Halophilic genetically engineered bacterium for synthesizing beta-carotene as well as construction method and application of halophilic genetically engineered bacterium

Abstract

The invention discloses a halophilic genetically engineered bacterium for synthesizing beta-carotene as well as a construction method and application of the halophilic genetically engineered bacterium. The halophilic genetically engineered bacterium H.volcanii is constructed by cloning a lycopene beta-cyclase gene Hma-LCYb coded by a halophilic archaea marisportui genome to an expression vector pTA1228 and then transforming the lycopene beta-cyclase gene Hma-LCYb into a halophilic archaea Haloferx volcanii cell. The engineering bacterium can be used for synthesizing beta-carotene, and the yield of the engineering bacterium is up to 0.26 mg / g of dry cell weight. The halophilic genetically engineered bacterium H.volcanii disclosed by the invention solves the problems of strain function deficiency, insufficient yield and safety in the production of natural beta-carotene, has high efficiency, economical efficiency and environmental protection property, and has remarkable industrial application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to a halophilic genetically engineered bacterium for synthesizing β-carotene, a construction method thereof, and an application thereof. Background Art

[0002] β-carotene is a natural pigment with a tetraterpene skeleton, composed of conjugated isoprene double bonds. Its molecular ends contain β-ionone ring structures, and central cleavage can generate two molecules of vitamin A. It is an important nutritional fortifier and antioxidant. Research shows that β-carotene exhibits anti-cancer, anti-inflammatory, immunomodulatory and other activities by scavenging free radicals, regulating the nuclear factor κB (NF-κB) signaling pathway and inhibiting the expression of pro-inflammatory factors (such as TNF-α, IL-6), and is widely used in the fields of functional foods, drug carriers and cosmetics. However, its commercial production still faces key technical bottlenecks.

[0003] Currently, the production of commercial β-carotene mainly relies on chemical synthesis methods, but this process has obvious defects: toxic chemical substances are likely to remain in the synthesis process, which may cause chromosomal mutations and carcinogenic risks, and the bioavailability of chemically synthesized products is significantly lower than that of β-carotene from natural sources. In contrast, natural β-carotene not only has high safety, but also exhibits stronger physiological effects due to the integrity of its bioactive structure.

[0004] However, the large-scale production of natural β-carotene still faces challenges. Traditional plant extraction methods are limited by high raw material costs, long cycles and low extraction efficiency, etc., and it is difficult to meet market demands. In recent years, biosynthesis using microorganisms has become a research hotspot. Currently, the microorganisms used for synthesizing β-carotene mainly include strains that naturally produce β-carotene and engineered strains modified by metabolic engineering, but there are still some problems to be solved urgently in the production process. The synthesis ability of microorganisms that naturally produce β-carotene is limited, and they are easily affected by environmental conditions (such as light, temperature, salinity), resulting in a long industrial production cycle and high costs. For example, the wild strain Xanthophyllomyces dendrorhous has a low yield; Blakeslea trispora is commonly used in industrial fermentation to produce β-carotene, and the yield can be increased by optimizing fermentation conditions, but there is a problem of poor strain stability; although microalgae such as Dunaliella salina and Haematococcus pluvialis can naturally accumulate β-carotene, the extraction cost is high due to restrictions such as light and nutritional conditions.

[0005] At present, the microbial synthesis of β-carotene mainly relies on metabolic engineering strains such as Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica. However, problems such as precursor supply, fermentation process, and separation cost need to be further solved. First, the regulation of metabolic engineering strains is complex: the synthesis efficiency of precursors such as isopentenyl pyrophosphate (IPP) directly affects the yield of β-carotene, which needs to be optimized by gene overexpression or knockout of competing pathways; the expression of heterologous genes (such as phytoene synthase gene (PSY) and lycopene cyclase gene (LCY) from plants) in the host may be restricted by factors such as codon preference and promoter strength, and codon optimization or promoter engineering is required. Moreover, there are also bottlenecks in the fermentation process and large-scale production: some engineering bacteria (such as Escherichia coli) are prone to accumulate toxic metabolites during high-density culture, inhibiting cell growth and product synthesis; β-carotene is an intracellular product, and it needs to be extracted by cell disruption and organic solvents, with cumbersome steps and easy product loss.

