Genetically engineered bacterium for catalyzing synthesis of ornamental blue from ornamental blue derivative as well as construction method and application of genetically engineered bacterium

Abstract

The invention discloses a genetically engineered bacterium for catalyzing synthesis of ornamental blue from an ornamental blue derivative as well as a construction method and application of the genetically engineered bacterium, and relates to the technical field of bioengineering. According to the invention, on the basis of a gene editing technology, CobB, NOX, katE, NAMPT, nadD, rbsk, prs and ppk genes are integrated onto an escherichia coli genome; a genetically engineered bacterium for catalyzing synthesis of the observation blue derivative (N-acetylobservation blue or N, N '-diacetyl-observation blue) is constructed, synthesis of NAD + can be enhanced, cyclic regeneration of NAD + can be realized, H2O2 which is generated in the synthesis process of NAD + and has a toxic effect on enzyme is eliminated, supply of cofactors PRPP and ATP is enhanced, deacetylase is activated, and the observation blue derivative (N-acetylobservation blue or N, N'-diacetyl-observation blue) is enabled to be converted into the observation blue derivative (N-acetylobservation blue or N, N '-diacetyl-observation blue) into the observation blue derivative (N-acetylobservation blue or N, N'-diacetyl-observation blue). N, N '-diacetyl-observation blue) is used for catalytic synthesis of observation blue.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioengineering, in particular to a gene engineering bacteria for catalyzing synthesis of indigoidine from indigoidine derivatives, and a construction method and application thereof. BACKGROUND

[0002] Indigoidine is a natural blue pigment with no toxic side effects, and is mainly applied in the dye industry. Indigoidine is mainly synthesized by indigoidine synthetase in the body, which combines two molecules of glutamine into one molecule of indigoidine. Indigoidine is a non-water-soluble blue pigment, which can be dissolved in a few organic solvents such as DMF, DMSO, tetrahydrofuran, pyridine, N-methyl pyrrolidone, etc., and can also be reduced to a leuco form by sodium hyposulfite. In the process of fermentation for synthesizing indigoidine by natural microorganisms or engineering bacteria, the production of N-acetyl indigoidine and N,N'-diacetyl-indigoidine derivatives often accompanies, which affects the product quality and leads to unstable color tone of the product. Therefore, it is necessary to develop a new method to convert these indigoidine derivatives into indigoidine as much as possible, so as to improve the yield and purity of indigoidine and obtain stable color tone of indigoidine dye. SUMMARY

[0003] The present application aims to overcome the shortcomings of the prior art, and provides a gene engineering bacteria for catalyzing synthesis of indigoidine from indigoidine derivatives, and a construction method and application thereof.

[0004] To achieve the above-mentioned object, the technical scheme adopted by the present application is as follows: a gene engineering bacteria for catalyzing synthesis of indigoidine from indigoidine derivatives, wherein the gene engineering bacteria is introduced with a deacetylase gene CobB, an NADH oxidase gene NOX and a catalase gene katE in a host bacteria; and / or introduced with a nicotinamide phosphoribosyltransferase gene NAMPT and a nicotinamide mononucleotide adenylyltransferase gene nadD; and / or introduced with a ribokinase gene rbsk, a phosphoribosyl pyrophosphate kinase gene prs and a polyphosphate kinase gene ppk; and the indigoidine derivatives include N-acetyl indigoidine and N,N'-diacetyl-indigoidine.

[0005] Based on the problem of more by-products (N-acetyl indigoidine and N,N'-diacetyl-indigoidine) in the production process of indigoidine in the prior art, the present inventors use gene editing technology to introduce deacetylase gene, NADH oxidase gene, catalase gene, nicotinamide phosphoribosyltransferase gene, nicotinamide mononucleotide adenylyltransferase gene, ribokinase gene, phosphoribosyl pyrophosphate kinase gene and polyphosphate kinase gene in the host bacteria to construct a metabolic engineering bacteria. The present inventors have found through a large number of researches and tests that the acetylated derivatives can be converted into deacetylated substances by expressing the deacetylase gene; NADH in the body can be converted into NAD by expressing the NADH oxidase gene and the catalase gene; and the NAD can be used to catalyze the synthesis of indigoidine from indigoidine derivatives. +This provides a cofactor for the deacetylation reaction and simultaneously removes the byproduct H2O2; by expressing the nicotinamide phosphoribosyltransferase gene and the nicotinamide mononucleotide adenylate transferase gene, the byproduct nicotinamide (Nam) can be recovered, and the energy from ATP can be used to drive NAD. + The regeneration of nicotinamide is achieved through the expression of ribokinase and phosphoribosylpyrophosphate kinase genes, providing sufficient 5-phosphoribose-1-pyrophosphate (PRPP) for nicotinamide recovery. Furthermore, the expression of polyphosphokinase provides more ATP for the above reactions. The genetically engineered bacteria of this invention can catalyze the synthesis of nicotinamide from N-acetylglucosamine or N,N'-diacetyl-nicotinosamine using whole-cell catalysis. The biocatalytic reaction conditions are mild, the cycle is short, and it is environmentally friendly. Simultaneously, it achieves the efficient conversion of nicotinamide derivatives (N-acetylglucosamine or N,N'-diacetyl-nicotinosamine) into nicotinamide.

[0006] In a preferred embodiment of the genetically engineered bacteria of the present invention, the host bacteria includes Escherichia coli.

