A method for biosynthesis of c-phycocyanin

Abstract

The application discloses a biosynthesis method of alginin. The method uses the whole cell of Escherichia coli with alginin synthetase and polyphosphate kinase functions, catalyzes the substrates aspartic acid and arginine to be combined into alginin, and simultaneously circulates ADP generated in the reaction to be ATP, so that the continuous and efficient synthesis reaction is guaranteed. Compared with the prior art, the application provides an effective and simple implementation method for the biosynthesis of renewable and clean polyphosphate-alginin, the total yield of alginin reaches 30.42 g / L, and the alginin has high industrialization value.

Description

Technical Field

[0001] This invention belongs to the field of biopolymer raw material production technology, specifically relating to a biosynthesis method of phycocyanin. Background Technology

[0002] Cyanocyanin (CGP), also known as phyanocyanin granule polypeptide or poly-L-arginine-poly(L-aspartic acid), is a non-ribosome synthesized natural amino acid polymer with a molecular weight between 8 and 100 kDa. The molecular structure of phyanocyanin consists of a poly(L-aspartic acid) backbone and arginine (or lysine, ornithine, citrulline) side chains. The arginine (or lysine, ornithine, citrulline) moieties are linked to the β-carboxyl groups of each aspartic acid group via α-amino groups, forming a long molecular chain polymer with up to several hundred dipeptide units. This structure contains highly charged, net-neutral zwitterions, and is only soluble under strong ionic conditions. It is a biopolymer with excellent chemical and material properties, suitable for industrial applications in food, medicine, cosmetics, nutrition, and agriculture.

[0003] Phycocyanin, an amino acid polymer composed of aspartic acid and arginine (or lysine, ornithine, or citrulline), has many potential applications. Phycocyanin is a natural amino acid polymer rich in nitrogen, serving as an excellent nitrogen source for organisms, as well as a rich source of carbon and energy. The hydrolysis product of phycocyanin—β-aspartic acid-arginine dipeptide—can be used as a supplement of arginine and aspartic acid in pharmaceuticals and food, with higher utilization than free amino acids. Polyaspartic acid extracted from phycocyanin possesses water solubility and biodegradability, making it suitable as an anti-scaling agent, water softener, and dispersant. Crosslinking phycocyanin with glutaraldehyde can synthesize smooth-surfaced films, which can serve as biocompatible materials with minimal irritation to organisms and animal cells, showing promise for applications in the medical device field.

[0004] Phycocyanin is mainly found in the vegetative cells of most cyanobacteria. Its synthesis does not depend on intracellular ribosomes, but is carried out by a specific phycocyanin synthase, CphA. Although cyanobacteria are the natural source strains of phycocyanin, they are not suitable for large-scale industrial production. The reasons are as follows: (1) Cyanobacteria grow slowly and require a long cultivation period, usually half a month or even longer, and can only obtain a low cell density; (2) Cyanobacteria produce a small amount of phycocyanin, which cannot meet the needs of large-scale industrial production; (3) The cultivation of cyanobacteria requires artificial adjustment of light and strict control of the growth environment, which is time-consuming and labor-intensive.

[0005] In recent years, the use of recombinant DNA technology to heterologously express phycocyanin synthase CphA in non-cyanobacteria (such as Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Pseudomonas aeruginosa, Corynebacterium glutamicum, etc.) and then fermenting it through microorganisms has become the preferred method to improve phycocyanin yield and production efficiency. Currently, domestic scholars have made many remarkable advances in heterologous synthesis of phycocyanin using different microorganisms.

[0006] Patent CN110791534A discloses a method for increasing the exogenous synthesis yield of water-soluble phycocyanin, which involves heterologous expression of phycocyanin synthase CphA in recombinant Escherichia coli. BP The addition of 2.4% yeast extract, 1 mM aspartic acid, 1 mM lysine, and phosphate to the culture medium during fermentation significantly increased the yield of water-soluble phycocyanin, with the optimal yield being 165.3 mg / g, which is 2.2 times higher than the yield of water-soluble phycocyanin produced by the control culture.

