Method for efficiently synthesizing violacein by using cell-free system

Abstract

The invention discloses a method for efficiently synthesizing violacein by using a cell-free system, which comprises the following steps: firstly, constructing recombinant plasmids of key enzymes VioA, VioB, VioC, VioD and VioE in a violacein synthesis route, artificially and exogenously adding substrates, energy, cofactors and the like required by a synthesis reaction based on an escherichia coli cell lysis buffer rich in VioA, VioB, VioC, VioD and VioE, and efficiently synthesizing violacein in vitro. According to the method, the cell-free system is applied to in-vitro synthesis of violacein for the first time, the substrate transfer distance and the distance between multiple enzymes are shortened, and the reaction rate and the catalytic efficiency are improved; operation is simple, stability is high, and reaction conditions are accurate and easy to control; the method has the advantages of simple operation, few side reactions, high production rate and high product yield, can conveniently and quickly detect violacein, and is suitable for detection and verification.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for efficiently synthesizing violacein using a cell-free system. Background Art

[0002] Violacein is formed by the oxidative condensation of two tryptophan molecules and belongs to indole derivatives. It is a non-polar blue-black pigment. Violacein is mainly produced by Gram-negative bacteria and is a broad-spectrum antibacterial pigment with various physiological activities. It has important biological activities such as anti-tumor, antibacterial, and antioxidant activities, and has broad application prospects in the food, medical, and printing and dyeing industries.

[0003] Complex indole derivatives such as violacein are all synthesized by a series of enzymes participating in multi-enzyme co-catalyzed reactions. At present, with the rapid development of genetic engineering technology and the introduction of metabolic engineering and synthetic biology methods, people are increasingly concerned about the production of violacein by microbial fermentation. Microbial fermentation is safe and non-toxic and can solve a series of problems such as low yield and productivity of violacein and high extraction cost. However, the artificial design of biosynthetic pathways for heterologous multi-enzyme cascade reactions in microbial cells is often restricted by various factors: firstly, it is necessary to balance metabolic pathways and multi-gene regulation in cells, which is time-consuming and cumbersome; secondly, the metabolism of the cell host itself is complex, and high-concentration intermediate metabolites or toxic end products will inhibit the cells; finally, the compatibility and adaptability of artificially synthesized biological elements with the host are low, and by-products of branch pathways will lead to low yield and productivity of the target product, etc. Summary of the Invention

[0004] Object of the Invention: Aiming at the deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a method for efficiently synthesizing violacein using a cell-free system. For the multi-enzyme synthesis system of violacein, the cell-free synthesis system is introduced into it, which has the advantages of short construction time, simple operation, controllable and stable reaction conditions; can tolerate high titers of products and is not affected by inhibition; can directly add substrates and extract products; and specifically synthesizes the target product, etc. Abandoning the traditional complex in vivo gene modification and the negative factors of various environmental interferences in cells enables the optimization of the reaction microenvironment in vitro and has strong application value.

[0005] Technical Solution: The present invention is realized by the following technical solutions: A method for efficiently synthesizing violacein using a cell-free system, comprising the following steps:

[0006] 1) Recombinant plasmids containing gene fragments vioA, vioB, vioC, vioD, and vioE are respectively constructed and transformed into Escherichia coli to obtain 5 kinds of recombinant Escherichia coli;

[0007] 2) Induce the expression of the 5 kinds of recombinant Escherichia coli respectively and perform ultrasonic disruption to obtain the cell lysates of VioA, VioB, VioC, VioD and VioE proteins;

[0008] 3) Mix the cell lysates of VioA, VioB, VioC, VioD and VioE proteins to obtain a crude extract mixture of VioA, VioB, VioC, VioD and VioE proteins, and add it to the substrate reaction mixture for reaction to obtain the product.

[0009] Among them, the vioA, vioB, vioC, vioD, and vioE genes in step 1) are all derived from the Janthinobacterium sp. B9-8 strain.

[0010] Among them, the recombinant plasmids in step 1) are pRSF-VioA, pRSF-VioB, pRSF-VioC, pRSF-VioD, and pRSF-VioE.

[0011] Among them, the temperature of the induced expression in step 2) is 18 - 30 °C, and the time is 4 - 6 h.

[0012] Among them, the volume ratios of the cell lysates of VioA, VioB, VioC, VioD and VioE proteins in the cell-free reaction system are 1% - 50% respectively, and the mass percentages of the five proteins of VioA, VioB, VioC, VioD and VioE in the total protein of the whole bacteria are 10% - 90% respectively. Among them, the substrate reaction mixture in step 3) includes a substrate and cofactors. The substrate is L-Trp, and the concentration is 4 - 20 mM. Preferably, the cofactors are CoA, NADPH and ATP, and the concentrations are all 0.5 - 1 mM.

[0013] Among them, the mass ratio of VioA:VioB:VioC:VioD:VioE proteins in step 3) is (1 - 2):(1 - 2):(1 - 2):(1 - 2):(1 - 2).

[0014] Among them, the reaction temperature for synthesizing violacein in step 3) is 18 - 37 °C, and the time is 2 - 24 h.