[0006] Therefore, developing a synthetic biology manufacturing platform with high yield, low cost, and environmental adaptability has become the core strategy to solve the above bottlenecks. Halophilic microorganisms are regarded as ideal chassis cells due to their natural stress resistance (tolerance to high osmotic pressure and resistance to phage contamination) and compatible metabolic networks (such as high flux of the MVA pathway in archaea). However, the systematic engineering transformation of high-efficiency synthesis of β-carotene by halophilic bacteria has not been achieved in the existing technology. Summary of the Invention

[0007] In view of the deficiencies of the existing technology, the present invention provides a halophilic genetically engineered bacterium for synthesizing β-carotene, its construction method and application. By cloning the lycopene β-cyclase gene Hma-LCYb encoded by the genome of the extreme halophilic archaeon Haloarcula marismortui from the Dead Sea into the expression vector pTA1228, and then transforming it into the model archaeon Haloferax volcanii cells, a halophilic genetically engineered bacterium is constructed. Since the starting strain lacks the lycopene β-cyclase gene and cannot synthesize β-carotene, while the genetically engineered bacterium constructed by the present invention can synthesize β-carotene, and its yield is as high as 0.26 mg / g cell dry weight. Therefore, the halophilic genetically engineered bacterium constructed by the present invention solves the problems of lack of strain function, insufficient yield, and safety in the production of natural β-carotene, and has high efficiency, economy, and environmental protection, and has significant industrial application value.

[0008] The present invention is achieved through the following technical solutions:

[0009] A method for constructing a halophilic genetically engineered bacterium for synthesizing β-carotene, comprising the following steps:

[0010] Step 1) Using the genomic DNA of halophilic archaeon H.marismortui as a template, and using a forward primer and a reverse primer, perform PCR amplification on the lycopene β-cyclase gene Hma-LCYb encoded by the genome of halophilic archaeon H.marismortui, and detect the PCR amplification result by agarose gel electrophoresis; the nucleotide sequences of the forward primer and the reverse primer are as shown in SEQ ID NO.1-2;

[0011] Step 2) Perform double digestion reactions on the PCR amplification product obtained in Step 1 and the pTA1228 vector with NdeI / EcoRI respectively;

[0012] Step 3) Recover the digested PCR product and pTA1228 vector fragment by agarose gel electrophoresis, and perform a ligation reaction;

[0013] Step 4) Transform the ligation product obtained in Step 3 into E.coli DH5α competent cells, coat them on an LB plate containing ampicillin, and culture overnight at 37°C; pick clones, and extract plasmids for sequencing verification to obtain recombinant plasmids;

[0014] Step 5) Transform the recombinant plasmid obtained in Step 4 into H.volcanii H1424 competent cells to obtain the halophilic genetically engineered bacterium.

[0015] Preferably, the nucleotide sequence of the lycopene β-cyclase gene Hma-LCYb in Step 1 is as shown in SEQ ID NO.3.

[0016] Preferably, the specific steps of the transformation in Step 4 are as follows: Pipette 5 μL of the ligation product into 50 μL of E.coli DH5α competent cells, mix well, and place on ice for 30 min; perform a static water bath at 42°C for 90 s, and continue to place on ice for 2 min; add 200 μL of LB liquid medium, culture on a shaker at 37°C at 150 rpm for 1 h; pipette 100 μL of the culture and coat it on an LB medium plate containing ampicillin with a final concentration of 50 μg / mL, and culture at 37°C for 12-16 h to obtain.