[0007] As a preferred embodiment of the genetically engineered bacteria of the present invention, the deacetylase gene CobB, NADH oxidase gene NOX, catalase gene katE, nicotinamide phosphoribosyltransferase gene NAMPT, nicotinamide mononucleotide adenylate transferase gene nadD, ribokinase gene rbsk, phosphoribosyl pyrophosphate kinase gene prs, and polyphosphate kinase gene ppk can be derived from any species.

[0008] In a preferred embodiment of the genetically engineered bacteria described in this invention, the deacetylase gene CobB, catalase gene katE, nicotinamide mononucleotide adenylate transferase gene nadD, ribokinase gene rbsk, phosphoribosyl pyrophosphate kinase gene prs, and polyphosphate kinase gene ppk are all derived from *Escherichia coli*. E. coli The NADH oxidase encoding gene NOX is derived from Streptococcus mutans ( ); Treptococcus mutans The nicotinamide phosphoribosyltransferase encoding gene NAMPT is derived from *Pinococcus pineae* (…). Chitinophaga pinensis ).

[0009] As a preferred embodiment of the genetically engineered bacterium, the nucleotide sequence of the deacetylase gene CobB is shown as SEQ ID NO. 1; the nucleotide sequence of the NADH oxidase gene NOX is shown as SEQ ID NO. 2; the nucleotide sequence of the catalase gene katE is shown as SEQ ID NO. 3; the nucleotide sequence of the nicotinamide phosphoribosyltransferase gene NAMPT is shown as SEQ ID NO. 4; the nucleotide sequence of the nicotinamide mononucleotide adenylyltransferase gene nadD is shown as SEQ ID NO. 5; the nucleotide sequence of the ribokinase gene rbsk is shown as SEQ ID NO. 6; the nucleotide sequence of the phosphoribosyl pyrophosphate kinase gene prs is shown as SEQ ID NO. 7; and the nucleotide sequence of the polyphosphate kinase gene ppk is shown as SEQ ID NO. 8.

[0010] The application further provides a method for constructing the genetically engineered bacterium for catalyzing the synthesis of obelin from obelin derivatives, comprising the following steps: S1. Integrating the deacetylase gene CobB, the NADH oxidase gene NOX and the catalase gene katE into E. coli by using gene editing technology to obtain a genetically engineered bacterium GL-01; S2. And / or integrating the nicotinamide phosphoribosyltransferase gene NAMPT and the nicotinamide phosphoribosyltransferase gene nadD into the genetically engineered bacterium GL-01 to obtain a genetically engineered bacterium GL-02; S3. And / or integrating the ribokinase gene rbsk, the phosphoribosyl pyrophosphate kinase gene prs and the polyphosphate kinase gene ppk into the genetically engineered bacterium GL-02 to obtain a genetically engineered bacterium GL-03, which is the genetically engineered bacterium for catalyzing the synthesis of obelin from obelin derivatives (N-acetyl obelin or N,N'-diacetyl-obelin).

[0011] Preferably, the NADH oxidase gene NOX in step S1 is codon-optimized and integrated into E. coli; and the nicotinamide phosphoribosyltransferase gene NAMPT in step S2 is codon-optimized and integrated into the genetically engineered bacterium GL-01.

[0012] The application further provides the use of the genetically engineered bacterium in the preparation of obelin.

[0013] The application further provides a method for synthesizing obelin, which comprises catalyzing the reaction of seed liquid of the genetically engineered bacterium and a catalytic medium.

[0014] The method for synthesizing oblate of the present application is to induce genetically engineered bacteria GL-03 to catalyze the preparation of oblate by taking oblate derivatives (N-acetyl oblate or N, N'-diacetyl-oblate) as substrates. The genetically engineered bacteria GL-03 used in the catalytic synthesis process can enhance the synthesis of NAD + , realize the cyclic regeneration of NAD + , eliminate the H2O2 which is toxic to enzymes generated in the synthesis process of NAD + , strengthen the supply of cofactors PRPP and ATP, and activate deacetylase, so that oblate derivatives (N-acetyl oblate or N, N'-diacetyl-oblate) can catalyze the synthesis of oblate. The molar conversion rate of the obtained oblate reaches more than 90%.

[0015] As a preferred embodiment of the method for synthesizing oblate of the present application, the catalytic culture medium comprises IPTG solution with a final concentration of 0.2-0.6 mM.

[0016] As a preferred embodiment of the method for synthesizing oblate of the present application, the catalytic reaction is specifically catalyzed at 28-32℃, 200-300 rpm for 24-48 h.

[0017] The present application has the following beneficial effects: Based on the gene editing technology, the present application integrates CobB, NOX, katE, NAMPT, nadD, rbsk, prs and ppk genes into the genome of Escherichia coli to construct a genetically engineered bacteria for catalyzing the synthesis of oblate by taking oblate derivatives (N-acetyl oblate or N, N'-diacetyl-oblate) as substrates. The genetically engineered bacteria can enhance the synthesis of NAD + , realize the cyclic regeneration of NAD + , eliminate the H2O2 which is toxic to enzymes generated in the synthesis process of NAD + , strengthen the supply of cofactors PRPP and ATP, and activate deacetylase, so that oblate derivatives (N-acetyl oblate or N, N'-diacetyl-oblate) can catalyze the synthesis of oblate. The present application also provides a method for synthesizing oblate. The genetically engineered bacteria of the present application are induced to catalyze the preparation of oblate by taking oblate derivatives (N-acetyl oblate or N, N'-diacetyl-oblate) as substrates. The molar conversion rate reaches more than 90%, the catalytic efficiency is high, the phenomenon of complex product components of oblate derivatives (N-acetyl oblate or N, N'-diacetyl-oblate) in the prior art can be effectively solved, and the method has the advantages of green environmental protection, safety and stability, and no chemical pollution. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The metabolic diagram of the genetically engineered bacteria of the present application for catalyzing the synthesis of oblate by taking oblate derivatives (N-acetyl oblate or N, N'-diacetyl-oblate) as substrates.