[0015] Among them, the concentration of the substrate in step 3) is 16 mM; the concentrations of the cofactors are all 1 mM; the mass ratio of VioA, VioB, VioC, VioD, and VioE in the cell lysate is 1:2:1:1:1.

[0016] Among them, the reaction temperature for synthesizing violacein in step 3) is 25 °C, and the reaction time is 24 h.

[0017] Specifically, the construction steps of the recombinant plasmid in step 1) specifically include: using the genome of Janthinobacterium sp. B9-8 as a template, performing PCR with the following primers respectively to obtain fragments vioA, vioB, vioC, vioD, and vioE. After gel extraction, use restriction enzymes BamHI / EcoRI, BamHI / HindIII, BamHI / EcoRI, BamHI / EcoRI, and BamHI / PstI to perform double digestion on the PCR fragments and the prokaryotic expression plasmid pRSFDuet-1, and ligate them to the plasmid pRSFDuet-1 respectively to obtain recombinant plasmids pRSF-VioA, pRSF-VioB, pRSF-VioC, pRSF-VioD, and pRSF-VioE.

[0018] The primer design is as follows:

[0019] (1) VioA primer design:

[0020] The upstream primer VioA F has the nucleotide sequence shown in SEQ ID NO.1 and a BamHI restriction enzyme site;

[0021] The downstream primer VioA R has the nucleotide sequence shown in SEQ ID NO.2 and an EcoRI restriction enzyme site;

[0022] (2) VioB primer design:

[0023] The upstream primer VioB F has the nucleotide sequence shown in SEQ ID NO.3 and a BamHI restriction enzyme site;

[0024] The downstream primer VioB R has the nucleotide sequence shown in SEQ ID NO.4 and a HindIII restriction enzyme site;

[0025] (3) VioC primer design:

[0026] The upstream primer VioC F has the nucleotide sequence shown in SEQ ID NO.5 and a BamHI restriction enzyme site;

[0027] The downstream primer VioC R has the nucleotide sequence shown in SEQ ID NO.6 and an EcoRI restriction enzyme site;

[0028] (4) VioD primer design:

[0029] The upstream primer VioD F has the nucleotide sequence shown in SEQ ID NO.7 and a BamHI restriction enzyme site;

[0030] The downstream primer VioD R has the nucleotide sequence shown in SEQ ID NO.8 and an EcoRI restriction site;

[0031] (5) Design of VioE primers:

[0032] The upstream primer VioE F has the nucleotide sequence shown in SEQ ID NO.9 and a BamHI restriction site;

[0033] The downstream primer VioE R has the nucleotide sequence shown in SEQ ID NO.10 and a PstI restriction site.

[0034] Among them, the method for synthesizing violacein in step 3) is specifically carried out by using a cell-free lysate, which specifically includes the following steps:

[0035] First, culture recombinant Escherichia coli that can express VioA, VioB, VioC, VioD, and VioE to overexpress the genes encoding vioA, vioB, vioC, vioD, and vioE; detect the total protein concentration using a BCA kit, detect the protein expression by SDS-PAGE, and detect the proportion of the protein expression mass concentration by ImageJ; collect the cells and use an ultrasonic cell disruptor to disrupt them at 210W, with a 2s on and 6s off cycle, under ice-water bath conditions for 5 - 10 minutes; centrifuge at 4°C and 12,000 rpm for 30 minutes to obtain the cell lysates of VioA, VioB, VioC, VioD, and VioE proteins respectively.

[0036] Second, carry out the reaction in a 100 mM Tris-HCl (pH 8) buffer system. The total amount of the five proteins in the crude extract solution obtained by mixing the cell lysates of VioA, VioB, VioC, VioD, and VioE proteins is 400 μg; 8 mM magnesium glutamate; 160 mM potassium glutamate; 1 mM NADPH; 1 mM ATP; 1 mM CoA; the enzyme mass ratio is VioA:VioB:VioC:VioD:VioE = 1:2:1:1:1, 16 mM L-Trp, and react at 25°C for 24 hours. The final yield reaches 189.22 mg / L, which is 35.57 times higher than that before optimization.

[0037] Beneficial effects: The present invention first applies a cell-free synthesis system to the in vitro synthesis of violacein. This method shortens the substrate transfer distance and the distance between enzymes, improves the reaction rate and catalytic efficiency, and enhances the protein stability. It not only explores the optimal enzyme ratio among the key enzymes in the violacein synthesis process, but also has simple operation and strong stability, laying a foundation for further optimizing the violacein synthesis. Description of the Drawings

[0038] Figure 1 Construction process of recombinant plasmids pRSF-VioA, pRSF-VioB, pRSF-VioC, pRSF-VioD, and pRSF-VioE in Example 1;

[0039] Figure 2 Colony PCR and double digestion detection results in Example 1; Figure 2 (a) Amplification of vioA, vioB, vioC, vioD, and vioE genes (lane 1 is vioA, lane 2 is vioB, lane 3 is vioC, lane 4 is vioD, lane 5 is vioE); Figure 2 (b) Diagram of double digestion verification of recombinant plasmids (lane 1 is recombinant plasmid pRSFDuet-VioA, lane 2 is recombinant plasmid pRSFDuet-VioB, lane 3 is recombinant plasmid pRSFDuet-VioC, lane 4 is recombinant plasmid pRSFDuet-VioD, lane 5 is recombinant plasmid pRSFDuet-VioE);