[0017] Preferably, the transformation steps in step 5) are specifically as follows: In a sterile environment, add 10 μL of 0.5 M EDTA to 100 μL of H. volcanii H1424 competent cells, gently tap to mix evenly, and place at room temperature for 10 min to form protoplasts; add 4 μL of 2 ng / μL recombinant plasmid, gently tap to mix evenly, and place at room temperature for 2 - 5 min to incubate the competent cells and the recombinant plasmid; add 100 μL of 60% PEG600, pipette to mix evenly, and place at room temperature for 20 min; add 1 mL of growth medium for resuscitation, centrifuge at 25 °C and 6000×g for 8 min, and aspirate the supernatant under sterile conditions; resuspend the cell pellet in 1 mL of freshly prepared growth medium, and culture on a shaker at 37 °C for 2 - 4 h; after the culture is completed, centrifuge at 6000×g for 2 min, pipette 100 μL and spread it on Hv-Ca medium, then place the plate at 45 °C and culture for one week to obtain the product.

[0018] Preferably, the preparation method of the Hv-Ca medium is specifically as follows: Add 600 mL of 30% SW, 5 g of acid hydrolysate casein to 400 mL of pure water. For solid medium, add 15 g of agar, autoclave at 121 °C, cool, and then it can be used.

[0019] Preferably, the preparation method of the H. volcanii H1424 competent cells in step 5) is specifically as follows: Streak the cells of the halophilic archaeon H. volcanii H1424 on a solid medium, then pick a single colony under sterile conditions into 10 mL of Hv-YPC, and shake culture overnight at 45 °C and 180 rpm; then, inoculate the bacterial solution into a container for culture at an inoculation amount of 10%, and shake culture at 45 °C and 175 rpm until the 600 OD value is 0.8 - 1.0, and at this time, the color of the cells is pink; at room temperature, centrifuge at 6000×g for 15 min to collect the cells, and discard the supernatant under sterile conditions; resuspend the cell pellet in 10 mL of buffered protoplast solution, centrifuge at 6000×g for 10 min, and discard the supernatant under sterile conditions; resuspend the cell pellet in buffered protoplast solution containing 15% glycerol, resuspend and mix evenly; finally, aliquot under sterile conditions and store at -80 °C.

[0020] Preferably, the preparation method of the Hv-YPC is specifically as follows: Add 600 mL of 30% SW, 5 g of yeast powder, 1 g of peptone, 1 g of acid hydrolysate casein to 400 mL of pure water. For solid medium, add 15 g of agar, autoclave at 121 °C, cool, and then it can be used.

[0021] Preferably, the method for configuring 30% SW is as follows: Add 1200 g of NaCl, 150 g of MgCl2·6H2O, 175 g of MgSO4·7H2O, 35 g of KCl, and 100 mL of 1 M Tris-HCl with pH = 7.5 to 4 L of pure water, and make up the volume to 5 L.

[0022] The halophilic genetically engineered bacterium obtained by the above construction method is the halophilic genetically engineered bacterium H. volcanii expressing lycopene β-cyclase protein, which is yellow; the amino acid sequence of the lycopene β-cyclase protein is as shown in SEQ ID NO. 4.

[0023] Application of the above-mentioned halophilic genetically engineered bacterium in the production of β-carotene.

[0024] The beneficial effects of the present invention are as follows:

[0025] (1) Compared with the existing chemical synthesis method or plant extraction method, the halophilic genetically engineered bacterium H. volcanii constructed in the present invention does not need to rely on toxic reagents or complex extraction processes, and can directly produce natural β-carotene efficiently through fermentation. Its yield level (0.26 mg / g cell dry weight) lays a high-yield foundation for subsequent process optimization and large-scale application, while reducing production costs and purification difficulties.

[0026] (2) The halophilic bacterium host selected in the present invention can grow in a high-salt environment and naturally has the ability to resist contamination by miscellaneous bacteria, greatly simplifying the sterilization requirements in the fermentation process and reducing energy consumption and operating costs. In addition, its characteristic of being resistant to extreme conditions helps to achieve high-density fermentation and further improve the total yield of β-carotene.

[0027] (3) The β-carotene synthesized by the halophilic genetically engineered bacterium H. volcanii constructed in the present invention has a natural structure, avoiding the toxic residual substances that may be introduced by the chemical synthesis method, with high safety and better bioavailability, and can be directly applied to fields with strict purity requirements such as medicine and food.

[0028] (4) The present invention provides an efficient strain resource for the green biological manufacturing of natural β-carotene, promotes its application in the development of functional products such as anti-cancer, antioxidant, and anti-inflammatory, and at the same time provides a technical reference for the synthesis of other high-value-added terpene compounds by halophilic microorganisms.

