Recombinant rhodobacter sphaeroides strain for synthesizing lycopene by fixing CO2 through photocatalysis as well as construction method and application of recombinant rhodobacter sphaeroides strain

Abstract

The invention discloses a recombinant rhodobacter sphaeroides strain for synthesizing lycopene by photocatalytic immobilization of CO2 and a construction method and application thereof, and the construction method comprises the following steps: knocking out an endogenous crtC gene of rhodobacter sphaeroides by using a homologous recombination method, and replacing a crtI3 gene of rhodobacter sphaeroides by using a crtI4 gene from rhodobacter rubrum to obtain a recombinant strain RS-1; a crtB gene and a crtE gene are connected to a pYYDT vector, transferred into escherichia coli S17 and then introduced into a recombinant rhodobacter sphaeroides RS-1 through conjugational transfer, and a recombinant strain RS-2 is obtained. The recombinant rhodobacter sphaeroides constructed by the invention can be used for de novo synthesis of lycopene by using CO2 as a carbon source through photocatalysis. The method is simple to operate, and meanwhile, high-yield lycopene can be obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of biosynthesis and bioenergy, and more particularly to a recombinant Rhodobacterium sphaeroides strain for photocatalytically fixing CO2 to synthesize lycopene, as well as a construction method and use thereof. Background Art

[0002] Lycopene is a natural red carotenoid produced exclusively by plants and microorganisms. Currently, lycopene is primarily synthesized through chemical synthesis, plant extraction, and microbial fermentation. However, direct plant extraction yields a low yield, making it unsuitable for industrial production. Chemical synthesis also produces numerous byproducts and exhibits low biological activity. Microbial production of lycopene, on the other hand, offers a shorter production cycle, lower costs, and is environmentally friendly and sustainable. More importantly, carotenoids produced through microbial fermentation are natural products with high biological activity. Summary of the Invention

[0003] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide a recombinant Rhodobacter sphaeroides strain for photocatalytically fixing CO2 to synthesize lycopene.

[0004] The second object of the present invention is to provide a method for constructing a recombinant Rhodobacterium sphaeroides strain for photocatalytic fixation of CO2 to synthesize lycopene.

[0005] The third object of the present invention is to provide the use of a recombinant Rhodobacterium sphaeroides strain in producing lycopene by photocatalytic fixation of CO2 to synthesize lycopene.

[0006] The technical solution of the present invention is summarized as follows:

[0007] The method for constructing a recombinant Rhodobacterium sphaeroides strain for photocatalytic CO2 fixation and lycopene synthesis comprises the following steps:

[0008] 1. The upstream 200 bp homology arm and the downstream 200 bp homology arm of the crtC gene of Rhodobacter sphaeroides were ligated to the pK18 vector to construct plasmid pC1; the crtC gene is the abbreviation of the Rhodobacter sphaeroides cretoidin hydroxylase gene;

[0009] The nucleotide sequence of the pC1 plasmid is shown in SEQ ID NO.1.

[0010] 2. Transform plasmid pC1 into Escherichia coli S17 to obtain recombinant strain S-1;

[0011] The recombinant Escherichia coli strain S-1 was constructed using the above construction method.

[0012] 3. The recombinant strain S-1 was conjugated with the wild-type Rhodobacter sphaeroides to obtain the recombinant Rhodobacter sphaeroides strain RS-1 that synthesizes lycopene;

[0013] The lycopene-synthesizing recombinant Rhodobacter sphaeroides strain RS-1 constructed by the above construction method.

[0014] 4. The upstream homology arm of the crtI3 gene from Rhodobacter sphaeroides, the crtI4 gene, and the downstream homology arm of the crtI3 gene were ligated into the pK18 vector to construct plasmid pI4; the crtI3 gene is an abbreviation for the phytoene three-step dehydrogenase gene from Rhodobacter sphaeroides; and the crtI4 gene is an abbreviation for the phytoene four-step dehydrogenase gene crtI3 from Rhodospirillum rubrum;

[0015] The nucleotide sequence of the pI4 plasmid is shown in SEQ ID NO.2.