[0040] Figure 3 Protein expression results of VioA, VioB, VioC, VioD, and VioE in Example 1;

[0041] Figure 4 Schematic diagram of the cell-free synthesis system of violacein in Example 2;

[0042] Figure 5 Relationship between violacein synthesis and reaction time in the cell-free system in Example 3;

[0043] Figure 6 Effect of the total amount of five proteins in the crude extract on violacein synthesis in Example 3;

[0044] Figure 7 Effect of substrate concentration on violacein synthesis in Example 3;

[0045] Figure 8 Effect of reaction temperature on violacein synthesis in Example 3;

[0046] Figure 9 Effect of enzyme ratio on violacein synthesis in Example 3;

[0047] Figure 10 Effect of cofactors and metal ions on violacein synthesis in Example 3. Detailed implementation methods

[0048] The technology of the present invention will be described in detail below in conjunction with the accompanying drawings and specific experiments. It should be understood that the following specific experimental examples are only used to help those skilled in the art understand the present invention, rather than limiting the present invention. The EcoRI of the present invention was purchased from Novoprotein Co., Ltd., with the product number C405-01, the BamHI was purchased from Novoprotein Co., Ltd., with the product number C401-01, the HindIII was purchased from Novoprotein Co., Ltd., with the product number C407-01, the PstI was purchased from Novoprotein Co., Ltd., with the product number C414-01, and the T4 DNA Ligase was purchased from Novoprotein Co., Ltd., with the product number C301-01.

[0049] Example 1 Construction and Expression of Recombinant Plasmids of Violacein Key Enzymes

[0050] 1. Construction of Recombinant Plasmids Containing vioA, vioB, vioC, vioD, and vioE Genes

[0051] The primers were designed as follows (as shown in Table 1):

[0052] Design of VioA Primers:

[0053] The upstream primer VioA F has the nucleotide sequence shown in SEQ ID NO.1 and a BamHI restriction site, specifically: 5’-CGGGGATCCGTGATAAACAACAAAG-3’.

[0054] The downstream primer VioA R has the nucleotide sequence shown in SEQ ID NO.2 and an EcoRI restriction site, specifically: 5’-CGGGAATTCTTAATTCATTTCTGCCGC-3’.

[0055] Design of VioB Primers:

[0056] The upstream primer VioB F has the nucleotide sequence shown in SEQ ID NO.3 and a BamHI restriction site, specifically: 5’-CCGGGATCCAATGAGTATTCTGAATTTTC-3’.

[0057] The downstream primer VioB R has the nucleotide sequence shown in SEQ ID NO.4 and a HindIII restriction site, specifically: 5’-CAAAAGCTTtCAGCCTTCCCGTGAAA-3’.

[0058] Design of VioC Primers:

[0059] The upstream primer VioC F has the nucleotide sequence shown in SEQ ID NO.5 and a BamHI restriction site, specifically: 5’-CGGGGATCCAATGAAAAAAATCATTATTGTTG-3’.

[0060] The downstream primer VioC R has the nucleotide sequence shown in SEQ ID NO.6 and an EcoRI restriction site, specifically: 5’-CGGGAATTCTTAGTTTACTCTCCCTAGTT-3’.

[0061] VioD primer design:

[0062] The upstream primer VioD F has the nucleotide sequence shown in SEQ ID NO.7 and a BamHI restriction site, specifically: 5’-GCCGGATCCAATGAAGATCTTAGTCG-3’.

[0063] The downstream primer VioD R has the nucleotide sequence shown in SEQ ID NO.8 and an EcoRI restriction site, specifically: 5’-CCGGAATTCTTAAGATTCCTTACGAG-3’.

[0064] VioE primer design:

[0065] The upstream primer VioE F has the nucleotide sequence shown in SEQ ID NO.9 and a BamHI restriction site, specifically: 5’-GCGGATCCCATGATTGCTGAGAAAATGTTACC-3’.

[0066] The downstream primer VioE R has the nucleotide sequence shown in SEQ ID NO.10 and a PstI restriction site, specifically: 5’-GCTGCAGTTAAATACTGCTTGAAAATATTTCGGC-3’.

[0067] Table 1 Artificial primer sequence table

[0068]

[0069]

[0070] Using the genome of Janthinobacterium sp. B9-8 strain in the Chinese patent application with the authorization number CN112195129B as a template, PCR was performed respectively with the primers designed in Table 1 to obtain gene fragments vioA, vioB, vioC, vioD, and vioE. After gel recovery, the PCR-amplified gene fragments and the prokaryotic expression plasmid pRSFDuet-1 (purchased from Novagen) were double digested with restriction enzymes BamHI / EcoRI, BamHI / HindIII, BamHI / EcoRI, BamHI / EcoRI, and BamHI / PstI at 37°C for 1 h (the enzyme digestion system is shown in Table 2). The digested fragments were respectively ligated to the corresponding digested plasmid pRSFDuet-1 (the ligation system is shown in Table 3), and ligated at 16°C for more than 3 h to obtain recombinant plasmids pRSF-VioA, pRSF-VioB, pRSF-VioC, pRSF-VioD, and pRSF-VioE (as Figure 1 shown).