[0029] (5) The yield of β-carotene synthesized by the halophilic genetically engineered bacterium H. volcanii constructed in the present invention is as high as 0.26 mg / g cell dry weight, which is higher than the yield of bacterioerythrin (0.06 mg / g cell dry weight), indicating that the introduction of the lycopene β-cyclase gene has changed the pathway of carotenoid synthesis in H. volcanii cells. Description of the Drawings

[0030] Figure 1 Flow chart for the construction of the halophilic genetically engineered bacterium H. volcanii;

[0031] Figure 2 Results of PCR amplification of the Hma-LCYb gene;

[0032] Figure 3 Comparison results of the colonies of the halophilic genetically engineered bacterium H. volcanii (A) and the initial strain (B);

[0033] Figure 4 Comparison results of the full-wavelength scans of carotenoid production by the halophilic genetically engineered bacterium H. volcanii and the initial strain;

[0034] Figure 5 Comparison of the carotenoid yields of the halophilic genetically engineered bacterium H. volcanii and the initial strain. Specific implementation mode

[0035] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0036] Unless otherwise specified, the technical means used in the following examples are all conventional means well known to those skilled in the art. The experimental methods without specific conditions indicated are all conventional methods in the art.

[0037] The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0038] The sources of the reagents and materials involved in the following examples are as follows: The OMEGA PCR kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; the pTA1228 vector was provided by Dr. Xipeng Liu of Shanghai Jiao Tong University; the OMEGA gel extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; the E. coli DH5α competent cells were from TransGen Biotech; the OMEGA plasmid extraction kit was from Tiangen Biochemical Technology (Beijing) Co., Ltd.; the H. volcanii H1424 cells (ATCC 29605) were from the American Type Culture Collection.

[0039] Example 1

[0040] A method for constructing a halophilic genetically engineered bacterium for synthesizing β-carotene, as Figure 1 shown, the specific steps are as follows:

[0041] 1. Design primers

[0042] Download the lycopene β-cyclase gene Hma-LCYb (NCBI: RR_RS13200, whose nucleotide sequence is shown in SEQ ID NO.3) encoded by the genome of the halophilic archaeon Haloarcula marismortui ATCC43049 that has been sequenced from GenBank, and design a pair of primers containing two different restriction endonuclease cleavage sites, where the cleavage sites are NdeI and EcoRI respectively. The nucleotide sequences of the forward primer and the reverse primer are as follows:

[0043] Forward primer (SEQ ID NO.1): 5’-GGG AAT TCC ATA TGC TGT CTA CCC TCA CGT AT-3’;

[0044] Reverse primer (SEQ ID NO.2): 5’-CCG GAA TTC TCA CTC CCA TCT GTC CAT GAG-3’.

[0045] 2. PCR amplification of the Hma-LCYb gene

[0046] (1) Using the above primer pair, perform PCR amplification with the genomic DNA of H. marismortui ATCC 43049 as the template. The PCR reaction system is shown in Table 1 below.

[0047] Table 1 PCR reaction system (50 μL)

[0048]

[0049] PCR reaction cycle parameters: 95 °C, 3 min; 95 °C, 30 s; 60 °C, 30 s; 72 °C, 1 min; cycle 34 times, 72 °C extension for 5 min.

[0050] (2) Detect the result of PCR amplification of the Hma-LCYb gene by agarose gel electrophoresis.

[0051] As Figure 2 shown, the Hma-LCYb gene was successfully amplified.

[0052] (3) Purification of the PCR product: Use the OMEGA PCR kit for recovery and purification. The specific steps are shown in its instruction manual. Use the Nanodrop 2000 ultra-micro spectrophotometer to measure the concentration of the PCR recovery product.

[0053] 3. Digest the PCR product of the Hma-LCYb gene and the expression plasmid vector

[0054] Perform double digestion (NdeI / EcoRI) reactions on the PCR product and the pTA1228 vector respectively. The digestion reaction system is shown in Table 2 below.