[0019] Figure 2 Figure of the results of the substrate and product in the process of synthesizing obelin from N-acetyl obelin by the genetically engineered bacteria GL-03.

[0020] Figure 3 Figure of the results of the substrate and product in the process of synthesizing obelin from N-acetyl obelin by the genetically engineered bacteria GL-03.

[0021] Figure 4 Figure of the results of the substrate and product in the process of synthesizing obelin from N-acetyl obelin, N,N'-diacetyl obelin and the mixture of N-acetyl obelin and N,N'-diacetyl obelin by the genetically engineered bacteria GL-03. DETAILED DESCRIPTION

[0022] The above content of the present application is further explained in detail by the following specific embodiments in the form of examples. However, it should not be understood that the above-mentioned subject matter of the present application is limited to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application.

[0023] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified, and the component raw materials used in each parallel experiment are the same.

[0024] Escherichia coli DH5a (hereinafter referred to as DH5a) was used for vector construction, and Escherichia coli BL21 (DE3) was purchased from Shanghai Beinuo Biotechnology Co., Ltd.; plasmids pACYCDuet-1, pETDuet-1 and pET-28a were purchased from BioWind; high-fidelity DNA polymerase, restriction endonuclease and In-Snap Assembly Master Mix were purchased from TAKARA; plasmid extraction kit, DNA purification kit, gel recovery kit and bacterial genomic DNA extraction kit were purchased from OMEGA.

[0025] The culture medium formula involved in the present application is as follows: LB (Luria-Bertani) liquid medium: 10 g / L of proteose peptone, 5 g / L of yeast extract, 10 g / L of NaCl, 121℃ sterilization for 20 min.

[0026] LB (Luria-Bertani) solid medium: 10 g / L of proteose peptone, 5 g / L of yeast extract, 10 g / L of NaCl, 15 g / L of agar powder, 121℃ sterilization for 20 min, the solid medium is cooled to about 50℃, the required antibiotic is added to the plate, and after solidification, it is placed at 4℃ for standby.

[0027] TB medium: 12 g / L tryptone, 24 g / L yeast extract, 4 mL / L glycerol, 2.313 g / L potassium dihydrogen phosphate, 12.54 g / L disodium hydrogen phosphate.

[0028] Catalytic medium: 20 g / L observed blue derivatives (N-acetyl observed blue or N, N'-diacetyl-observed blue), 5 g / L citric acid, 10 g / L D-ribose, 40 g / L glucose, 5 g / L yeast powder, 11.9 g / L disodium hydrogen phosphate, 2.68 g / L potassium dihydrogen phosphate, 3 g / L magnesium sulfate, 50 mM sodium hexametaphosphate.

[0029] In the following examples, unless otherwise specified, the method of transforming plasmids into E. coli DH5α is chemical transformation, which includes the following steps: adding the transformation system to the thawed DH5α transformation competent on ice, ice bath for 30 min, 42℃ heat shock for 1 min, ice bath again for 2 min, then add 900 μL pre-cooled LB, 37℃, 220 rpm, incubate for 60 min; take 100 μL of the incubated bacterial solution and add it to the LB medium containing the corresponding resistance, and incubate at 30℃-37℃ overnight.

[0030] Electroporation includes the following steps: take 200-500 μg of the fragment to be transformed and add it to the prepared electrocompetent cells, mix gently and uniformly, then add it to the electrotransformation cup, ice bath for 5-10 min; 1.8KV electric shock 2-3 times, then quickly add 42℃ preheated LB medium, heat shock in a 42℃ water bath for 1 min; then place the heat-shocked transformation solution in a 37℃ incubator for 2 hours; after incubation, take 100 μL of the transformation solution and spread it on an LB solid plate, and incubate in a 37℃ incubator for 48 h.

[0031] Gibson assembly technology (hereinafter referred to as Gibson) is operated according to the following references: Gibson, D. G., et al. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343-345. Gibson, D. G., et al. (2010). Creation of a bacterial cell controlled by a chemically synthesized genome. Science, 329(5987), 52-56. The primers involved in the following examples are shown in Table 1.

[0032] Table 1 Construction of genetically engineered bacteria GL-01 This example provides a genetically engineered bacteria GL-01, and the specific construction method is as follows: 1) The deacetylase encoding gene CobB (nucleotide sequence as shown in SEQ ID NO. 1), the catalase encoding gene katE (nucleotide sequence as shown in SEQ ID NO. 3), and the NADH oxidase encoding gene NOX (nucleotide sequence as shown in SEQ ID NO. 2) were synthesized by GenScript Biotech Corporation after codon optimization.

[0033] 2) The commercially available plasmid pET-28a on the market was used as a template, and PCR amplification was performed using p28-F and p28-R primers to obtain a pET-28a linearized vector. The endogenous gene CobB was amplified using primers CobB-p28-F and CobB-p28-R with homologous arms, and the target gene fragment was obtained after purification. The CobB-28a linearized vector and the CobB gene with homologous arms were connected using a seamless cloning kit, and the ligation product was transformed into E. coli DH5α using a chemical transformation method. After recovery and culture, it was plated on LB solid medium containing 50 μg / mL kanamycin resistance and cultured in a 37°C incubator for about 16 h. Colony PCR was performed using primers CobB-YZ-F and CobB-YZ-R, and the correct strain was cultured for extraction of the recombinant plasmid, which was sent to GenScript Biotech Corporation for sequencing. The correct sequencing was pET-28a-CobB plasmid.