[0071] Table 2 Enzyme digestion system

[0072]

[0073] Table 3 Ligation system

[0074]

[0075] The ligation solution was transferred into Escherichia coli BL21(DE3) competent cells by heat shock method. Through colony PCR, sequencing, and double digestion verification, as Figure 2 shown in a, specific fragments vioA, vioB, vioC, vioD, and vioE were amplified at the corresponding band positions of 1287 bp in lane 1, 3024 bp in lane 2, 1290 bp in lane 3, 1131 bp in lane 4, and 582 bp in lane 5, respectively, proving that the gene had been ligated to the vector in the selected monoclonal. As Figure 2 shown in b, the positive clones were sequenced and verified by enzyme digestion. The bands in lanes 1-5 of each lane were consistent with the size of the target fragment, proving that the recombinant plasmid was successfully constructed. Recombinant plasmids pRSF-VioA, pRSF-VioB, pRSF-VioC, pRSF-VioD, and pRSF-VioE were obtained. Then, the recombinant plasmids pRSF-VioA, pRSF-VioB, pRSF-VioC, pRSF-VioD, and pRSF-VioE were respectively transformed into BL21(DE3) for expression.

[0076] 2. Expression and verification of VioA, VioB, VioC, VioD, and VioE

[0077] LB + Kana liquid medium: Tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, kanamycin 25 μg / mL.

[0078] The BL21(DE3) strains containing recombinant plasmids pRSF-VioA, pRSF-VioB, pRSF-VioC, pRSF-VioD, and pRSF-VioE were respectively inoculated into 50 mL of LB + Kana liquid medium and cultured overnight at 37 °C and 180 rpm as the mother liquor. The next day, 2% of the mother liquor was inoculated into fresh LB + Kana liquid medium and cultured at 37 °C and 200 rpm until the OD 600 reached about 0.8, then isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.5 mM was added, and the target protein was induced to express for 4 h at 28 °C and 180 rpm. After the induction ended, the cells were centrifuged at 8500 rpm and 4 °C for 5 min to obtain the bacterial cells. 3 mL of 20 mM Tris-HCl (pH 8.0) and 200 μL of 2 mM dithiothreitol (DTT) were added to wash the cells twice; then the cells were resuspended in 20 mM Tris-HCl buffer (resuspended according to 1 mL of 20 mM Tris-HCl buffer per g of wet bacterial sludge). The total protein concentration was detected using a BCA kit, and the protein expression was detected by SDS-PAGE. The protein expression was as Figure 3 shown.

[0079] Figure 3 shown that a large amount of the target protein was expressed at 48 kDa, 111 kDa, 48 kDa, 42 kDa, and 22 kDa respectively, which was consistent with the sizes of VioA, VioB, VioC, VioD, and VioE proteins, proving that VioA, VioB, VioC, VioD, and VioE have all achieved soluble overexpression in the recombinant Escherichia coli. Example 2 Construction of a cell-free synthesis system for violacein based on a lysate rich in key enzymes

[0080] Escherichia coli containing recombinant plasmids pRSF-VioA, pRSF-VioB, pRSF-VioC, pRSF-VioD, and pRSF-VioE were respectively cultured in LB + Kana liquid medium overnight at 37 °C and 180 rpm; inoculated into fresh LB + Kana liquid medium at an inoculation amount of 2%, and cultured at 200 rpm and 37 °C until the OD 600When it is 0.6 - 0.8, add IPTG with a final concentration of 0.5 mM to induce for 4 h, and collect the cells. Add 3 mL of pre-cooled 20 mM Tris-HCl (pH 8.0) and 200 μL of 2 mM DTT; wash the bacterial cells twice, and then resuspend them in 20 mM Tris-HCl buffer (resuspend 1 g of wet bacterial cells with 1 mL of Tris-HCl buffer); break the bacterial cells with an ultrasonic cell disruptor. Under the condition of an ice-water bath, at 210 W, turn on for 2 s and turn off for 6 s, and break for 5 - 10 min; centrifuge at 4 °C and 12,000 rpm for 30 min to obtain the cell lysates of VioA, VioB, VioC, VioD, and VioE proteins respectively.

[0081] Preliminary construction based on cell-free metabolic engineering of violacein from crude extracts:

[0082] A schematic diagram of the violacein synthesis method is as Figure 4 shown.

[0083] After the cell lysates of Escherichia coli induced with recombinant plasmids pRSF-VioA, pRSF-VioB, pRSF-VioC, pRSF-VioD, and pRSF-VioE are subjected to SDS-PAGE electrophoresis, use Image J to analyze the proportions of VioA, VioB, VioC, VioD, and VioE proteins in the total protein of the whole bacteria respectively, and calculate the addition amounts of each enzyme.

