An engineered bacterium for producing lycopene and a system construction method and application thereof

Abstract

The present invention belongs to the field of biosynthesis technology, and specifically relates to an engineering bacterium for producing lycopene and a system construction method and application thereof. The engineering bacterium for producing lycopene is Escherichia coli, carrying two plasmids, namely, pCDF-Duet-MKI4 plasmid and pET28a-crtBI plasmid; the pCDF-Duet plasmid is used to express the MKI4 gene, and a five-carbon compound substrate is converted into a diterpene precursor geranylgeranyl pyrophosphate (GGPP); the pET28a plasmid is used to express the crtB and crtI genes, and the GGPP is converted into phytoene, and then into lycopene. The present invention constructs a biosynthesis system of lycopene in Escherichia coli, which can efficiently convert a five-carbon compound substrate into lycopene, and the shake flask fermentation yield can be as high as 900 mg / L.

Description

Technical Field

[0001] The invention belongs to the technical field of biosynthesis, and specifically relates to an engineering bacterium for producing lycopene and a system construction method and application thereof. Background Art

[0002] Lycopene is a carotenoid found in plant foods and is also a red pigment. It is a dark red needle-shaped crystal that is soluble in chloroform, benzene and oils but not in water. It is unstable to light and oxygen and turns brown when it comes into contact with iron. It does not have the physiological activity of vitamin A, but has a strong antioxidant function. It is found in high levels in mature red plant fruits, especially in tomatoes, carrots, watermelons, papayas and guavas. It can be used as a pigment in food processing and is also often used as a raw material for antioxidant health foods.

[0003] In industrial production, tomato fruits and waste materials generated when processing tomato products, such as tomato skins, seeds and juice, are usually used to extract lycopene, but these sources cannot meet the industry's demand for lycopene. More sustainable and efficient methods of producing lycopene need further research.

[0004] Lycopene is a chain-like unsaturated olefin compound with 11 conjugated double bonds and 2 non-conjugated double bonds, so its stability is very poor, and it is easy to undergo cis-trans isomerization and oxidative degradation. Therefore, the product obtained by chemical synthesis has a complex structure and is not easy to purify to obtain pure lycopene. Synthetic biology is the construction of cell factories or in vitro enzyme catalysis based on the analysis of biosynthetic pathways, and the green and efficient synthesis of target lead compounds avoids the danger, toxicity and inefficiency of chemical synthesis, and the reaction conditions are relatively mild, making it easier to obtain pure monomer compounds. Through the analysis of the biosynthesis of related compounds and the construction of cell factories, the target compounds can be produced continuously and efficiently, solving the problem of the source of lycopene. Chinese invention patent CN108588106B discloses a lycopene high-yield strain, its preparation method and application, and provides a lycopene high-yield strain of Escherichia coli. The construction process is relatively complicated, and after optimization, the shake flask yield is 393.2 mg / L, and further research is needed to produce lycopene in a simpler, higher-yield, and lower-cost technology. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides an engineering bacterium for producing lycopene and a method for constructing a system thereof and applications thereof.

[0006] The specific technical solutions are as follows:

[0007] The first object of the present invention is to provide an engineered bacterium for producing lycopene, wherein the engineered bacterium for producing lycopene is Escherichia coli, carrying two plasmids, namely, pCDF-Duet-MKI4 plasmid and pET28a-crtBI plasmid; the engineered bacterium for producing lycopene converts a five-carbon compound substrate into a diterpene precursor GGPP, and then converts GGPP into phytoene, and then into lycopene;

[0008] Specifically, the pCDF-Duet plasmid was used to express the MKI4 gene, which converted the five-carbon compound substrate into the diterpene precursor geranylgeranyl pyrophosphate (GGPP), and the pET28a plasmid was used to express the crtB and crtI genes, which converted GGPP into phytoene and then into lycopene.

[0009] Furthermore, the expression process of the engineering bacteria producing lycopene is:

[0010] (1) Overexpression of the MKI4 gene specifically recognizes five-carbon compound substrates with a hydroxyl structure and converts the five-carbon compound substrates into the diterpene precursor GGPP;

[0011] (2) Overexpression of the crtB gene converts GGPP into phytoene;

[0012] (3) Overexpression of the crtI gene converts phytoene into lycopene.

[0013] Furthermore, the Escherichia coli is an Escherichia coli B / r type strain.

[0014] Furthermore, the Escherichia coli is BL21Gold (DE3).

[0015] Furthermore, the five-carbon compound substrate has a hydroxyl structure.