[0016] 5. Transform plasmid pI4 into Escherichia coli S17 to obtain recombinant strain S-2;

[0017] The recombinant Escherichia coli strain S-2 was constructed using the above construction method.

[0018] 6. The recombinant strain S-2 and the recombinant Rhodobacter sphaeroides RS-1 were conjugated and transferred to obtain the recombinant Rhodobacter sphaeroides RS-2 that synthesizes lycopene;

[0019] The lycopene-synthesizing recombinant Rhodobacter sphaeroides strain RS-2 was constructed using the above construction method.

[0020] 7. Ligate the crtB gene and crtE gene into plasmid pYYDT to construct plasmid pBE; the crtB gene is the abbreviation of the phytoene synthase gene; the crtE gene is the abbreviation of the geranyl diphosphate synthase gene;

[0021] The nucleotide sequence of the pBE plasmid is shown in SEQ ID NO.3.

[0022] 8. Transform the plasmid pBE into Escherichia coli S17 to obtain the recombinant strain S-3;

[0023] The recombinant Escherichia coli strain S-3 was constructed using the above construction method.

[0024] 9. The recombinant strain S-3 and the recombinant Rhodobacter sphaeroides strain RS-2 were conjugated and transferred to obtain the lycopene-producing recombinant Rhodobacter sphaeroides strain RS-3;

[0025] The recombinant Escherichia coli strain S-3 constructed by the above construction method.

[0026] The lycopene-synthesizing recombinant Rhodobacter sphaeroides strain RS-3 constructed by the above construction method;

[0027] The use of the above-mentioned recombinant Rhodobacter sphaeroides strain RS-3 for synthesizing lycopene.

[0028] Advantages of the present invention:

[0029] The recombinant Rhodobacter sphaeroides strains RS-2 and RS-3 constructed in this invention can, through metabolic engineering, synthesize lycopene de novo under light using CO2 as the sole carbon source. This method of synthesizing lycopene using microorganisms is simple to operate and improves the utilization rate of CO2. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Various other advantages and benefits will become apparent to those skilled in the art upon reading the detailed description of the preferred embodiment below. The accompanying drawings are for illustration purposes only and are not to be considered as limiting the present invention. The same reference symbols are used throughout the drawings to represent the same components. In the drawings:

[0031] Figure 1 This is the lycopene metabolic pathway of Rhodobacterium sphaeroides.

[0032] Figure 2 Schematic diagram of the construction of recombinant plasmid pC1.

[0033] Figure 3 Schematic diagram of the construction of recombinant plasmid pI4.

[0034] Figure 4 Schematic diagram of the construction of recombinant plasmid pBE.

[0035] Figure 5 This is a comparison chart of lycopene production by wild-type Rhodobacter sphaeroides WT, recombinant Rhodobacter sphaeroides strain RS-1, and RS-1 using CO2 under light.

[0036] Figure 6 This is a comparison chart of the lycopene production produced by recombinant Rhodobacter sphaeroides strains RS-2 and RS-3 using CO2 under light. DETAILED DESCRIPTION

[0037] The following is a detailed description of the present invention by way of specific embodiments, but this should not be construed as the entire content of the present invention. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0038] Rhodobacter phaeroides was purchased from the China Center for Microbial Culture Collection (https: / / www.biobw.org / ) on September 6, 2020, with the number bio-53235.

[0039] The present invention will be further described below with reference to specific embodiments.

[0040] Example 1

[0041] The construction of the recombinant sphaeroides bacteria strain RS-1 for synthesizing lycopene comprises the following steps:

[0042] 1. The crtC gene of the Rhodobacter sphaeroides crtC hydroxylase was obtained from the NCBI (National Center for Biotechnology Information, https: / / www.ncbi.nlm.nih.gov / ) database. Primers C-5O and C-5I were designed and synthesized to amplify the upstream homology arm of the crtC gene of the Rhodobacter sphaeroides crtC gene, and primers C-3I and C-3O were designed to amplify the downstream homology arm of the crtC gene of the Rhodobacter sphaeroides crtC gene. The upstream homology arm and the downstream homology arm of the crtC gene were ligated by overlap PCR. The above fragments were ligated into the pK18 vector by enzyme digestion and ligation to construct the recombinant plasmid pC1, as shown in FIG. Figure 2 As shown;