[0034] 3) Using pET-28a-CobB plasmid as template, using p28-CobB-F and p28-CobB-R to carry out PCR amplification, and purifying the product to obtain pET-28a-CobB linearized vector. Using primers p28-CobB-NOX-F and p28-CobB-NOX-R with homologous arms to amplify the synthesized gene NOX, and obtaining the target gene fragment after purifying the product. Using the seamless cloning kit to connect the pET-28a-CobB linearized vector and the NOX gene with homologous arms, and using the chemical transformation method to transform the connection product into E. coli DH5α, after recovering and culturing, coating on LB solid medium plate containing 50 μg / mL kanamycin resistance, and culturing in a 37°C incubator for about 16 h. Using primers CobB-NOX-YZ-F and CobB-NOX-YZ-R to carry out colony PCR verification, culturing the strains verified by PCR, extracting the recombinant plasmid, and sending it to the Bioengineering Technology Company for sequencing. The correct sequencing is the pET-28a-CobB-NOX plasmid.

[0035] 4) Using pET-28a-CobB-NOX plasmid as template, using p28-CobB-NOX-RBS-F and p28-CobB-NOX-RBS-R to carry out PCR amplification, and purifying the product to obtain pET-28a-CobB-NOX linearized vector. Using primers p28-CobB-NOX-katE-F and p28-CobB-NOX-katE-R with homologous arms to amplify the endogenous gene katE, and obtaining the target gene fragment after purifying the product. Using the seamless cloning kit to connect the pET-28a-CobB-NOX linearized vector and the katE gene with homologous arms, and using the chemical transformation method to transform the connection product into E. coli DH5α, after recovering and culturing, coating on LB solid medium plate containing 50 μg / mL kanamycin resistance, and culturing in a 37°C incubator for about 16 h. Using primers CobB-NOX-katE-YZ-F and CobB-NOX-katE-YZ-R to carry out colony PCR verification, culturing the strains verified by PCR, extracting the recombinant plasmid, and sending it to the Bioengineering Technology Company for sequencing. The correct sequencing is the pET-28a-CobB-NOX-katE plasmid.

[0036] 5) The pET-28a-CobB-NOX-katE plasmid was transformed into E. coli BL21 (DE3) by chemical transformation method. After recovery and culture, the bacteria were spread on LB solid medium plates containing 50 μg / mL kanamycin resistance and cultured in a 37°C incubator for about 16 h. The single colonies grown on the LB solid medium plates were the recombinant E. coli BL21 (DE3) / pET-28a-CobB-NOX-katE, named GL-01.

[0037] Example 2 Construction of genetically engineered bacteria GL-02 This example provides a genetically engineered bacteria GL-02, and the specific construction method is as follows: 1) The codon-optimized nicotinamide phosphoribosyltransferase encoding gene NAMPT (nucleotide sequence as shown in SEQ ID NO. 4) was synthesized by GenScript Biotech Corporation. The pETDuet-NAMPT-F and pETDuet-NAMPT-R primers were used for PCR amplification to obtain the nicotinamide phosphoribosyltransferase encoding gene NAMPT fragment with homologous arms. The commercially available plasmid pETDuet-1 was used as the substrate, and the restriction endonuclease EcoRI was used for enzyme digestion to recover the pETDuet-1 linearized vector.

[0038] 2) The pETDuet-1 linearized vector and the NAMPT gene fragment with homologous arms were connected using the Seamless Cloning Kit from Bao Biological Corporation. The ligation product was transformed into E. coli DH5α by chemical transformation method. After recovery and culture, the bacteria were spread on LB solid medium plates containing 50 μg / mL ampicillin resistance and cultured in a 37°C incubator for about 16 h. Colony PCR was performed using the primers NAMPT-YZ-F and NAMPT-YZ-R. The correct strains were cultured, the recombinant plasmid was extracted, and sent to GenScript Biotech Corporation for sequencing. The correct sequencing was the pETDuet-NAMPT plasmid.

[0039] 3) Using pETDuet-NAMPT plasmid as substrate, enzyme digestion was performed using restriction endonuclease Sla I to recover pETDuet-NAMPT linearized vector. Using E. coli BL21 (DE3) genome as template, PCR amplification was performed using primers pETDuet-nadD-F and pETDuet-nadD-R to obtain nicotinamide mononucleotide adenylyltransferase encoding gene nadD fragment (nucleotide sequence as shown in SEQ ID NO. 5). Connection was performed using seamless cloning connection kit, and the connection product was transformed into E. coli DH5α using chemical transformation method. After recovery and culture, coating was performed on LB solid culture medium plate containing 50 μg / mL ampicillin resistance, and culture was performed in a 37°C incubator for about 16 h. Colony PCR was performed using primers nadD-YZ-F and nadD-YZ-R, and strains that passed PCR verification were cultured, and the recombinant plasmid was extracted and sent to Qianke Biotechnology Company for sequencing. The one that passed sequencing was pETDuet-NAMPT-nadD plasmid.