[0016] Furthermore, the five-carbon compound substrate is a five-carbon compound of 3-methyl-2-butene-1-ol or 3-methyl-3-butene-1-ol.

[0017] Furthermore, the chemical structural formula of lycopene is as follows:

[0018] .

[0019] The second object of the present invention is to provide a method for constructing a system of engineered bacteria for producing lycopene as described above, comprising the following steps:

[0020] The genes ThiM (NCBI accession NO.: WP 001195564.1), IPK (NCBI accession NO.: WP 001192822.1), Idi (NCBI accession NO.: WP 001192822.1), and Bnd3 (NCBI accession NO.: MCG8971285.1) obtained by gene synthesis were integrated into a plasmid pCDF-Duet to obtain the pCDF-Duet-MKI4 plasmid; the plasmid was transformed into the Escherichia coli BL21Gold (DE3) strain, and the recombinant Escherichia coli transformed with the plasmid could convert the five-carbon compound substrate into GGPP;

[0021] The codon-optimized genes crtB (UniProt accession NO.: Q9ACU1) and crtI (UniProt accession NO.: P21685) obtained by gene synthesis were integrated into a plasmid pET28a to obtain the pET28a-crtBI plasmid;

[0022] The pET28a-crtBI plasmid and the pCDF-Duet-MKI4 plasmid are co-transformed into the Escherichia coli BL21Gold (DE3) strain to obtain an engineered bacterium that produces lycopene.

[0023] Further, the operation method of the co-transformation is as follows:

[0024] S1. Take the BL21Gold (DE3) E. coli competent cells stored in a -80 ºC refrigerator and place them on ice for about 2 minutes to thaw;

[0025] S2. Place a PCR tube on ice, add 70 μL of sterilized ultrapure water, 20 μL of 5× KCM solution, 5 μL of pCDF-Duet-MKI4 plasmid and 5 μL of pET28a-crtBI plasmid, and cool the PCR tube in an ice bath for 1 min;

[0026] S3. Use a 200 μL pipette to mix the solution in the PCR tube, add it to 100 μL of thawed BL21Gold (DE3) E. coli competent cells, and place on ice for 30 min;

[0027] S4. Insert the centrifuge tube into the float and place it in a 42°C water bath for 90 seconds. After 90 seconds, immediately insert the centrifuge tube into ice and place it in an ice bath for 2 minutes.

[0028] S5. Add 600 μL of SOC solution to a centrifuge tube in a clean bench, place in a 250 mL shake flask, and shake at 37 °C and 250 rpm for 1 h; then place the centrifuge tube in a centrifuge and centrifuge at 7500 rpm for 2 min;

[0029] S6. After centrifugation, pour out the liquid in the centrifuge tube in the clean bench. There will be some bacterial liquid left in the centrifuge tube. Use the remaining bacterial liquid at the bottom of the tube to resuspend the precipitate; use a 200 μL pipette to aspirate the resuspended bacterial liquid, add it to the LB plate with kanamycin and streptomycin sulfate resistance, and spread it. After the bacterial liquid is dried, cover it with a lid, seal it with a sealing film, and invert it in a 37 ℃ constant temperature incubator for overnight culture;

[0030] S7. Pick a single colony from the LB plate and add it to the LB medium containing kanamycin and streptomycin sulfate, and culture it in a shaker at 250 rpm for about 8 h to obtain a culture solution;

[0031] S8. In a clean bench, pipette 600 μL of the above culture solution into a 1.5 mL centrifuge tube, then add 600 μL of sterilized 50% glycerol, mix the liquid in the centrifuge tube, freeze it in liquid nitrogen, and then store it in a -80 ºC refrigerator.

[0032] The above-mentioned result is an engineered strain capable of producing lycopene, which is named XU-BG-0001.

[0033] The engineered bacteria for producing lycopene can be reproduced according to the above method and have stable expression.

[0034] The third object of the present invention is to provide a use of the engineered bacteria for producing lycopene as described above in producing lycopene.

[0035] Furthermore, the method for producing lycopene comprises the following steps: adding LB liquid culture medium, kanamycin and streptomycin sulfate into a shake flask, adding the engineered bacteria for producing lycopene as described above into the shake flask and culturing until the OD value reaches 0. 600 When the value reaches 1.5-5, cool to room temperature; at 28 °C, add isopropyl- β -D-thiogalactoside (Isopropyl- β -D-thiogalactopyranoside, IPTG) and 1-10 mM of a five-carbon compound substrate, cultured for 16-72 h, to obtain lycopene.

[0036] Further, OD 600 When the value reaches 2, the induced yield is higher.

[0037] Furthermore, the optimal IPTG concentration was 0.5 mM.