[0043] The nucleotide sequence of the pC1 plasmid is shown in SEQ ID NO.1;

[0044] 2. Transform plasmid pC1 into Escherichia coli S17 to obtain recombinant strain S-1;

[0045] The DNA PCR enzyme used in this invention is Fast Pfu polymerase from Quanshijin Biotechnology Co., Ltd. A 50 μL PCR amplification system is as follows: 1 μL DNA template; 2.5 μL each of a forward primer (10 μM) and a backward primer (10 μM); 5 μL of dNTPs (10 mM); 10 μL of 5× Pfu Buffer; 1 μL of Fast Pfu polymerase; and finally, the mixture is made up to 50 μL with double-distilled water.

[0046] PCR fragment amplification: amplification conditions were as follows: pre-denaturation at 95°C for 2 min (1 cycle); denaturation at 95°C for 20 s, annealing at 55°C for 20 s, and extension at 72°C for 30 s (35 cycles); and extension at 72°C for 5 min (1 cycle).

[0047] Each pair of homologous upper and lower arm fragments obtained by PCR amplification was ligated with the pK18 linearized suicide plasmid vector cut with HindⅢ and BamhI restriction enzymes using the BM seamless ligation kit (Bomaide Biotechnology Co., Ltd.) under the conditions of 50°C for 15 min and 16°C for 10 min.

[0048] The process of making S17 competent cells is as follows:

[0049] Take S17 glycerol bacteria, streak inoculate on LB solid medium, place the solid medium in a 37 ° C incubator for 24 hours, then use an inoculation loop to pick a single colony on the solid medium, transfer it to a test tube filled with LB antibiotic-free medium for primary inoculation culture, place the test tube in a shaker at 37 ° C, 220 rpm and culture for 12 hours, then take out 1 mL of bacterial solution from the test tube and transfer it to a flask filled with LB antibiotic-free medium for secondary culture, place the flask in a shaker at 37 ° C, 220 rpm and culture until OD 600 The value was about 0.6, and then the culture medium was ice-bathed for 20 minutes, and the bacterial solution was centrifuged, the supernatant was poured out, and the precipitate was resuspended with the competent preparation reagent, and the resuspended solution was divided into aliquots and stored in a -80℃ refrigerator.

[0050] The specific steps of S17 chemical transformation are as follows:

[0051] (1) Take 100 μL of S17 competent cells from the -80°C refrigerator and place on ice for 10 minutes;

[0052] (2) Mix the pC1 plasmid and S17 competent cells in a 1.5 mL centrifuge tube and incubate on ice for 30 minutes;

[0053] (3) Place the centrifuge tube after ice bath in a 42°C water bath for 90 seconds and ice bath again for 2 minutes;

[0054] (4) Add 1 mL of LB medium to the above system and incubate at 37°C, 200 rpm in a shaker for 1 h;

[0055] (5) Place the centrifuge tube after shaking culture in a centrifuge and centrifuge at 5000 rpm for 5 min. Pour off the supernatant and resuspend the bacterial pellet in approximately 100 μL of LB culture medium. Spread the resuspended solution on an LB plate (containing 1 mM kanamycin) and incubate at 30°C for 24 h.

[0056] (6) Pick a single colony for PCR verification.

