasing glutamine levels. Additionally, transforming the integrative expression plasmid p1929-R5bpsA into engineered bacteria allows for transcription and translation of bpsA without reagent induction, further improving fermentation stability and enabling efficient succinate production.

[0188] Several byproducts occur during the biosynthesis of sage: pyruvate is converted to lactate by lactate dehydrogenase, isocitrate is converted to succinate by isocitrate lyase, and glutamine is converted to glutamate by glutaminase. As shown in Table 7, the sage yield of the engineered bacteria obtained in Example 1 was higher than that of the engineered bacteria obtained in Comparative Examples 1-6, indicating that this application reduces the generation of byproducts and increases the flux of sage-related pathways by knocking out the encoding genes of lactate dehydrogenase, isocitrate lyase, glutamate 5-kinase, and glutaminase, thereby increasing the yield of sage.

[0189] 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 high-yield engineered bacterium for producing blue-green flowers, characterized in that, The engineered bacteria express isocitrate dehydrogenase ICD, blue pigment synthase bpsA, and glutamine synthase glnA. Y405F And glutamate dehydrogenase GDHA; The engineered bacteria knocked out aconitase inhibitor acnR, glutamate mechanosensitive channel yggB, glutaminase glsK, isocitrate lyase aceA, and lactate dehydrogenase ldh. The engineered bacteria that produce high-yield blue-green foliage are mainly prepared through the following steps: A1. Knock out glutaminase glsK in Corynebacterium glutamicum and replenish glutamin synthase glnA. Y405F Engineered strain I was obtained by applying the glsK site; A2. Knock out lactate dehydrogenase ldh in engineered bacteria I obtained in step A1, and add glutamate dehydrogenase gdhA to the ldh site to obtain engineered bacteria II. A3. Knock out the aconitase inhibitor acnR in the engineered strain II obtained in step A2, and add isocitrate dehydrogenase icd to the acnR site to obtain engineered strain III; A4. Knock out the glutamate mechanosensitive channel yggB in the engineered bacteria III obtained in step A3 to obtain engineered bacteria IV; A5. Knock out the isocitrate lyase aceA encoding gene in engineered bacteria IV obtained in step A4 to obtain engineered bacteria V; A6. The plasmid containing the encoding gene of blue pigment synthase bpsA is transferred into the engineered bacteria V obtained in step A5 to obtain engineered bacteria VI. The engineered bacteria VI is a high-yield blue pigment synthase. The plasmid containing the encoding gene of blue pigment synthase bpsA also contains the p1929 promoter. The sequence of the p1929 promoter is shown in SEQ ID NO.

5. The isocitrate dehydrogenase ICD is derived from Corynebacterium glutamicum; the coding sequence of the isocitrate dehydrogenase ICD is shown in SEQ ID NO.1; The glutamine synthase glnA Y405F It is encoded by glutamine synthase glnA containing a mutation site, wherein glutamine synthase glnA Y405F The encoded sequence is shown in SEQ NO.2; The glutamate dehydrogenase gdhA is derived from Corynebacterium glutamicum; the coding sequence of the glutamate dehydrogenase gdhA is shown in SEQ ID NO.3; The blue pigment synthase bpsA is derived from *Streptomyces lilacinus*. Streptomyceslavendulae The coding sequence of the blue pigment synthase bpsA is shown in SEQ ID NO.

4.

2. The method for constructing engineered bacteria as described in claim 1, characterized in that, Includes the following steps: A1. Knock out glutaminase glsK in Corynebacterium glutamicum and replenish glutamin synthase glnA. Y405F Engineered strain I was obtained by applying the glsK site; A2. Knock out lactate dehydrogenase ldh in engineered bacteria I obtained in step A1, and add glutamate dehydrogenase gdhA to the ldh site to obtain engineered bacteria II. A3. Knock out the aconitase inhibitor acnR in the engineered strain II obtained in step A2, and add isocitrate dehydrogenase icd to the acnR site to obtain engineered strain III; A4. Knock out the glutamate mechanosensitive channel yggB in the engineered bacteria III obtained in step A3 to obtain engineered bacteria IV; A5. Knock out the isocitrate lyase aceA encoding gene in engineered bacteria IV obtained in step A4 to obtain engineered bacteria V; A6. Transfer the plasmid containing the encoding gene of blue pigment synthase bpsA into the engineered bacteria V obtained in step A5 to obtain engineered bacteria VI. The engineered bacteria VI obtained is a high-yield blue pigment engineered bacteria. In step A6, the plasmid containing the gene encoding the blue pigment synthase bpsA also contains the p1929 promoter, the sequence of which is shown in SEQ ID NO.5; The isocitrate dehydrogenase ICD is derived from Corynebacterium glutamicum; the coding sequence of the isocitrate dehydrogenase ICD is shown in SEQ ID NO.1; The glutamine synthase glnA Y405F It is encoded by glutamine synthase glnA containing a mutation site, wherein glutamine synthase glnA Y405F The encoded sequence is shown in SEQ NO.2; The glutamate dehydrogenase gdhA is derived from Corynebacterium glutamicum; the coding sequence of the glutamate dehydrogenase gdhA is shown in SEQ ID NO.3; The blue pigment synthase bpsA is derived from *Streptomyces lilacinus*. Streptomyceslavendulae The coding sequence of the blue pigment synthase bpsA is shown in SEQ ID NO.

4.

3. The construction method as described in claim 2, characterized in that, In step A6, the gene encoding the blue pigment synthase bpsA is integrated into engineered bacteria V after codon optimization.

4. The application of the engineered bacteria as described in claim 1 in the production of bluebells.

5. A method for producing indigo ornaments, characterized in that, The seed culture of the engineered bacteria described in claim 1 is obtained by fermenting it with a fermentation culture medium in a bioreactor.

6. The production method as described in claim 5, characterized in that, The fermentation conditions are 18-22℃ and 180-220rpm for 72-96h.

7. The production method as described in claim 5, characterized in that, The fermentation medium comprises the following components: 68-72 g / L glucose, 18-22 g / L ammonium sulfate, 0.5-1.5 g / L potassium dihydrogen phosphate, 0.01-0.1 g / L ferric sulfate, 0.01-0.10 g / L manganese sulfate, 2-6 μg / L biotin, 10-30 μg / L thiamine, 12-17 mL / L soy sauce, and 45-55 g / L calcium carbonate.

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