[0038] Furthermore, the optimal concentration of the five-carbon compound substrate was 2 mM.

[0039] Furthermore, the optimal culture time was 18 h.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention constructs a lycopene biosynthesis system in Escherichia coli. Using the strain constructed by the present invention and according to the method of the present invention, continuous and efficient production of lycopene can be achieved, and the shake flask yield can be as high as 900 mg / L. DETAILED DESCRIPTION

[0042] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0043] Unless otherwise specified, the materials and reagents used in the examples can be obtained from commercial sources. The competent cells used were purchased from Beijing Tsingke Biotechnology Co., Ltd., and the plasmids and gene synthesis used were purchased from Suzhou Genewise Biotechnology Co., Ltd.

[0044] The chemical structural formula of lycopene referred to in the embodiment is as follows:

[0045] .

[0046] Example 1

[0047] A method for constructing a system of engineered bacteria for producing lycopene comprises the following steps:

[0048] The genes ThiM (NCBI accession NO.: WP 001195564.1), IPK (NCBI accession NO.: WP 001192822.1), Idi (NCBI accession NO.: WP 001192822.1), and Bnd3 (NCBI accession NO.: MCG8971285.1) obtained by gene synthesis were integrated into a plasmid pCDF-Duet to obtain a plasmid pCDF-Duet-MKI4; the plasmid was transformed into the Escherichia coli BL21Gold (DE3) strain, and the recombinant Escherichia coli transformed with the plasmid could convert 3-methyl-2-butene-1-ol or 3-methyl-3-butene-1-ol into GGPP;

[0049] The synthesized codon-optimized genes crtB (UniProt accession NO.: Q9ACU1) and crtI (UniProt accession NO.: P21685) were integrated into a plasmid pET28a to obtain the pET28a-crtBI plasmid;

[0050] The pET28a-crtBI plasmid and the pCDF-Duet-MKI4 plasmid are co-transformed into the Escherichia coli BL21Gold (DE3) strain to obtain lycopene engineering bacteria.

[0051] The operation method of the above-mentioned joint transformation is as follows:

[0052] S1. Take the BL21Gold (DE3) E. coli competent cells stored in a -80 ºC refrigerator and place them on ice for about 2 minutes to thaw;

[0053] S2. Place a PCR tube on ice, add 70 μL of sterilized ultrapure water, 20 μL of 5× KCM solution, 5 μL of pCDF-Duet-MKI4 plasmid and 5 μL of pET28a-crtBI plasmid, and cool the PCR tube in an ice bath for 1 min;

[0054] S3. Use a 200 μL pipette to mix the solution in the PCR tube, add it to 100 μL of thawed BL21Gold (DE3) E. coli competent cells, and place on ice for 30 min;

[0055] S4. Insert the centrifuge tube into the float and place it in a 42°C water bath for 90 seconds. After 90 seconds, immediately insert the centrifuge tube into ice and place it in an ice bath for 2 minutes.

[0056] S5. Add 600 μL of SOC solution to a centrifuge tube in a clean bench, place in a 250 mL shake flask, and shake at 37 °C and 250 rpm for 1 h; then place the centrifuge tube in a centrifuge and centrifuge at 7500 rpm for 2 min;

[0057] S6. After centrifugation, pour out the liquid in the centrifuge tube in the clean bench (do not shake it, as some liquid will remain in the centrifuge tube), and resuspend the precipitate with the remaining bacterial solution at the bottom of the tube; use a 200 μL pipette to aspirate the resuspended bacterial solution, add it to the LB plate with kanamycin (working concentration 50 mg / L) and streptomycin sulfate (working concentration 50 mg / L) resistance, and spread it. After the bacterial solution is dried, cover it with a lid, seal it with a sealing film, and invert it in a 37 ℃ constant temperature incubator for overnight culture;

[0058] S7. Pick a single colony from the LB plate and add it to LB medium added with kanamycin (working concentration 50 mg / L) and streptomycin sulfate (working concentration 50 mg / L), and culture it in a shaker at 250 rpm for about 8 h to obtain a culture solution;

[0059] S8. In a clean bench, pipette 600 μL of the above culture solution into a 1.5 mL centrifuge tube, then add 600 μL of sterilized 50% glycerol, mix the liquid in the centrifuge tube, freeze it in liquid nitrogen, and then store it in a -80 ºC refrigerator.

[0060] The strain obtained above is an engineered strain capable of producing lycopene, and is named XU-BG-0001.

[0061] The engineered bacteria for producing lycopene can be reproduced according to the above method and have stable expression.