[0084] The total volume of the cell-free synthesis system for violacein is prepared to be 200 μL:

[0085] (1) Protein mixture part: The total amount of the five proteins is 100 μg (control the mass ratio of proteins VioA:VioB:VioC:VioD:VioE = 1:1:1:1:1). By calculation, the mass percentage of VioA protein in the total protein of the whole bacteria is 16.71%, the mass percentage of VioB protein in the total protein of the whole bacteria is 14.11%, the mass percentage of VioC protein in the total protein of the whole bacteria is 24.56%, the mass percentage of VioD protein in the total protein of the whole bacteria is 22.40%, and the mass percentage of VioE protein in the total protein of the whole bacteria is 19.11%. By calculation, the addition amount of the VioA protein lysate with an initial concentration of 14.1 μg / μL is 8.5 μL, the addition amount of the VioB protein lysate with an initial concentration of 11.1 μg / μL is 12.8 μL, the addition amount of the VioC protein lysate with an initial concentration of 15.1 μg / μL is 5.4 μL, the addition amount of the VioD protein lysate with an initial concentration of 10.7 μg / μL is 8.3 μL, and the addition amount of the VioE protein lysate with an initial concentration of 11.2 μg / μL is 9.3 μL.

[0086] (2) Energy and substrate components (both refer to the final concentrations in the cell-free synthesis system): containing magnesium glutamate with a final concentration of 8 mM (Aladdin Biochemical Technology Co., Ltd., Shanghai); potassium glutamate with a final concentration of 160 mM (Aladdin Biochemical Technology Co., Ltd., Shanghai); NADPH with a final concentration of 1 mM (Sinopharm Chemical Reagent Co., Ltd.); ATP with a final concentration of 1 mM (Solarbio Science & Technology Co., Ltd.); CoA with a final concentration of 1 mM (Bio-Engineering (Shanghai) Co., Ltd.); L-Trp (substrate) with a final concentration of 4 mM (Aladdin Biochemical Technology Co., Ltd., Shanghai). For the remaining insufficient volume, it was made up with 100 mM Tris-HCl (pH = 8) buffer to make the total volume of the violacein cell-free synthesis system 200 μL. After preparation on ice, it was vortexed and mixed evenly and placed on ice.

[0087] (3) Mix the above-prepared 200 μL violacein cell-free synthesis system in a 1.5 mL centrifuge tube and react in the dark at 30 °C. Measure the yield of violacein at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 18 h, 24 h, 30 h, and 36 h respectively. As Figure 5 shown, it was found that the synthesis rate of violacein in the system increased significantly within 12 h, and the synthesis rate gradually leveled off after 24 h, with a final yield of 5.32 mg / L.

[0088] Example 3 Optimization of the violacein cell-free synthesis system in Example 2

[0089] Based on the research in Example 2, in this example, the above system was optimized from aspects such as the total amount of five proteins in the crude extract of the reaction, substrate concentration, reaction temperature, enzyme ratio, cofactors, and metal ions.

[0090] 1. Effect of the total amount of five proteins in the protein mixture on violacein synthesis

[0091] The total volume of the cell-free synthesis system of violacein is 200 μL, containing magnesium glutamate at a final concentration of 8 mM; potassium glutamate at a final concentration of 160 mM; NADPH at a final concentration of 1 mM; ATP at a final concentration of 1 mM; CoA at a final concentration of 1 mM; 100 mM (pH = 8) Tris-HCl; L-Trp (substrate) at a final concentration of 4 mM. The mass ratio of the initial five enzymes is controlled as VioA:VioB:VioC:VioD:VioE = 1:1:1:1:1. According to the Image J software, calculate the mass percentage ratio of the five enzymes VioA, VioB, VioC, VioD, and VioE in the total protein of the whole bacteria in each protein expression lysate. The proportion of VioA protein is 16.71%, the proportion of VioB protein is 14.11%, the proportion of VioC protein is 24.56%, the proportion of VioD protein is 22.40%, and the proportion of VioE protein is 19.11%. Select 315 μg, 400 μg, 450 μg, and 500 μg as the exploration range of the total amount of the five proteins in the crude extract of the system. According to the total amount of the above protein crude extract, the mass percentage of the five proteins, and the measured protein concentration of each protein lysate, calculate the added volume of each protein lysate. The specific data are shown in Table 4. The volume of each reaction system less than 200 μL is made up with 100 mM Tris-HCl (pH = 8) buffer. React at 30 °C for 24 h to compare the differences in the synthesis efficiency of violacein among the total amounts of the five proteins. As Figure 6 shown, as the total amount of the five proteins in the crude extract of the system increases, the yield of violacein in the system increases. When it is 400 μg, the highest yield of violacein is 48.61 mg / L. However, when the total amount of the five proteins in the crude extract of the system continues to increase, such as 450 or 500 μg, the yield of violacein decreases instead. As the cell-free system reaction proceeds, the initially added proteins can directly catalyze the rate-limiting step, reduce the reaction activation energy, and significantly increase the reaction rate. When the protein content in the system is too high, on the one hand, the reaction products or by-products cannot be dialyzed out of the reaction system, breaking the dynamic balance of the reaction system and reducing the synthesis of violacein; on the other hand, the substrates and energy substances in the system can only meet the reactions of a certain amount of proteins. Excessive protein addition makes the system insufficient to provide the reaction for progress, thus affecting the synthesis efficiency of the system.