[0040] 4) The pETDuet-NAMPT-nadD plasmid constructed above was transformed into strain GL-01 using chemical transformation method. After recovery and culture, coating was performed on LB solid culture medium plate containing 50 μg / mL ampicillin resistance, and culture was performed in a 37°C incubator for about 16 h. The single colony grown on the LB solid culture medium plate was recombinant E. coli BL21 (DE3) / pET-28a-CobB-NOX-katE+ pETDuet-NAMPT-nadD, named GL-02.

[0041] Example 3 Construction of genetically engineered bacterium GL-03 The present example provides a genetically engineered bacterium GL-03, and the specific construction method is as follows: 1) Using the *E. coli* BL21(DE3) genome as a template, PCR amplification was performed using pACYCDuet-rbsk-F and pACYCDuet-rbsk-R primers to obtain the rbsk fragment encoding the ribokinase gene (nucleotide sequence shown in SEQ ID NO. 6). The commercially available plasmid pACYCDuet-1 was used as a substrate, and the pACYCDuet-1 linearized vector was obtained by restriction endonuclease EcoRI digestion. The pACYCDuet-1 linearized vector and the rbsk gene fragment with homologous arms were ligated using a seamless cloning ligation kit from Takara Bio Inc. The ligation product was transformed into *E. coli* DH5α using chemical transformation. After resuscitation and culture, the ligation product was plated onto LB agar plates containing 50 μg / mL chloramphenicol-resistant medium and incubated at 37°C for approximately 16 h. Colony PCR verification was performed using primers rbsk-YZ-F and rbsk-YZ-R. Strains that were correctly verified by PCR were cultured, recombinant plasmids were extracted, and sent to Qingke Biotechnology Co., Ltd. for sequencing. The plasmid that was correctly sequenced was pACYCDuet-rbsk.

[0042] The pACYCDuet-rbsk plasmid was used as a substrate, and linearized with restriction endonuclease Notl to obtain the pACYCDuet-rbsk linearized vector. The E. coli BL21 (DE3) genome was used as a template, and the primer pACYCDuet-prs-F and pACYCDuet-prs-R were used for PCR amplification to obtain the phosphoribosyl pyrophosphokinase coding gene prs fragment (nucleotide sequence as shown in SEQ ID NO. 7). The ligation product was transformed into E. coli DH5a by chemical transformation after ligation using a seamless cloning kit, and after recovery and culture, it was plated on LB solid medium containing 50 μg / mL chloramphenicol resistance and cultured in a 37°C incubator for about 16 h. Colony PCR was performed using primers prs-YZ-F and prs-YZ-R, and the correct strain was cultured, and the recombinant plasmid was extracted and sent to the Bosik Biological Technology Company for sequencing. The correct sequencing was the pACYCDuet-rbsk-prs plasmid. The pACYCDuet-rbsk-prs plasmid was used as a substrate, and linearized with restriction endonuclease Kpnl to obtain the pACYCDuet-rbsk-prs linearized vector. The E. coli BL21 (DE3) genome was used as a template, and the primer pACYCDuet-ppk-F and pACYCDuet-ppk-R were used for PCR amplification to obtain the polyphosphate kinase coding gene ppk fragment (nucleotide sequence as shown in SEQ ID NO. 8). The ligation product was transformed into E. coli DH5a by chemical transformation after ligation using a seamless cloning kit, and after recovery and culture, it was plated on LB solid medium containing 50 μg / mL chloramphenicol resistance and cultured in a 37°C incubator for about 16 h. Colony PCR was performed using primers ppk-YZ-F and ppk-YZ-R, and the correct strain was cultured, and the recombinant plasmid was extracted and sent to the Bosik Biological Technology Company for sequencing. The correct sequencing was the pACYCDuet-rbsk-prs-ppk plasmid.

[0043] 5) The pACYCDuet-rbsk-prs-ppk plasmid constructed in the above step was transformed into strain GL-02 by chemical transformation, and after recovery and culture, it was plated on LB solid medium containing 50 μg / mL chloramphenicol resistance and cultured in a 37°C incubator for about 16 h. The single colony grown on the LB solid medium plate was the recombinant BL21 (DE3) / pET-28a-CobB-NOX-katE + pETDuet-NAMPT-nadD + pACYCDuet-rbsk-prs-ppk, named GL-03.

[0044] Example 4 The embodiment provides a flask catalysis method for synthesizing oculin by catalyzing N-acetyloculin, which comprises the following steps. (1) the constructed genetically engineered bacteria are cultured in LB solid medium at 37°C for 48 h, single colonies are picked and inoculated into 5 mL LB medium, and then the medium is cultured at 37°C and 220 rpm for 12-16 h to obtain a first-stage seed liquid; (2) 350 μL of the first-stage seed liquid obtained in step (1) is inoculated into 35 mL TB medium, and then the medium is cultured at 37°C and 220 rpm until the OD600 reaches 0.6-0.8, then 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) is added, and the medium is induced to express at 30°C for 8 h. (2) 350 μL of the first-stage seed liquid obtained in step (1) is inoculated into 35 mL TB medium, and then the medium is cultured at 37°C and 220 rpm until the OD600 reaches 0.6-0.8, then 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) is added, and the medium is induced to express at 30°C for 8 h.

[0045] (3) the bacteria collected in step (2) are transferred into a 250 mL baffle flask containing 30 mL catalytic medium (oculin derivative is 10 g / L N-acetyloculin), and then the medium is cultured at 30°C and 220 rpm for 24 h to obtain oculin catalytic liquid.