[0062] Example 2

[0063] A method for producing lycopene using the above-mentioned engineered strain XU-BG-0001 comprises the following steps:

[0064] (1) Add 5 mL of LB liquid medium, kanamycin (working concentration 50 mg / L) and streptomycin sulfate (working concentration 50 mg / L) to the shaking tube, pick a single clone from the plate of XU-BG-0001 engineered strain, or pick a frozen strain, add it to the shaking tube, and culture overnight at 37°C and 250 rpm;

[0065] (2) Add 100 mL of LB liquid medium, kanamycin (working concentration 50 mg / L) and streptomycin sulfate (working concentration 50 mg / L) to a 250 mL shake flask. Take 1 mL of bacterial solution from a shake tube and add it to the 250 mL shake flask. Incubate at 37 °C and 250 rpm for 8 h.

[0066] (3) Add 1 L of LB liquid medium, kanamycin (working concentration 50 mg / L) and streptomycin sulfate (working concentration 50 mg / L) to each of two 2 L shaking flasks. Take 10 mL of the culture solution in (2) and add it to each of the 2 L shaking flasks. Cultivate at 37 °C and 220 rpm until the OD reaches 0. 600 When the value reaches 2, take out the shake bottle and cool it down to room temperature;

[0067] (4) Adjust the temperature of the shaker to 28 °C. When the temperature drops to the set temperature, add 0.5 mM IPTG and 2 mM 3-methyl-2-butene-1-ol to one of the two shake flasks; add 0.5 mM IPTG and 2 mM 3-methyl-3-butene-1-ol to the other one. Both are cultured at 28 °C and 220 rpm for 18 h.

[0068] (5) Take the bacterial solution from (4) and add it to two 1 L centrifuge buckets respectively. Centrifuge at 4000 rpm and 4 °C for 10 min. After centrifugation, discard the bacterial solution and retain the bacterial cells.

[0069] (6) The cells in (5) were extracted with acetone in an ultrasonic cleaner for 10 min, and then centrifuged at 4000 rpm and 4°C for 5 min. The supernatant was added to a rotary evaporator. The cells were extracted three times, and the extracts were combined and concentrated under reduced pressure.

[0070] (7) Fill the glass column with 200-300 mesh silica gel for about 30 cm. Mix the sample with an equal weight of 100-200 mesh silica gel powder. After loading the sample, rinse it with pure petroleum ether. Find the desired point on the spot plate (the Rf value of pure petroleum ether is 0.95). After combining the samples, perform NMR identification.

[0071] Example 3

[0072] A method for producing lycopene using the engineered strain XU-BG-0001 is different from that in Example 2 in that, in step (3), the culture is performed until OD 600 The value reaches 5; in step (4), in two shaking flasks, add IPTG with a working concentration of 1 mM and 3-methyl-2-butene-1-ol with a working concentration of 1 mM to one of them; add IPTG with a working concentration of 1 mM and 3-methyl-3-butene-1-ol with a working concentration of 1 mM to the other one, and culture for 72 h;

[0073] The remaining technical features refer to Example 2.

[0074] Example 4

[0075] A method for producing lycopene using the engineered strain XU-BG-0001 is different from that in Example 2 in that, in step (3), the culture is performed until OD 600 The value reaches 1.5; in step (4), in two shaking flasks, add IPTG with a working concentration of 0.1 mM and 3-methyl-2-butene-1-ol with a working concentration of 1 mM to one of them; add IPTG with a working concentration of 0.1 mM and 3-methyl-3-butene-1-ol with a working concentration of 1 mM to the other one, and culture for 16 h;

[0076] The remaining technical features refer to Example 2.

[0077] Lycopene obtained in Example 2-4 1 H NMR data and 13 C NMR data are as follows:

[0078] 1 H NMR (CDCl3, 600 MHz) d 1.61 (6H, s, CH3), 1.69 (6H, s, CH3), 1.82 (6H, s, CH3), 1.97 (12H, s, CH3), 2.12 (8H, overlap, CH2), 4.17 (1H, d, J = 6.9Hz, CH2), 5.11 (2H, m, vinyl H) 5.95 (2H, d, J = 11.0 Hz, vinyl H), 6.18 (2H,d, J = 11.5 Hz, vinyl H), 6.25 (4H, overlap, vinyl H), 6.35 (2H, d, J = 15.1 Hz, vinyl H), 6.49 (2H, dd, J = 15.1, 11.0 Hz, vinyl H), 6.63 (4H, overlap, vinylH);

[0079] 13 C NMR (CDCl3, 150 MHz) d 139.7, 139.7, 137.5, 137.5, 136.7, 136.7,136.3, 136.3, 135.6, 135.6, 132.8, 132.8, 131.9, 131.9, 131.7, 131.7, 130.2,130.2, 125.9, 125.9, 125.3, 125.3, 125.0, 125.0, 124.1, 124.1, 40.4, 40.4,26.8, 26.8, 25.9, 25.9, 17.9, 17.9, 17.1, 17.1, 13.1, 13.1, 13.0, 13.0.