[0057] 3. The recombinant strain S-1 and the wild-type Rhodobacter sphaeroides were conjugated and transferred to obtain the recombinant Rhodobacter sphaeroides strain RS-1. The conjugation and transfer process is as follows:

[0058] (1) Bacteria activation

[0059] The Rhodobacterium sphaeroides RS was taken out of the -80°C refrigerator and cultured overnight in NH1 liquid medium at 32°C, 200 rpm to activate the strain; the recombinant Escherichia coli strain S-1 was taken out of the -80°C refrigerator and cultured overnight in LB liquid medium (kanamycin sulfate was added to a final concentration of 100 μg / mL when culturing the strain S-1) at 37°C, 220 rpm. After the strain was activated once, the overnight culture S-1 was transferred to 100 mL LB liquid medium at a 1% inoculum volume and cultured at 37°C, 200 rpm for 2-3 h until the OD 600 The value is 0.4-0.6 for secondary activation;

[0060] (2) Construction and screening of single-exchange Rhodobacterium sphaeroides strains

[0061] Take 1 mL each of the activated Rhodobacter sphaeroides RS and Escherichia coli S-1 and wash the bacteria twice with antibiotic-free NH1 liquid culture medium. Mix Rhodobacter sphaeroides RS and Escherichia coli S-1 in a ratio of 5:1. Take 20 μL of the mixed bacterial solution and directly spot it on the antibiotic-free NH1 culture medium with a 0.22 μm pore size water filter membrane. After inverted incubation at 32°C for 20 hours, use a sterilized pipette tip to take an appropriate amount of plaques and resuspend them in 100 μL of antibiotic-free NH1 culture medium before coating. On NH1 solid culture medium containing kanamycin sulfate and nalidixic acid (kanamycin sulfate and nalidixic acid were added to the NH1 culture medium at a final concentration of 100 μg / mL), single colonies grew after inverted culture at 32°C for 4-6 days; primers 1-crtI-u and 1-crtI-d, 2-crtI-u and 2-crtI-d were designed to perform colony PCR on the single colonies to screen for the correct single-exchange Rhodobacterium sphaeroides conjugate.

[0062] (3) Construction and verification of double-exchange Rhodobacter sphaeroides strains

[0063] The correct single exchange Rhodobacter sphaeroides conjugate was cultured in NH1 medium (with kanamycin sulfate added at a final concentration of 100 μg / mL) at 32°C and 200 rpm overnight. After the strain was activated once, the overnight culture was transferred to 100 mL of antibiotic-free NH1 liquid medium at a 1% inoculum volume and cultured at 32°C and 200 rpm for 6-8 h until the OD 600 After the value is 0.4-0.5, secondary activation is carried out; 100 μL of the secondary activated bacterial liquid is spread on NH1 solid culture medium (with 1% sucrose added) and inverted culture at 32°C for 4-6 days to grow single colonies, and colony PCR is performed on the single colonies using 1-crtI-u and 2-crtI-d to screen out the correct double-exchange sphaeroides conjugate.

[0064] 4. Construction of the recombinant sphaeroides strain RS-2 for lycopene synthesis, comprising the following steps:

[0065] The crtI4 gene was synthesized, and the phytoene three-step dehydrogenase gene crtI3 from Rhodobacter sphaeroides was obtained from the NCBI database. Primers I-5O and I-5I were designed and synthesized to amplify the upstream homology arm of the crtI3 gene of Rhodobacter sphaeroides, and I-3I and I-3O were designed to amplify the downstream homology arm of the crtI3 gene of Rhodobacter sphaeroides. The upstream homology arm of the crtI3 gene, crtI4, and the downstream homology arm of the crtI3 gene were ligated using the overlap PCR method. The three fragments were ligated into the pK18 vector by enzyme digestion and ligation to construct the recombinant plasmid pI4. Figure 3 As shown; the gene crtI3 is the abbreviation of the phytoene three-step dehydrogenase gene of Rhodobacterium sphaeroides; the gene crtI4 is the abbreviation of the phytoene four-step dehydrogenase gene from Rhodospirillum rubrum;

[0066] The nucleotide sequence of the pI4 plasmid is shown in SEQ ID NO.2;

[0067] 5. Transform plasmid pI4 into E. coli S17 competent cells to obtain recombinant strain S-2;

[0068] 6. The recombinant strain S-2 and the recombinant Rhodobacter sphaeroides RS-1 were transferred by conjugation to obtain the recombinant Rhodobacter sphaeroides strain RS-2.