[0092] Table 4 Added volume of each protein expression lysate

[0093]

[0094]

[0095] 2. Effect of substrate concentration on violacein synthesis

[0096] The total volume of the cell-free synthesis system for violacein is 200 μL, containing 8 mM magnesium glutamate, 160 mM potassium glutamate, 1 mM NADPH, 1 mM ATP, 1 mM CoA, and 100 mM (pH = 8) Tris-HCl. After preparation on ice, vortex thoroughly and place on ice. According to the analysis and calculation by Image J software, the mass percentage of five enzymes, namely VioA, VioB, VioC, VioD, and VioE, in the crude enzyme extract to the total protein of the whole bacteria was determined. The proportion of VioA protein was 16.71%, VioB protein was 14.11%, VioC protein was 24.56%, VioD protein was 22.40%, and VioE protein was 19.11%. The total amount of the five proteins in the system crude extract was 400 μg, and the enzyme mass ratio was VioA:VioB:VioC:VioD:VioE = 1:1:1:1:1. 34 μL of VioA protein lysate with a concentration of 14.1 μg / μL was added, 51.1 μL of VioB protein lysate with a concentration of 11.1 μg / μL was added, 21.6 μL of VioC protein lysate with a concentration of 15.1 μg / μL was added, 33.4 μL of VioD protein lysate with a concentration of 10.7 μg / μL was added, and 37.4 μL of VioE protein lysate with a concentration of 11.2 μg / μL was added. The concentrations of the substrate L-Trp were 4 mM, 8 mM, 12 mM, 16 mM, and 20 mM respectively. For each reaction system with a volume less than 200 μL, it was made up to 200 μL with 100 mM Tris-HCl (pH = 8) buffer. The reaction was carried out at 30 °C for 24 h.

[0097] As Figure 7 shown, when the substrate concentration was 16 mM, the highest yield of violacein was 87.53 mg / L. However, when the total substrate concentration was 12 mM or 20 mM, there was no significant difference in the yield of violacein, probably because the L-tryptophan substrate does not need to consider the toxicity and restrictive effects on cells in the cell-free system. When the substrate concentrations were 4 mM and 8 mM, the energy factors or enzyme activities provided in the system were quickly consumed for the synthesis of violacein, and the yield of violacein gradually increased. When the substrate concentration was greater than 16 mM, there were no energy factors and active proteins available to utilize the substrate in the total system. Therefore, the substrate concentration of 16 mM had reached the limit of full utilization.

[0098] 3. Effect of reaction temperature on violacein synthesis

[0099] The most crucial component in the cell-free reaction is the key enzyme in the synthesis pathway, and temperature has a significant impact on enzyme activity. Considering that the optimal catalytic temperatures of the 5 enzymes may be different, in order to further increase the production of violacein. Therefore, an attempt was made to optimize the reaction temperature. The reaction system: 8 mM magnesium glutamate; 160 mM potassium glutamate; 1 mM NADPH; 1 mM ATP; 1 mM CoA; 100 mM (pH = 8) Tris-HCl. After preparation on ice, vortex thoroughly and place on ice. According to the analysis and calculation by Image J software, the mass percentage ratios of the 5 enzymes, namely VioA, VioB, VioC, VioD, and VioE, in the crude enzyme extract to the total protein of the whole bacterium were as follows: the proportion of VioA protein was 16.71%, the proportion of VioB protein was 14.11%, the proportion of VioC protein was 24.56%, the proportion of VioD protein was 22.40%, and the proportion of VioE protein was 19.11%. The total amount of the five proteins in the system crude extract was 400 μg, and the enzyme mass ratio was VioA:VioB:VioC:VioD:VioE = 1:1:1:1:1. 34 μL of the VioA protein lysate with a concentration of 14.1 μg / μL was added, 51.1 μL of the VioB protein lysate with a concentration of 11.1 μg / μL was added, 21.6 μL of the VioC protein lysate with a concentration of 15.1 μg / μL was added, 33.4 μL of the VioD protein lysate with a concentration of 10.7 μg / μL was added, and 37.4 μL of the VioE protein lysate with a concentration of 11.2 μg / μL was added. The concentration of the substrate L-Trp was 16 mM. When the volume of the reaction system was less than 200 μL, it was made up with 100 mM Tris-HCl (pH = 8) buffer. Five reaction temperatures of 18, 25, 28, 30, and 37 °C were set respectively, and the reaction was carried out for 24 h. As Figure 8 shown, when the temperature is relatively low, due to the reduced thermal motion of the enzyme itself, the rate of the enzymatic reaction will decrease, reducing the enzyme activity. Therefore, as the temperature increases, the production of violacein slowly increases, and the production of violacein is the highest at 25 °C, with the highest production being 81.63 mg / L. When the reaction temperature exceeds 30 °C, the production of violacein decreases as the temperature increases. Especially when the reaction temperature rises to 37 °C, it may be due to the inhibition of enzyme activity by high temperature. Enzymes will denature and even inactivate under high-temperature conditions, which is because high temperature destroys the spatial structure of the enzyme, leading to permanent inactivation of the enzyme. At the same time, since violacein is usually a product of extreme psychrophiles, it is not conducive to its synthesis at higher temperatures, resulting in a significant reduction in the production of violacein in the system.