[0055] Table 2 Digestion Reaction System (20 μL)

[0056]

[0057] Incubate in a 37 °C water bath for 2 h. After digestion, perform 1.0% agarose gel electrophoresis on the digestion products. Use the OMEGA Gel Extraction Kit for gel extraction and purification. The specific steps can be found in its instruction manual. Measure its concentration using a Nanodrop 2000 ultra-micro spectrophotometer.

[0058] 4. Ligate the digested PCR product and the vector

[0059] Recover the digested PCR product and the pTA1228 vector by agarose gel electrophoresis and perform a ligation reaction. The ligation reaction system is shown in Table 3 below.

[0060] Table 3 Ligation Reaction System (10 μL)

[0061]

[0062] React at 22 °C for 2 h.

[0063] 5. Transformation of the recombinant plasmid

[0064] Transform the ligation product into E.coli DH5α competent cells, and spread them on an LB plate containing ampicillin, and culture overnight at 37 °C. Pick colonies and extract plasmids for sequencing verification. The LB plate containing ampicillin is prepared by adding 10 g of sodium chloride, 10 g of peptone, 5 g of yeast extract, and 15 g of agar powder to 1 L of pure water, autoclaving at 121 °C, and waiting for it to cool.

[0065] Aspirate 5 μL of the ligation product into 50 μL of E.coli DH5α competent cells, mix well, and place on ice for 30 min. Incubate in a static water bath at 42 °C for 90 s, and quickly return to ice and continue to ice-bath for 2 min. Add 200 μL of LB liquid medium, and culture on a shaker at 37 °C at 150 rpm for 1 h. Aspirate 100 μL of the culture and spread it on an LB medium plate containing ampicillin at a final concentration of 50 μg / mL, and culture at 37 °C for 12 - 16 h to obtain 100 - 200 colonies.

[0066] LB liquid medium: Add 10 g of sodium chloride, 10 g of peptone, and 5 g of yeast extract to 1 L of pure water, autoclave at 121 °C, and it can be used after cooling.

[0067] Subsequently, the positive clones were verified: Four clones were selected and inoculated into test tubes containing 5 mL of LB medium with 50 μg / mL ampicillin, and cultured overnight at 150 rpm on a shaker at 37°C. The plasmid was extracted using an OMEGA plasmid extraction kit and sequenced. The sequencing results were compared with the sequences annotated in NCBI to verify the positive clones and obtain the recombinant plasmid.

[0068] 6. Transformation of the recombinant plasmid into H. volcanii H1424 cells

[0069] The cloned plasmid with the correct gene sequence was transformed into competent H. volcanii H1424 cells.

[0070] The method for preparing competent H. volcanii H1424 cells is as follows: The cells of the halophilic archaeon H. volcanii H1424 were streaked on a solid medium, and then a single colony was picked under sterile conditions into 10 mL of Hv-YPC and cultured overnight with shaking at 45°C and 180 rpm. Then, with an inoculation amount of 10%, the bacterial solution was inoculated into a 150 mL Erlenmeyer flask and cultured with shaking at 45°C and 175 rpm until the OD 600 value was approximately 0.8 - 1.0, and at this time, the color of the bacterial cells was seen to be pink. At room temperature, the bacterial cells were collected by centrifugation at 6000×g for 15 min, and the supernatant was discarded under sterile conditions. The bacterial cells were resuspended in 10 mL of buffered protoplast solution, centrifuged at 6000×g for 10 min, and the supernatant was discarded under sterile conditions. The bacterial cells were resuspended in buffered protoplast solution containing 15% glycerol, resuspended and mixed well, ensuring gentle and slow operation. Finally, it was aliquoted under sterile conditions and stored at -80°C for later use.

[0071] Preparation of Hv-YPC: Add 600 mL of 30% SW, 5 g of yeast powder, 1 g of peptone, 1 g of acid-hydrolyzed casein to 400 mL of pure water. For solid medium, add 15 g of agar powder, and it can be used after autoclaving at 121°C and cooling.