[0069] 7. Construction of a recombinant sphaeroides strain RS-3 for lycopene synthesis, comprising the following steps:

[0070] The phytoene synthase gene crtB and the phytoene diphosphate synthase gene crtE from Rhodobacter sphaeroides were obtained from the NCBI database, and primers crtB-U and crtB-D, and crtE-U and crtE-D were designed and synthesized, respectively. The Rhodobacter sphaeroides genome was used as a template, and PCR amplification was performed with primers crtB-U and crtB-D, and crtE-U and crtE-D, respectively, to obtain fragments crtB and crtE. Primers pYYDT-U and PYYDT-D were designed, and pCR amplification was performed with primers pYYDT-U and PYYDT-D using the in-house pYYDY plasmid as a template to obtain the pYYDT linear vector. The crtB fragment and crtE fragment were sequentially ligated to the pYYDT linear vector by seamless cloning to obtain the recombinant plasmid pBE. Figure 4 shown.

[0071] The nucleotide sequence of the pBE plasmid is shown in SEQ ID NO.3;

[0072] 8. Transform the plasmid pBE into E. coli S17 competent cells to obtain the recombinant strain S-3;

[0073] 9. The recombinant strain S-3 and the recombinant Rhodobacter sphaeroides strain RS-2 were conjugated and transferred to obtain the recombinant Rhodobacter sphaeroides strain RS-3 that synthesizes lycopene.

[0074] Example 2

[0075] Recombinant Rhodobacterium sphaeroides RS-1, RS-2, and RS-3 synthesize lycopene using CO2 under light, including the following process:

[0076] 1. Bacteria activation

[0077] The wild-type Rhodobacter sphaeroides WT, recombinant Rhodobacter sphaeroides RS-1, RS-2 and RS-3 were taken out of the -80°C refrigerator and cultured overnight at 32°C and 200 rpm in an NH1-free liquid medium and an NH1 liquid medium with kanamycin sulfate added at a final concentration of 100 μg / mL to activate the strains; then, the overnight cultured bacterial liquid was transferred to 100 mL of NH1-free liquid medium and a NH1 liquid medium with kanamycin sulfate added at a final concentration of 100 μg / mL at a 2% inoculation rate, and cultured at 32°C and 200 rpm for 24 hours to activate the strains.

[0078] The formula of NH1 medium is: yeast extract 8 g / L, glucose 3 g / L, NaCl 2 g / L, KH2PO4 1.3 g / L, MgSO4 0.125 g / L, thiamine hydrochloride 1 mg / L, niacin 1 mg / L, and biotin 15 μg / L.

[0079] 2. Photocatalytic synthesis of lycopene using CO2

[0080] The activated strain culture solution was measured by OD 600 =1 were transferred into 40 mL photoreactors, nitrogen was introduced for 10 min to remove oxygen in the reactor, and cultured at 32°C and 250 rpm for 72 h. The culture solution was taken for product analysis.

[0081] The formula of the fermentation medium is: Na2HPO4 16g / L, NaH2PO4 6.8g / L, NaHCO3 2.5g / L, MgSO40.58g / L, CaCl2 0.067g / L.

[0082] 3. Extraction and Analysis of Lycopene

[0083] 1 mL of fermentation broth was transferred to a 2 mL EP tube and centrifuged at 12,000 g for 2 minutes. The supernatant was discarded and the cells were washed twice with ddH2O. The washed cells were resuspended in 1 mL of acetone (containing 1% butylated hydroxytoluene (BHT) antioxidant), vortexed for 3 minutes, and incubated at 55°C in the dark for 15 minutes, vortexing for 30 seconds every 3 minutes. The lycopene in the upper acetone layer was collected. The lycopene was then filtered through a 0.45 μm pore size organic filter into a brown injection bottle. The fermentation products were monitored and analyzed by high-performance liquid chromatography (HPLC) on a Thermo U3000 instrument using a Diamonsil SBC18 column (4.6 mm x 250 mm). The column temperature was 30°C, and the mobile phase consisted of methanol:acetonitrile:isopropanol (30:50:20 v / v). The elution was performed at an isocratic flow rate of 1 mL / min and a wavelength of 470 nm for 20 minutes. Lycopene was qualitatively determined by referring to the peak time of the corresponding lycopene standard, and the amount of lycopene was quantitatively determined by referring to the lycopene peak area.