[0100] 4. Effect of enzyme ratio on the synthesis of violacein

[0101] To study the effect of the mass concentration ratio of five enzymes, namely VioA, VioB, VioC, VioD, and VioE, on the synthesis of violacein, 28 groups of mass concentration ratios of the five enzymes were selected, which were: Group 1: 1:1:1:1:1; Group 2: 2:1:1:1:1; Group 3: 1:2:1:1:1; Group 4: 1:1:2:1:1; Group 5: 1:1:1:2:1; Group 6: 1:1:1:1:2; Group 7: 2:1:2:2:2; Group 8: 2:2:1:2:2; Group 9: 2:2:2:1:2; Group 10: 2:2:2:2:1; Group 11: 1:1:1:2:2; Group 12: 1:1:2:2:1; Group 13: 1:2:2:1:1; Group 14: 2:2:1:1:1; Group 15: 1:1:2:2:2; Group 16: 1:2:2:2:2; Group 17: 2:1:2:1:1; Group 18: 2:1:1:2:1; Group 19: 2:1:1:1:2; Group 20: 1:2:1:2:1; Group 21: 1:1:2:1:2; Group 22: 1:2:1:1:2; Group 23: 2:1:1:2:1; Group 24: 2:2:2:1:1; Group 25: 2:2:1:2:1; Group 26: 1:2:2:2:1; Group 27: 2:1:1:2:2; Group 28: 2:1:2:1:2. The total volume of the cell-free synthesis system for violacein was 200 μL, containing 8 mM magnesium glutamate; 160 mM potassium glutamate; 1 mM NADPH; 1 mM ATP; 1 mM CoA; 100 mM (pH = 8) Tris-HCl; and the substrate L-Trp concentration was 16 mM. According to the analysis and calculation of Image J software, the mass percentage ratio of the five enzymes, VioA, VioB, VioC, VioD, and VioE, in the crude enzyme extract to the total protein of the whole bacteria was as follows: the proportion of VioA protein was 16.71%, the proportion of VioB protein was 14.11%, the proportion of VioC protein was 24.56%, the proportion of VioD protein was 22.40%, and the proportion of VioE protein was 19.11%. The total amount of the five proteins in the system crude extract was 400 μg, the concentration of the VioA protein lysate was 14.1 μg / μL, the concentration of the VioB protein lysate was 11.1 μg / μL, the concentration of the VioC protein lysate was 15.1 μg / μL, the concentration of the VioD protein lysate was 10.7 μg / μL, and the concentration of the VioE protein lysate was 11.2 μg / μL. When the volume of each reaction system was less than 200 μL, it was made up with 100 mM Tris-HCl (pH = 8) buffer. After preparation on ice, it was vortexed and mixed evenly, placed on ice, and reacted at 25 °C for 24 h. By exploring the effect of separately increasing the total amount of a certain key enzyme in the system on the synthesis of violacein. It can be seen from the violacein yields of each group of enzyme ratios that increasing the total amount of a certain key enzyme can increase the yield of violacein. Such as Figure 9As shown in the figure, the enzyme mass ratio of VioA:VioB:VioC:VioD:VioE=1:2:1:1:1 has the highest yield of purple bacteriocin, which can reach 192.78mg / L. We speculate that this may be because VioB may be the rate-limiting enzyme in the entire synthesis process of purple bacteriocin, so it has the greatest impact on the entire synthesis pathway of purple bacteriocin. VioC enzyme is second, probably because in the entire synthesis pathway of purple bacteriocin, VioC enzyme catalyzes both deoxychromoviridans and purple bacteriocin precursors. Once it is missing in the body, only purple bacteriocin precursors and deoxychromoviridans, chromoviridans, and oxychromoviridans can be produced. Therefore, increasing the proportion of VioC enzyme can avoid insufficient power for the synthesis of the target product due to the diversion of by-products. However, the increase in the proportion of VioA enzyme leads to a slight decrease in the yield of purple bacteriocin. We speculate that this may be caused by insufficient supply of precursors. The yield of purple bacteriocin is increased by increasing the proportion of VioD and VioE enzymes.