[0007] The paper Kyle Swain, et al. Biochemical Engineering Journal (2023) (DOI:10.1016 / j.bej.2023.108916) describes a heterologous phycocyanin synthesis method. The phycocyanin synthase gene TmCphA1 from Tatumella morbirosei was introduced into E. coli BL21(DE3) for heterologous expression. Phycocyanin was synthesized in a laboratory-scale bioreactor, achieving a yield of up to 1.9 grams per liter of culture medium within 48 hours. The yield of insoluble phycocyanin was about 2 times higher than that of other tested enzymes, and 10.8 times higher than that of CphA1 from Synechocystis sp. PCC6308.

[0008] The literature Lars Wiefel, et al. Appl Microbiol Biotechnol (2011) (DOI:10.1007 / s00253-011-3224-4) reported the expression of phycocyanin synthase from Synechocystis PCC 6308 in *Pseudomonas putida* ATCC 4359. After optimized culture medium, the cell dry weight of this strain was 1.09 ± 0.05 g / L. The highest percentage of insoluble CGP synthesized was 14.7 ± 0.7% (mass fraction) of cell dry weight, and the highest percentage of soluble CGP was 28.7 ± 0.8% (mass fraction). The total CGP content of the cells reached 43.4% (mass fraction) of cell dry weight. After conversion, the yield of insoluble CGP was 0.16 g / L, the yield of soluble CGP was 0.31 g / L, and the yield of total CGP was 0.47 g / L.

[0009] Current methods for heterologous synthesis of phycocyanin, such as fermentation processes using microorganisms like *Escherichia coli*, yeast, *Pseudomonas*, and *Corynebacterium glutamicum*, suffer from low yields (the highest currently achieved is 1.9 g / L), severely limiting its industrial application. Therefore, there is still a need to develop an economical, convenient, and efficient biosynthetic method to significantly improve the synthesis efficiency and final yield of phycocyanin, thereby meeting the demands of industrial production. Summary of the Invention

[0010] The purpose of this invention is to provide a method for the biosynthesis of phycocyanin. This method co-expresses a primer-independent phycocyanin synthase TeCphA from *Thermosynechococcus elongatus* BP-1 and a polyphosphate kinase RsPPK from *Rhodobacter sphaeroides* using engineered *Escherichia coli* strain TeCphA-RsPPK. The phycocyanin synthase TeCphA catalyzes the synthesis of phycocyanin from aspartic acid and arginine substrates in the reaction system, simultaneously consuming adenosine triphosphate (ATP) to generate adenosine diphosphate (ADP). The polyphosphate kinase RsPPK catalyzes the regeneration of ADP into ATP, providing energy for the catalytic reaction of phycocyanin synthase TeCphA, ensuring the continuous, efficient, and economical synthesis of phycocyanin, thereby significantly increasing the yield of phycocyanin. Figure 1 ).

[0011] To achieve the above objectives, this invention utilizes whole-cell Escherichia coli for the biosynthesis of phycocyanin, comprising the following steps:

[0012] (1) Prepare the phycocyanin synthesis reaction solution, which contains amino acid substrate, polyP, ATP, MgCl2, KCl and whole cells of engineered bacteria (TeCphA-RsPPK);

[0013] (2) Pour the reaction solution into the bioreactor to continuously synthesize phycocyanin, detect the concentration of amino acid substrate in the reaction solution, and add substrate in a timely manner according to the reaction requirements;

[0014] (3) Phycocyanin was extracted and purified by acid treatment-ethanol precipitation method.