[0084] 4. Results Analysis

[0085] like Figure 5 As shown, since the endogenous crtI3 gene of the wild-type Rhodobacter sphaeroides strain WT is a three-step phytoene dehydrogenase, the wild-type strain WT and the recombinant strain RS-1 cannot synthesize lycopene; the recombinant Rhodobacter sphaeroides RS-2, in which the crtC gene of the Rhodobacter sphaeroides chain hydroxylase was knocked out and the crtI4 gene from Rhodospirillum rubrum replaced the endogenous crtI3 gene of Rhodobacter sphaeroides, was able to synthesize and accumulate lycopene. Under light, the RS-2 strain was able to use CO2 to synthesize lycopene with a yield of 8.51 mg / g DCW; Figure 6 The results showed that the recombinant Rhodobacter sphaeroides RS-33, which overexpressed the phytoene synthase gene crtB and the geranyl diphosphate synthase gene crtE based on RS-2, had a strong ability to synthesize lycopene. The shake flask fermentation culture produced 30.73 mg / g DCW of lycopene, which was 3.6 times higher than that of strain RS-2.

[0086] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

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Claims

1. A recombinant Rhodobacterium sphaeroides strain for photocatalytic CO2 fixation to synthesize lycopene and its construction method, characterized by The steps include: (1) The upstream homology arm and the downstream homology arm of the crtC gene of Rhodobacter sphaeroides were ligated to the pK18 vector to construct a plasmid pC1; the crtC gene is the abbreviation of the endogenous streptozotocin hydroxylase gene of Rhodobacter sphaeroides; The nucleotide sequence of the pC1 plasmid is shown in SEQ ID NO.1; (2) Plasmid pC1 was transformed into Escherichia coli S17 to obtain recombinant strain S-1; (3) The recombinant strain S-1 and the wild-type strain of Rhodobacter sphaeroides were conjugated and transferred to obtain the recombinant Rhodobacter sphaeroides strain RS-1 that synthesizes lycopene. (4) Connecting the upstream homology arm of the crtI3 gene of Rhodobacter sphaeroides, the crtI4 gene, and the downstream homology arm of the crtI3 gene to the pK18 vector to construct plasmid pI4; the crtI3 gene is the abbreviation of the phytoene three-step dehydrogenase gene of Rhodobacter sphaeroides; the crtI4 gene is the abbreviation of the phytoene four-step dehydrogenase gene from Rhodospirillum rubrum; The nucleotide sequence of the pI4 plasmid is shown in SEQ ID NO.2; (5) Plasmid pI4 was transformed into Escherichia coli S17 to obtain recombinant strain S-2; (6) The recombinant strain S-2 was conjugated with Rhodobacter sphaeroides to obtain the recombinant Rhodobacter sphaeroides strain RS-2 that synthesizes lycopene. (7) The crtB gene and the crtE gene were ligated into the pYYDT vector to construct the plasmid pBE; the crtB gene is the abbreviation of the phytoene synthase gene; the crtE gene is the abbreviation of the geranyl diphosphate synthase gene; The nucleotide sequence of the pBE plasmid is shown in SEQ ID NO.3; (8) Plasmid pBE was transformed into Escherichia coli S17 to obtain recombinant strain S-3; (9) The recombinant strain S-3 and the recombinant Rhodobacter sphaeroides strain RS-1 were conjugated and transferred to obtain the recombinant Rhodobacter sphaeroides strain RS-2 that synthesizes lycopene.

2. The recombinant Escherichia coli strains S-1, S-3 and S-3 are constructed by the construction method of claim 1.

3. The construction method of claim 1 is used to construct recombinant Rhodobacter sphaeroides strains RS-1 and RS-2 for photocatalytic CO2 synthesis of lycopene.

4. Use of the recombinant Rhodobacter sphaeroides strains RS-1 and RS-2 according to claim 3 for photocatalytic fixation of CO2 to synthesize lycopene.

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