[0046] Example 5 The embodiment provides a flask catalysis method for synthesizing oculin by catalyzing N-acetyloculin, which comprises the following steps. (1) the constructed genetically engineered bacteria are cultured in LB solid medium at 37°C for 48 h, single colonies are picked and inoculated into 5 mL LB medium, and then the medium is cultured at 37°C and 220 rpm for 12-16 h to obtain a first-stage seed liquid; (2) 350 μL of the first-stage seed liquid obtained in step (1) is inoculated into 35 mL TB medium, and then the medium is cultured at 37°C and 220 rpm until the OD600 reaches 0.6-0.8, then 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) is added, and the medium is induced to express at 30°C for 8 h. (2) 350 μL of the first-stage seed liquid obtained in step (1) is inoculated into 35 mL TB medium, and then the medium is cultured at 37°C and 220 rpm until the OD600 reaches 0.6-0.8, then 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) is added, and the medium is induced to express at 30°C for 8 h.

[0047] (3) the bacteria collected in step (2) are transferred into a 250 mL baffle flask containing 30 mL catalytic medium (oculin derivative is 10 g / L N-acetyloculin), and then the medium is cultured at 30°C and 220 rpm for 24 h to obtain oculin catalytic liquid.

[0048] Example 6 The embodiment provides a 5L bioreactor catalysis method for synthesizing oculin by catalyzing N-acetyloculin, which comprises the following steps. (1) The constructed engineering bacteria were cultured in LB solid medium at 37°C for 48 h, and single colonies were inoculated into 5 mL of LB medium and cultured at 37°C and 220 rpm for 12-16 h to obtain a first-stage seed solution; (2) 1 mL of the first-stage seed solution obtained in step (1) was inoculated into 200 mL of TB medium and cultured at 37°C and 220 rpm for 8-10 h to obtain a second-stage seed solution.

[0049] (3) The second-stage seed solution obtained in step (2) was inoculated into a 5 L bioreactor (TB medium, liquid volume 2 L) by flame inoculation method, and cultured at 37°C, 300 rpm and aeration amount 1.0 vvm until OD600 was 0.6-0.8, then 0.5 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and the culture was induced at 30°C for 8-12 h. After the induction expression was completed, the bacterial cells were collected under the centrifugal conditions of 4°C, 5000 rpm and 10 min.

[0050] (4) The bacterial cells collected in step (3) were transferred into a 5 L bioreactor (catalytic medium containing 20 / L N-acetyl acylanine, liquid volume 2.5 L), and cultured at 30°C and 300 rpm for 24 h to obtain an acylanine catalytic solution.

[0051] Example 7 The present embodiment provides a 5 L bioreactor catalytic method for synthesizing acylanine from N, N'-diacetyl-acylanine, which comprises the following steps: (1) The constructed engineering bacteria were cultured in LB solid medium at 37°C for 48 h, and single colonies were inoculated into 5 mL of LB medium and cultured at 37°C and 220 rpm for 12-16 h to obtain a first-stage seed solution; (2) 1 mL of the first-stage seed solution obtained in step (1) was inoculated into 200 mL of TB medium and cultured at 37°C and 220 rpm for 8-10 h to obtain a second-stage seed solution.

[0052] (3) The second-stage seed solution obtained in step (2) was inoculated into a 5 L bioreactor (TB medium, liquid volume 2 L) by flame inoculation method, and cultured at 37°C, 300 rpm and aeration amount 1.0 vvm until OD600 was 0.6-0.8, then 0.5 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and the culture was induced at 30°C for 8-12 h. After the induction expression was completed, the bacterial cells were collected under the centrifugal conditions of 4°C, 5000 rpm and 10 min.

[0053] ​​(4) The bacteria collected in step (3) were transferred to a 5L bioreactor (catalytic medium containing 20 g / L N-acetyl-oxazine, liquid volume 2.5 L), and cultured at 30°C and 300 rpm for 24 h to obtain an oxazine fermentation liquor.

[0054] Example 8 The present example provides a 5L bioreactor catalytic method for catalyzing a mixture of N-acetyl-oxazine, N,N'-diacetyl-oxazine and oxazine, which comprises the following steps: (1) The constructed engineering bacteria were cultured in LB solid medium at 37°C for 48 h, and a single colony was inoculated into 5 mL LB medium and cultured at 37°C and 220 rpm for 12-16 h to obtain a primary seed liquor; (2) 1 mL of the primary seed liquor obtained in step (1) was inoculated into 200 mL TB medium and cultured at 37°C and 220 rpm for 8-10 h to obtain a secondary seed liquor.

[0055] (3) The secondary seed liquor obtained in step (2) was inoculated into a 5L bioreactor (TB medium, liquid volume 2 L) by flame inoculation method, and after the OD600 was 0.6-0.8, 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and the culture was induced at 30°C for 8-12 h. After the induction expression was completed, the bacteria were collected under the conditions of 4°C, 5000 rpm and 10 min centrifugation.

[0056] (4) The bacteria collected in step (3) were transferred to a 5L bioreactor (catalytic medium containing 20 g / L N-acetyl-oxazine, liquid volume 2.5 L), and cultured at 30°C and 300 rpm for 24 h to obtain an oxazine fermentation liquor.

[0057] Example 1 The genetically engineered bacteria GL-01-GL-03 of Examples 1-3 were used to synthesize oxazine according to the methods of Examples 4 and 5, and the synthesis route of the genetically engineered bacteria catalyzing oxazine derivatives (N-acetyl-oxazine or N,N'-diacetyl-oxazine) to synthesize oxazine is shown in Figure 1 , and the content of oxazine, N-acetyl-oxazine and N,N'-diacetyl-oxazine in the catalytic liquor was detected by high performance liquid chromatography.