[0015] Preferably, the preparation of the reaction solution in step (1) includes the following steps: 1.5 L of phycocyanin synthesis reaction solution is prepared using pure water, which contains 80 mM aspartic acid (Asp), 80 mM arginine (Arg), 16 mM sodium hexametaphosphate (polyP), 8 mM adenosine triphosphate (ATP), 30 mM MgCl2, 50 mM KCl and 20 OD engineered bacteria (TeCphA-RsPPK) whole cells, and the pH is adjusted to 7.0;

[0016] Preferably, the preparation of whole cells of the engineered bacteria (TeCphA-RsPPK) in step (1) includes the following steps: Prepare 100 mL of seed culture medium containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 0.05 g / L kanamycin, 0.03 g / L chloramphenicol, with the remainder being purified water, and adjust the pH to 7.0 with ammonia; after sterilizing in a 250 mL Erlenmeyer flask, inoculate with a single colony of the engineered bacteria (TeCphA-RsPPK), and culture in a shaker at 37°C and 200 rpm for 8 hours; then inoculate into a 1000 mL Erlenmeyer flask containing 200 mL of induction expression medium containing 12 g / L peptone, 24 g / L yeast extract, 4 g / L glycerol, 2.3 g / L potassium dihydrogen phosphate, 12.5 g / L dipotassium hydrogen phosphate, 0.05 g / L kanamycin, 0.03 g / L chloramphenicol, and the remainder being purified water, and adjust the pH to 7.0 with ammonia; after sterilization in a 250 mL Erlenmeyer flask, inoculate with a single colony of the engineered bacteria (TeCphA-RsPPK) and culture in a shaker at 37°C and 200 rpm for 8 hours; then inoculate into a 1000 mL Erlenmeyer flask containing 200 mL of induction expression medium containing 12 g / L peptone, 24 g / L yeast extract, 4 g / L glycerol, 2.3 g / L potassium dihydrogen phosphate, 12.5 g / L dipotassium hydrogen phosphate, 0.05 g / L kanamycin, 0.03 g / L chloramphenicol, and the The solution was prepared with chloramphenicol at a concentration of g / L, with the remainder being purified water. The shake flask was incubated on a shaker at 37°C and 200 rpm for 3 hours. The temperature was then lowered to 25°C, and 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) was added. The culture was continued for 12 hours. After the culture was completed, the culture medium was centrifuged at 4000 rpm for 20 minutes at 4°C. The supernatant was discarded to obtain whole cells containing phycocyanin synthase and polyphosphate kinase.

[0017] Preferably, the biosynthesis of phycocyanin in step (2) includes the following steps: the reaction solution is poured into a bioreactor to synthesize phycocyanin, the temperature is controlled at 40°C, the rotation speed is 500 rpm, and the concentration of the two amino acid substrates is measured at 2-hour intervals. The initial concentration of phycocyanin is replenished to 80 mM according to the consumption.

[0018] Preferably, the detection of amino acid concentration in the reaction solution in step (2) includes the following steps: take 0.5 mL of the reaction solution and centrifuge at 10000 rpm for 10 min, and dilute the supernatant 100 times with pure water; take 0.25 mL of the diluted solution and mix it with 0.3 mL of 0.1 M sodium borate solution (pH 9.5) and 0.2 mL of OPA derivatizing agent, and let it stand in the dark for 2 min; filter the mixture with a 0.45 µm filter membrane and transfer it into a sample vial, detect the peak area of ​​the two amino acids in the sample vial by high performance liquid chromatography, and calculate the concentration of the two amino acids in the reaction solution according to the amino acid standard curve; the working conditions of high performance liquid chromatography are: C-18 column, column temperature 35℃, flow rate 0.8 mL / min, mobile phase A (pH 7.0 0.05 M sodium acetate solution): mobile phase B (chromatographic grade methanol) = 70%: 30%, detection wavelength 334 nm, and injection volume 5 µL.

[0019] Preferably, the extraction and purification of phycocyanin in step (3) includes the following steps: adding 3 M HCl to adjust the pH of the reaction solution to 1.5, and treating it with acid at 25 ℃ and 250 rpm for 20 h on a shaker; centrifuging the reaction solution at 9000 rpm and 4 ℃ for the first time for 20 min; taking the supernatant from the first centrifugation, adding 5 M NaOH to adjust the pH to 7.0; centrifuging the reaction solution at 9000 rpm and 4 ℃ for the second time for 20 min, and collecting the precipitate from the second centrifugation, which is the insoluble phycocyanin; collecting the supernatant from the second centrifugation, adding two volumes of anhydrous ethanol and mixing evenly, centrifuging at 9000 rpm and 4 ℃ for the third time for 20 min, and collecting the precipitate from the third centrifugation, which is the soluble phycocyanin; washing the two phycocyanin solutions with acetone equal to the volume of the precipitate, centrifuging at 9000 rpm and 4 ℃ for 20 min, collecting the two phycocyanin precipitates, and freeze-drying them in a pre-cooled vacuum freeze dryer for 24 hours. h, two pure phycocyanin products were obtained.