[0072] Method for preparing 30% SW: Add 1200 g of NaCl, 150 g of MgCl2·6H2O, 175 g of MgSO4·7H2O, 35 g of KCl, 100 mL of 1M Tris-HCl, pH = 7.5 to 4 L of pure water, and make up the volume to 5 L.

[0073] The transformation method is as follows: Take out the prepared competent cells of H. volcanii H1424 from the -80°C refrigerator, thaw them at room temperature and place them on ice for use. The whole process is operated in a sterile environment. Add 10 μL of 0.5 M EDTA (pH = 8.0) to 100 μL of H. volcanii H1424 competent cells, gently tap and mix well, and let them form protoplasts at room temperature for 10 min. Add 4 μL of the recombinant plasmid with a concentration of 2 ng / μL, gently tap and mix well, and incubate the competent cells and the recombinant plasmid at room temperature for 2 - 5 min. Add 100 μL of 60% PEG600, gently pipette and mix well, and let it stand at room temperature for 20 min. Add 1 mL of growth medium for resuscitation, centrifuge at 6000×g at 25°C for 8 min, and aspirate the supernatant under sterile conditions. Resuspend the cell pellet in 1 mL of freshly prepared growth medium and culture it on a shaker at 37°C for 2 - 4 h. After the culture is completed, centrifuge at 6000×g for 2 min, aspirate 100 μL and spread it on Hv-Ca medium, and then place the plate at 45°C for about one week of culture. The halophilic genetically engineered bacterium H. volcanii (HVO-pTA1228-Hma-LCYb) is obtained.

[0074] Hv-Ca medium: Add 600 mL of 30% SW, 5 g of acid hydrolysate casein to 400 mL of pure water. For solid medium, add 15 g of agar, and it can be used after autoclaving at 121°C and cooling.

[0075] 7. Extraction and determination of β-carotene

[0076] Some studies have shown that β-carotene has absorption at a wavelength of 453 nm, so the spectrophotometer method is used to determine the content of pigments. The specific method is as follows:

[0077] Use pure β-carotene to prepare acetone solutions with standard gradient concentrations to plot the standard curve of OD 453 versus pigment concentration: y = 0.1382x + 0.0339 (R 2 = 0.9913, where x is the pigment concentration and y is the OD 453 ). Take 3 mL of the halophilic genetically engineered bacterium H. volcanii bacterial liquid cultured above into a centrifuge tube, centrifuge at 12000 rpm for 5 min, and remove the supernatant. Resuspend the cell pellet in 3 mL of equal-volume acetone, ultrasonically vibrate at 55°C for 15 min, after the vibration, centrifuge at 12000 rpm for 3 min, aspirate the supernatant into a cuvette to measure the OD 453 value, record the reading, and then calculate the pigment yield using the standard curve. Define the ratio (mg / g) of the measured yield of the extracted β-carotene to the dry cell weight (DCW) as the yield of β-carotene synthesized by the strain.

[0078] 8. Extraction and Determination of Bacterioruberin

[0079] Bacterioruberin is a natural C-50 carotenoid with antibacterial activity. Research shows that bacterioruberin synthesized by halophilic archaea can absorb at a wavelength of 494 nm. Therefore, in this example, the spectrophotometer method is used to determine the content of bacterioruberin. The specific method is as follows:

[0080] Take 3 mL of the halophilic genetically engineered bacterium H. volcanii bacterial solution after the above induction culture into a centrifuge tube, centrifuge at 12000 rpm for 2 min, and remove the supernatant. Resuspend the bacterial cells in 3 mL of methanol and acetone with a volume ratio of 1:1, shake for 10 min, and then centrifuge at 12000 rpm for 3 min after shaking. Absorb the supernatant into a cuvette to measure the OD 494 value and record the reading. Use the following formula to convert the measured OD 494 value to mg:

[0081]

[0082] The ratio (mg / g) of the yield of the extracted bacterioruberin to the dry cell weight (DCW) of the cells is defined as the yield of bacterioruberin synthesized by the strain.