[0058] The chromatographic conditions of the high performance liquid chromatography detection method are as follows: Mobile phase: methanol and pure water gradient elution, and the gradient conditions are shown in Table 2. ​

[0059] Wavelength 600 nm, flow rate 1.0 mL / min, sample solution: DMSO, sample volume: 10 μL, column temperature 35℃, running time 20 min.

[0060] Chromatographic column: Galasil EF-C18M 4.6 mm id x 250 mm L (SN B06211801).

[0061] Specific experimental method: 1 mg / mL of observed blue, N-acetyl observed blue and N-N'-diacetyl-observed blue standard solution was prepared respectively, and gradient concentration standard solution was obtained by dilution, high performance liquid chromatography analysis was carried out, and the observed blue peak area, N-acetyl observed blue peak area and N, N'-diacetyl-observed blue were taken as the ordinate, and the observed blue concentration, N-acetyl observed blue concentration and N, N'-diacetyl-observed blue were taken as the abscissa to draw the standard curve, the obtained observed blue standard curve was Y=165835X+14358, R 2 =0.9996; the obtained N-acetyl observed blue standard curve was Y=195587X+23189, R 2 =0.9998; and the obtained N, N'-diacetyl-observed blue standard curve was Y=175587X+33759, R 2 =0.9992.

[0062] Table 2 Gradient elution method for liquid phase detection The peak graph of each group of catalytic liquid samples was detected by high performance liquid chromatography, and the contents of observed blue, N-acetyl observed blue and N, N'-diacetyl-observed blue were calculated according to the standard curve, and the results are shown in Table 3.

[0063] Table 3 Different genetically engineered bacteria catalyze observed blue derivatives (N-acetyl observed blue or N, N'-diacetyl-observed blue) to synthesize observed blue As can be seen from Table 3, the genetically engineered bacteria GL-01~GL-03 of the present application can catalyze a certain amount of observed blue derivatives (N-acetyl observed blue or N, N'-diacetyl-observed blue) to observed blue under IPTG induction. Among them, the strain GL-03 has the strongest ability to catalyze observed blue derivatives (N-acetyl observed blue or N, N'-diacetyl-observed blue) to synthesize observed blue, and 10 g / L of observed blue derivatives (N-acetyl observed blue or N, N'-diacetyl-observed blue) can obtain 7.96 g / L and 6.77 g / L of observed blue respectively after 24 h of catalysis. Comparison can be made that the integration of NAD + dependent deacetylase and NADH oxidase can enable the strain to have the ability to convert observed blue derivatives (N-acetyl observed blue or N, N'-diacetyl-observed blue) to observed blue, but it cannot continuously supply NAD+ This resulted in weak conversion ability, with molar conversion rates of only 38% and 30.3% after 24 hours; further, by constructing NAD... + The conversion efficiency was significantly improved after the recycling system and the enhancement of ATP supply, reaching 93% and 90.6% respectively, which fully demonstrates the effectiveness of maintaining the cofactor NAD in this catalytic system. + A sustained supply of ATP is an important rate-limiting step.

[0064] Depend on Figure 1 It is known that the genetically engineered bacteria constructed in this invention can synthesize lanthanum using lanthanum derivatives (N-acetylanthanum or N,N'-diacetyl-lanthanum) as substrates through whole-cell catalysis: firstly, excess intracellular reducing power NADH is converted into the key cofactor NAD by NADH oxidase. + Then NAD + The cyclic regeneration is achieved through the combined action of nicotinamide phosphoribosyltransferase and nicotinamide mononucleotide adenylate transferase, continuously providing the cofactor NAD for the deacetylation reaction. + In this process, the key intermediate PRPP in the circulatory system is continuously produced through the synergistic action of ribokinase and phosphoribosylpyrokinase. This process consumes a large amount of ATP and produces ADP and AMP. The expression of polyphosphokinase can catalyze the synthesis of ATP from ADP / AMP, providing sufficient energy for this process. Finally, NAD... + NAD-dependent deacetylases + With the assistance of catalytic synthesis, lanthanum derivatives (N-acetyllanthanum or N,N'-diacetyllanthanum) are synthesized into lanthanum.

[0065] Example 2 The genetically engineered bacterium GL-03 from Example 3 was used to synthesize styrax according to the methods of Examples 6 and 7, respectively. The contents of styrax, N-acetyrax, and N,N'-diacetyl-styrax in the catalytic solution were detected using the high-performance liquid chromatography method of Example 1. After 24 hours of catalysis, the contents of styrax, N-acetyrax, and N,N'-diacetyl-styrax during the catalytic process were calculated based on the standard curve. The results are as follows: Figure 2 and Figure 3 As shown.

[0066] Depend on Figure 2 and Figure 3It was found that 20 g / L of N-acetylglucosamine or N,N'-diacetyl-lglucosamine as substrates could synthesize 16.25 g / L and 13.82 g / L of glucosamine, respectively, in a 5 L reactor after 24 h of catalysis, with molar conversion rates exceeding 90%. N,N'-diacetyl-lglucosamine, due to its two acetyl groups, consistently showed lower conversion efficiency than N-acetylglucosamine, but its conversion rate remained high, indicating that the genetically engineered bacterium GL-03 possesses highly efficient deacetylation capabilities. The synthesis rate in the first 12 h (1.07 g / L / h or 0.88 g / L / h) was significantly higher than that in the latter 12 h (0.29 g / L / h or 0.27 g / L / h), suggesting that in practical applications, increasing the substrate concentration can lead to faster glucosamine production.