[0020] This invention also provides an engineered strain (TeCphA-RsPPK) for biosynthesizing phycocyanin, the method for constructing the engineered strain comprising the following steps:

[0021] (1) DNA fragments of the commercially synthesized phycocyanin synthase gene TeCphA and polyphosphate kinase gene RsPPK, respectively, with nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2;

[0022] (2) The DNA fragment of the phycocyanin synthase gene TeCphA and the pCOLADuet-1 vector were digested with SalI and NotI respectively. The gene DNA fragment and the vector fragment were ligated with T4 ligase. The ligation product was transformed into the host strain of Escherichia coli DH5α by heat shock method. The transformed cells were plated on LB solid medium containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder and 0.05 g / L kanamycin. Transformants were grown. Single colonies were picked and transformed into LB liquid medium containing kanamycin. After being cultured at 37°C overnight, the recombinant plasmid pCOLADuet-TeCphA was extracted and sent to a commercial biotechnology company for sequencing to confirm that it contained the correct gene fragment.

[0023] (3) The DNA fragment of the polyphosphate kinase gene RsPPK and the pACYCDuet-1 vector were digested with NdeI and XhoI, respectively. The gene DNA fragment and the vector fragment were ligated with T4 ligase. The ligation product was transformed into the host strain of Escherichia coli DH5α by heat shock method. The transformed cells were plated on LB solid medium containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 1.5% agar powder and 0.03 g / L chloramphenicol. Transformants were grown. Single colonies were picked and transformed into LB liquid medium containing chloramphenicol. After being cultured at 37°C overnight, the recombinant plasmid pACYCDuet-RsPPK was extracted and sent to a commercial biotechnology company for sequencing to confirm that it contained the correct gene fragment.

[0024] (4) The recombinant plasmids pCOLADuet-TeCphA and pACYCDuet-RsPPK were co-transformed into the Escherichia coli host strain BL21(DE3) by heat shock method. The transformed strains were spread on LB solid medium containing kanamycin and chloramphenicol and transformed. Single colonies were picked to obtain the engineered strain (TeCphA-RsPPK).

[0025] Beneficial effects

[0026] Compared with existing technologies, this invention provides an economical and efficient method for the biosynthesis of phycocyanin, and has high industrialization value.

[0027] The existing technology has the following technical defects:

[0028] (1) The yield is low. The yield of phycocyanin synthesized by cyanobacteria or by Escherichia coli, yeast, Pseudomonas and other heterologous sources is generally low. The reported yield is 2 g / L, or only 40-50% w / w of cell dry weight, which cannot meet the needs of industrial application.

[0029] (2) The production cycle is long. The production cycle of natural cyanobacteria synthesizing phycocyanin is about 96 hours, while the production cycle of heterologous strains such as Escherichia coli, yeast, and Pseudomonas also reaches 30-40 hours, which greatly increases the time cost of production.

[0030] (3) Homologous or heterologous expression of phycocyanin synthase in the production strain, and then phycocyanin insoluble particles are synthesized and accumulated in the cell through fermentation culture. This synthesis and accumulation of phycocyanin is limited by cell growth metabolism and space capacity, and cannot break through the current production bottleneck.

[0031] (4) Phycocyanin synthesis requires ATP as energy, but cells are limited by their own growth and metabolism and cannot provide enough ATP to maintain the high efficiency and continuous progress of the reaction, resulting in low efficiency of existing production technologies.

[0032] (5) The proportion of insoluble phycocyanin is much higher than that of soluble phycocyanin. The content of soluble phycocyanin can only be increased by adding lysine or increasing the synthesis temperature, and the yield is limited.