[0102] 5. Effects of cofactors and metal ions on the synthesis of violacein

[0103] The total volume of the cell-free synthesis system of purple bacteriocin is 200μL: 8mM magnesium glutamate; 160mM potassium glutamate; 100mM (pH=8) Tris-HCl; the concentration of substrate L-Trp is 16mM. After preparation on ice, vortex to mix and place on ice. According to Image J software analysis, the mass percentage ratio of the five enzymes VioA, VioB, VioC, VioD and VioE in the crude enzyme extract to the total protein of the whole bacteria was calculated. The total amount of the five proteins in the cell-free synthesis system is 400μg, and the enzyme mass ratio VioA:VioB:VioC:VioD:VioE=1:2:1:1:1. The optimization condition group number of the cofactor and metal salt ion is according to Figure 10 Ten groups were set up. By adding some cofactors, salt ions (such as glutamate) and metal ions to the cell-free system, the enzyme activity can be activated and the reaction can be promoted. Figure 10As shown in the figure, first, cofactors NADPH, CoA, and ATP were investigated. Group 1 was the original system, containing 1 mM NADPH; 1 mM ATP; 1 mM CoA. It was found that the yield of prodigiosin in the original system was the highest, reaching 189.22 mg / L, indicating that additional cofactors ATP, CoA, and NADPH were required for the cell-free metabolic engineering synthesis system of prodigiosin. By comparing the yield differences of prodigiosin among groups, it was found that when CoA, ATP, and NADPH were sequentially removed in groups 2-4, the absence of ATP and NADPH had a more obvious impact on the yield of prodigiosin. In particular, the absence of NADPH in group 4 resulted in the prodigiosin yield reaching only 128.19 mg / L, which was 67.75% of that in group 1. When ATP and NADPH were added to group 2 (CoA was absent), it was found that when CoA was removed in group 2, the yield of prodigiosin decreased, which was 94.07% of the prodigiosin yield in group 1. Subsequently, the addition of glutamate (magnesium glutamate, potassium glutamate) was further reduced (group 9), which further reduced the synthesis of prodigiosin. In group 10, the concentration of Mg 2+ was further reduced, and it was found that the yield of prodigiosin decreased. Based on this, it was speculated that the metal ion content in the crude extract was insufficient to maintain the activity of key enzymes, and additional supplementation was also required in the reaction for constructing the cell-free system of prodigiosin.

[0104] In this example, through the optimization of the system in Example 3, a prodigiosin yield of 189.22 mg / L was finally obtained, which was 35.57 times higher than the yield before optimization.

Claims

1. A method for efficiently synthesizing violacein using a cell-free system, characterized in that, It includes the following steps: 1) Recombinant plasmids containing gene fragments were constructed respectively, and after transforming Escherichia coli respectively, five kinds of recombinant Escherichia coli were obtained; vioA, vioB, vioC, vioD, vioE ​ 2) Induce the expression of the 5 recombinant Escherichia coli respectively and perform ultrasonic disruption to obtain cell lysates of VioA, VioB, VioC, VioD and VioE proteins; 3) Mix the cell lysates of VioA, VioB, VioC, VioD and VioE proteins to obtain a crude extract mixture of VioA, VioB, VioC, VioD and VioE proteins, add it to the substrate reaction mixture to obtain a cell-free reaction system, and then the reaction is carried out to obtain the product.

2. The method for efficiently synthesizing violacein using a cell-free system according to claim 1, wherein as described in Step 1) vioA, vioB, vioC, vioD, vioE All genes are derived from Janthinobacterium sp. B9-8 strain 3. A method for efficiently synthesizing violacein using a cell-free system according to claim 1, characterized in that, The recombinant plasmids described in step 1) are pRSF-VioA, pRSF-VioB, pRSF-VioC, pRSF-VioD, pRSF-VioE.

4. A method for efficiently synthesizing violacein using a cell-free system according to claim 1, characterized in that, The temperature of the induced expression described in step 2) is 18 - 30 °C, and the time is 4 - 6 h.

5. A method for efficiently synthesizing violacein using a cell-free system according to claim 1, characterized in that, In step 3), the volume ratios of the cell lysates of VioA, VioB, VioC, VioD and VioE proteins in the cell-free reaction system are 1% - 50% respectively, and the mass percentages of the five proteins VioA, VioB, VioC, VioD and VioE in the total bacterial protein are 10% - 90% respectively.

6. A method for efficiently synthesizing violacein using a cell-free system according to claim 1, characterized in that, The substrate reaction mixture described in step 3) includes a substrate and cofactors. The substrate is L-Trp, and the concentration is 4 - 20 mM. Preferably, the cofactors are CoA, NADPH and ATP, and the concentrations are all 0.5 - 1 mM.

7. A method for efficiently synthesizing violacein using a cell-free system according to claim 1, characterized in that, In step 3), the mass ratio of VioA:VioB:VioC:VioD:VioE proteins is (1 - 2):(1 - 2):(1 - 2):(1 - 2):(1 - 2).

8. A method for efficiently synthesizing violacein using a cell-free system according to claim 1, characterized in that, The reaction temperature for synthesizing violacein in step 3) is 18 - 37 °C, and the time is 2 - 24 h.

9. A method for efficiently synthesizing violacein using a cell-free system according to claim 6, characterized in that, In step 3), the concentration of the substrate is 16 mM; the concentrations of the cofactors are all 1 mM; the mass ratio of VioA, VioB, VioC, VioD, VioE in the cell lysate is 1:2:1:1:

1.

10. A method for efficiently synthesizing violacein using a cell-free system according to claim 6, characterized in that, In step 3), the reaction temperature for synthesizing violacein is 25 °C, and the reaction time is 24 h.

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