[0083] 9. Experimental Results

[0084] The comparison results of the colony colors between the halophilic genetically engineered bacterium H. volcanii (HVO-pTA1228-Hma-LCYb) constructed in this example and the initial strain (HVO-pTA1228) are as Figure 3 shown. The color of the HVO-pTA1228-Hma-LCYb strain in the medium is yellow ( Figure 3 in A), while the color of the HVO-pTA1228 strain is red ( Figure 3 in B).

[0085] The comparison results of the full-wavelength scans of carotenoid production between the halophilic genetically engineered bacterium H. volcanii (HVO-pTA1228-Hma-LCYb) constructed in this example and the initial strain (HVO-pTA1228) are as Figure 4As shown, the maximum absorption wavelength of the carotenoids produced by the HVO-pTA1228-Hma-LCYb strain is 453 nm, which coincides with the maximum absorption wavelength of β-carotene, indicating that the main type of carotenoids produced by the halophilic genetically engineered bacterium H. volcanii constructed in this example is β-carotene; while the maximum absorption wavelength of the carotenoids produced by the HVO-pTA1228 strain is 494 nm, which coincides with the maximum absorption wavelength of bacterioruberin, indicating that the main type of carotenoids produced by the initial strain is bacterioruberin.

[0086] The comparison of the carotenoid production yields between the halophilic genetically engineered bacterium H. volcanii (HVO-pTA1228-Hma-LCYb) constructed in this example and the initial strain (HVO-pTA1228) is as Figure 5 shown. The production yield of β-carotene by the HVO-pTA1228-Hma-LCYb strain is 0.26 ± 0.001 mg / g cell dry weight, and its production yield of bacterioruberin is only 0.06 ± 0.003 mg / g cell dry weight, which is lower than the production yield of bacterioruberin by the HVO-pTA1228 strain (0.27 ± 0.003 mg / g cell dry weight).

[0087] The above experimental data show that the halophilic genetically engineered bacterium H. volcanii constructed in the present invention can express lycopene β-cyclase protein (GenBank: AAV46922.1, whose amino acid sequence is shown in SEQ ID NO.4), and then synthesize β-carotene, indicating that the introduction of the lycopene β-cyclase gene has changed the pathway of carotenoid synthesis in H. volcanii cells.

[0088] The embodiments described above are only a part of the embodiments of the present invention, rather than all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the present invention claimed, but merely represents the selected embodiments of the present invention. The scope of protection of the present invention shall be subject to the scope claimed in the claims. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

Claims

1. A method for constructing a halophilic genetically engineered bacterium for synthesizing β-carotene, characterized in that, It includes the following steps: Step 1) Using the genomic DNA of halophilic archaeon H. marismortui as a template, and using a forward primer and a reverse primer, perform PCR amplification on the lycopene β-cyclase gene Hma-LCYb encoded by the genome of halophilic archaeon H. marismortui, and detect the PCR amplification result by agarose gel electrophoresis; the nucleotide sequences of the forward primer and the reverse primer are as shown in SEQ ID NO.1-2; Step 2) Perform NdeI / EcoRI double digestion reactions on the PCR amplification product obtained in Step 1 and the pTA1228 vector respectively; Step 3) Recover the digested PCR product and pTA1228 vector fragment by agarose gel electrophoresis, and perform a ligation reaction; Step 4) Transform the ligation product obtained in Step 3 into E. coli DH5α competent cells, coat it on an LB plate containing ampicillin, and culture it overnight at 37°C; Pick clones, extract plasmids and perform sequencing verification to obtain recombinant plasmids; Step 5) Transform the recombinant plasmid obtained in Step 4 into H. volcanii H1424 competent cells to obtain the halophilic genetically engineered bacterium.

2. The construction method of a halophilic genetically engineered bacterium for synthesizing β-carotene according to claim 1, characterized in that, In Step 1), the nucleotide sequence of the lycopene β-cyclase gene Hma-LCYb is as shown in SEQ ID NO.

3.

3. The construction method of a halophilic genetically engineered bacterium for synthesizing β-carotene according to claim 1, characterized in that, The specific steps of the transformation in Step 4 are as follows: Pipette 5 μL of the ligation product into 50 μL of E. coli DH5α competent cells, mix well, and place on ice for 30 min; Incubate in a static water bath at 42°C for 90 s, and continue to place on ice for 2 min; Add 200 μL of LB liquid medium, culture at 150 rpm on a shaker at 37°C for 1 h; Pipette 100 μL of the culture and coat it on an LB medium plate containing ampicillin with a final concentration of 50 μg / mL, and culture at 37°C for 12-16 h to obtain.