[0080] After the products were confirmed by NMR identification, the masses of the compounds obtained in Examples 2-4 were weighed. In Example 2, when the feeding substrate was 3-methyl-2-butene-1-ol, the lycopene yield was 960.2 mg, and when the feeding substrate was 3-methyl-3-butene-1-ol, the lycopene yield was 920.3 mg; in Example 3, when the feeding substrate was 3-methyl-2-butene-1-ol, the lycopene yield was 610.7 mg, and when the feeding substrate was 3-methyl-3-butene-1-ol, the lycopene yield was 690.6 mg; in Example 4, when the feeding substrate was 3-methyl-2-butene-1-ol, the lycopene yield was 521.4 mg, and when the feeding substrate was 3-methyl-3-butene-1-ol, the lycopene yield was 519.5 mg; it can be seen that the ability of the engineered strain to produce lycopene can be as high as 900 mg / L.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An engineered bacterium for producing lycopene, characterized in that: The engineered bacteria for producing lycopene is Escherichia coli, which carries two plasmids, namely pCDF-Duet-MKI4 plasmid and pET28a-crtBI plasmid; the engineered bacteria for producing lycopene converts the five-carbon compound substrate into the diterpene precursor GGPP, then converts GGPP into phytoene, and then converts it into lycopene; The method for constructing a system of engineered bacteria for producing lycopene comprises the following steps: The ThiM, IPK, Idi, and Bnd3 genes obtained by gene synthesis were integrated into a plasmid pCDF-Duet to obtain the pCDF-Duet-MKI4 plasmid; The codon-optimized genes crtB and crtI obtained by gene synthesis were integrated into a plasmid pET28a to obtain a pET28a-crtBI plasmid; the protein encoded by crtB is UniProt accession NO.: Q9ACU1, and the protein encoded by crtI is UniProt accession NO.: P21685; The pET28a-crtBI plasmid and the pCDF-Duet-MKI4 plasmid were co-transformed into an Escherichia coli strain to obtain an engineered bacterium that produces lycopene; The Escherichia coli is BL21Gold (DE3).

2. The engineered bacteria for producing lycopene according to claim 1, characterized in that: The expression process of the engineering bacteria producing lycopene is as follows: (1) Overexpression of the MKI4 gene specifically recognizes five-carbon compound substrates with a hydroxyl structure and converts the five-carbon compound substrates into the diterpene precursor GGPP; (2) Overexpression of the crtB gene converts GGPP into phytoene; (3) Overexpression of the crtI gene converts phytoene into lycopene.

3. The engineered bacteria for producing lycopene according to claim 1, characterized in that: The five-carbon compound substrate has a hydroxyl structure.

4. The engineered bacteria for producing lycopene according to claim 3, characterized in that: The five-carbon compound substrate is a five-carbon compound of 3-methyl-2-butene-1-ol or 3-methyl-3-butene-1-ol.

5. A method for constructing a system of engineered bacteria for producing lycopene according to any one of claims 1 to 4, characterized in that: The steps include: The ThiM, IPK, Idi, and Bnd3 genes obtained by gene synthesis were integrated into a plasmid pCDF-Duet to obtain the pCDF-Duet-MKI4 plasmid; The codon-optimized genes crtB and crtI obtained by gene synthesis were integrated into a plasmid pET28a to obtain the pET28a-crtBI plasmid; The pET28a-crtBI plasmid and the pCDF-Duet-MKI4 plasmid were co-transformed into the Escherichia coli strain to obtain an engineered bacterium that produces lycopene.

6. Use of the engineered bacteria for producing lycopene as claimed in any one of claims 1 to 4 in producing lycopene.

7. The use according to claim 6, characterized in that: The method for producing lycopene comprises the following steps: adding LB liquid culture medium, kanamycin and streptomycin sulfate into a shaking flask, adding the engineered bacteria for producing lycopene according to any one of claims 1 to 4 into the shaking flask and culturing until OD 600 When the value reaches 1.5-5, cool to room temperature; at 28 °C, add isopropyl- β -D-thiogalactoside and 1-10 mM of a five-carbon compound substrate, cultured overnight, lycopene was obtained.