[0067] Example 3 The genetically engineered bacterium GL-03 from Example 3 was used to synthesize styrax according to the method in Example 8. The contents of styrax, N-acetyrax, and N,N'-diacetyl-styrax in the catalytic solution were detected using the high-performance liquid chromatography method of Effect Example 1. After 24 hours of catalysis, the contents of styrax, N-acetyrax, and N,N'-diacetyl-styrax during the catalytic process were calculated based on the standard curve. The results are as follows: Figure 4 As shown.

[0068] Depend on Figure 4 It can be seen that when the catalytic solution containing 10 g / L N-acetylasin, 10 g / L N,N'-diacetyl-lasin, and 10 g / L lasin is catalyzed in a 5L reactor for 24 h, 25.32 g / L lasin can be obtained. The molar conversion rates of N-acetylasin and N,N'-diacetyl-lasin reach 98.3% and 92.5%, respectively. This proves that the genetically engineered bacterium GL-03 can effectively convert lasin derivatives (N-acetylasin or N,N'-diacetyl-lasin) and lasin mixtures into lasin, which has certain application value.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention 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 the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A genetically engineered bacterium that catalyzes the synthesis of blueberry derivatives, characterized in that, The genetically engineered bacteria are obtained by introducing the deacetylase gene CobB, the NADH oxidase gene NOX, and the catalase gene katE into the host bacteria; and / or introducing the nicotinamide phosphoribosyltransferase gene NAMPT and the nicotinamide mononucleotide adenylate transferase gene nadD; and / or introducing the ribokinase gene rbsk, the phosphoribosyl pyrophosphate kinase gene prs, and the polyphosphate kinase gene pppk; the guanlan derivatives include N-acetylanlan and N,N'-diacetyl-guanlanlan.

2. The genetically engineered bacterium according to claim 1, characterized in that, The host bacteria include Escherichia coli.

3. The genetically engineered bacterium according to claim 1, characterized in that, The deacetylase gene CobB, NADH oxidase gene NOX, catalase gene katE, nicotinamide phosphoribosyltransferase gene NAMPT, nicotinamide mononucleotide adenylate transferase gene nadD, ribokinase gene rbsk, phosphoribosyl pyrophosphate kinase gene prs, and polyphosphate kinase gene pppk can be derived from any species.

4. The genetically engineered bacteria according to claim 3, characterized in that, The deacetylase gene CobB, catalase gene katE, nicotinamide mononucleotide adenylate transferase gene nadD, ribokinase gene rbsk, phosphoribosyl pyrophosphate kinase gene prs, and polyphosphate kinase gene ppk are all derived from *Escherichia coli*; the NADH oxidase encoding gene NOX is derived from *Streptococcus mutans*; and the nicotinamide phosphoribosyltransferase encoding gene NAMPT is derived from *Chitinobacterium pinnatifida*.

5. The genetically engineered bacterium according to claim 1, characterized in that, The nucleotide sequence of the deacetylase gene CobB is shown in SEQ ID NO.1; the nucleotide sequence of the NADH oxidase gene NOX is shown in SEQ ID NO.2; the nucleotide sequence of the catalase gene katE is shown in SEQ ID NO.3; the nucleotide sequence of the nicotinamide phosphoribosyltransferase gene NAMPT is shown in SEQ ID NO.4; the nucleotide sequence of the nicotinamide mononucleotide adenylate transferase gene nadD is shown in SEQ ID NO.5; the nucleotide sequence of the ribokinase gene rbsk is shown in SEQ ID NO.6; the nucleotide sequence of the phosphoribosyl pyrophosphate kinase gene prs is shown in SEQ ID NO.7; and the nucleotide sequence of the polyphosphate kinase gene pppk is shown in SEQ ID NO.

8.

6. The method for constructing the genetically engineered bacterium for synthesizing safflower derivatives according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Using gene editing technology, the deacetylase gene CobB, the NADH oxidase gene NOX, and the catalase gene katE were integrated into Escherichia coli to obtain the genetically engineered bacterium GL-01. S2. and / or integrate the nicotinamide phosphoribosyltransferase gene NAMPT and the nicotinamide phosphoribosyltransferase gene nadD into the genetically engineered bacterium GL-01 to obtain the genetically engineered bacterium GL-02; S3. And / or integrate the ribokinase gene rbsk, the phosphoribosyl pyrophosphate kinase gene prs, and the polyphosphate kinase gene pppk into the genetically engineered bacterium GL-02 to obtain the genetically engineered bacterium GL-03, which is the genetically engineered bacterium that catalyzes the synthesis of lanthanum from the lanthanum derivative (N-acetylanthanum or N,N'-diacetyl-lanthanum).

7. The use of the genetically engineered bacteria according to any one of claims 1-5 in the preparation of blue violet.

8. A method for synthesizing blue pigment, characterized in that, The method is obtained by catalytic reaction of the seed liquid of the genetically engineered bacteria according to any one of claims 1-5 with a catalytic culture medium.

9. The method for synthesizing blue violet according to claim 8, characterized in that, The catalytic culture medium comprises an IPTG solution with a final concentration of 0.2–0.6 mM.

10. The method for synthesizing blue violet according to claim 8, characterized in that, The catalytic reaction is specifically carried out at 28~32℃ and 200~300 rpm for 24~48 h.

Images