4. The construction method of a halophilic genetically engineered bacterium for synthesizing β-carotene according to claim 1, characterized in that, The specific steps of the transformation in Step 5 are as follows: In a sterile environment, add 10 μL of 0.5 M EDTA to 100 μL of H. volcanii H1424 competent cells, gently tap and mix well, and place at room temperature for 10 min to form protoplasts; Add 4 μL of 2 ng / μL recombinant plasmid, gently tap and mix well, and place at room temperature for 2-5 min to incubate the competent cells and the recombinant plasmid; Add 100 μL of 60% PEG600, pipette and mix well, and place at room temperature for 20 min; Add 1 mL of growth medium for resuscitation, centrifuge at 25°C and 6000×g for 8 min, and aspirate the supernatant under sterile conditions; Resuspend the cell pellet in 1 mL of freshly prepared growth medium, culture on a shaker at 37°C for 2-4 h; After the culture is completed, centrifuge at 6000×g for 2 min, pipette 100 μL and coat it on Hv-Ca medium, and then place the plate at 45°C and culture for one week to obtain.

5. The construction method of a halophilic genetically engineered bacterium for synthesizing β-carotene according to claim 4, characterized in that The preparation method of the Hv-Ca medium is as follows: Add 600 mL of 30% SW, 5 g of acid-hydrolyzed casein to 400 mL of pure water. For solid medium, add 15 g of agar, autoclave at 121°C, cool and then it can be used.

6. The construction method of a halophilic genetically engineered bacterium for synthesizing β-carotene according to claim 1, characterized in that, The preparation method of the competent cells of H. volcanii H1424 described in step 5) is as follows: Streak the cells of the halophilic archaeon H. volcanii H1424 on a solid medium, and then pick a single colony under sterile conditions into 10 mL of Hv-YPC, and culture it overnight with shaking at 45 °C and 180 rpm; then, inoculate the bacterial liquid into a container for culture at an inoculation amount of 10%, and culture it with shaking at 45 °C and 175 rpm until the OD 600 value is 0.8-1.0, and at this time, the color of the cells is pink; at room temperature, centrifuge at 6000×g for 15 min to collect the cells, and discard the supernatant under sterile conditions; resuspend the cells in 10 mL of buffered protoplast solution, centrifuge at 6000×g for 10 min, and discard the supernatant under sterile conditions; resuspend the cells in buffered protoplast solution containing 15% glycerol, resuspend and mix well; finally, aliquot under sterile conditions and store at -80 °C.

7. The construction method of a halophilic genetically engineered bacterium for synthesizing β-carotene according to claim 6, characterized in that, The preparation method of the Hv-YPC is as follows: Add 600 mL of 30% SW, 5 g of yeast powder, 1 g of peptone, and 1 g of acid hydrolyzed casein to 400 mL of pure water. If it is a solid medium, 15 g of agar needs to be added. It can be used after autoclaving at 121 °C and cooling.

8. The construction method of a halophilic genetically engineered bacterium for synthesizing β-carotene according to claim 5 or 7, characterized in that, The preparation method of the 30% SW is as follows: Add 1200 g of NaCl, 150 g of MgCl2·6H2O, 175 g of MgSO4·7H2O, 35 g of KCl, and 100 mL of 1 M Tris-HCl with pH = 7.5 to 4 L of pure water, and make up the volume to 5 L.

9. The halophilic genetically engineered bacterium obtained by the construction method according to any one of claims 1-8, characterized in that, The engineered bacterium is the halophilic genetically engineered bacterium H. volcanii expressing the lycopene β-cyclase protein, which is yellow; the amino acid sequence of the lycopene β-cyclase protein is as shown in SEQ ID NO.

4.

10. Use of the halophilic genetically engineered bacterium according to claim 9 in the production of β-carotene.

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