[0033] Compared with the above phycocyanin synthesis technologies, this invention obtains whole cells that co-express phycocyanin synthase and polyphosphokinase by inducing the culture of engineered Escherichia coli (TeCphA-RsPPK), and then uses the substrates aspartic acid and arginine in the whole-cell catalytic reaction system to efficiently synthesize phycocyanin, which has the following beneficial effects:

[0034] (1) The yield reached 30.42 g / L, which is the highest level reported to date;

[0035] (2) The reaction cycle is about 24 hours. This technology is a whole-cell catalytic reaction. Compared with fermentation synthesis, the equipment requirements are lower and the control operation is simpler. There is no need to consider the problem of cell contamination during the reaction process.

[0036] (3) Phycocyanin synthesized by whole-cell catalysis is not affected by cell growth and metabolism, and is not limited by space capacity;

[0037] (4) Co-expressed polyphosphokinase can continuously regenerate ADP into ATP, providing a continuous source of energy and phosphorylation donors for the synthesis reaction of phycocyanin, thereby significantly increasing the rate of the synthesis reaction.

[0038] (5) The soluble phycocyanin yield of this invention is 20.26 g / L, and the soluble phycocyanin content is twice that of the insoluble phycocyanin content. For existing technologies, it is difficult to achieve the same yield. Soluble phycocyanin has relatively high solubility, which can accelerate the reaction rate of molecules in the system and improve production efficiency in industrial applications. This invention uses a whole-cell biosynthesis method for phycocyanin. After reacting at 40℃ for 24 h, the yield of insoluble phycocyanin is 10.16 g / L, and the yield of soluble phycocyanin is 20.26 g / L, reaching a total yield of 30.42 g / L, which is the highest yield level reported to date. The phycocyanin biosynthesis method provided by this invention has high yield, fast reaction, and low cost, and has great industrialization potential. Attached Figure Description

[0039] Figure 1 Schematic diagram of the phycocyanin synthesis reaction.

[0040] Figure 2 Expression diagram of phycocyanin synthase TeCphA and polyphosphate kinase RsPPK

[0041] Figure 3 This is a high-performance liquid chromatography (HPLC) chromatogram of aspartic acid and arginine standard solutions.

[0042] Figure 4 This is a standard curve for aspartic acid and arginine.

[0043] Figure 5 Figure showing the effect of reaction conditions on the synthesis efficiency of phycocyanin

[0044] Figure 6 This is a high-performance liquid chromatography (HPLC) chromatogram of aspartic acid and arginine in the reaction solution.

[0045] Figure 7 Detection graph of soluble and insoluble phycocyanin synthesized from 80 mM substrate. Specific Implementation

[0046] The present invention will be further described in detail below with reference to the embodiments. The experimental methods in the embodiments were carried out under conventional conditions, referring to the experimental methods described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (4th Edition, 2017, Science Press).

[0047] The materials and reagents used in the examples are all commercially available.

[0048] The DNA fragments of the phycocyanin synthase TeCphA and polyphosphate kinase RsPPK in the examples have nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing. The DNA and plasmids pCOLADuet-1 and pACYCDuet-1 were either artificially synthesized or purchased from commercial biotechnology companies such as Genscript Biotech Inc.

[0049] Example 1: Construction and Identification of Recombinant Strains

[0050] Plasmid pCOLADuet-1, 3719 bp in size, contains the kanamycin resistance gene; plasmid pACYCDuet-1, 4008 bp in size, contains the chloramphenicol resistance gene. Gene DNA fragments of the phycocyanin synthase TeCphA and polyphosphate kinase RsPPK were artificially synthesized by commercial companies, and their nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing.

[0051] The DNA fragment of the phycocyanin synthase gene TeCphA and the pCOLADuet-1 vector were digested with restriction endonucleases SalI and NotI, respectively. The gene DNA fragment and the vector fragment were ligated using T4 ligase. The ligation product was transformed into Escherichia coli host strain DH5α by heat shock. The transformed cells were plated on LB solid medium containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder and 0.05 g / L kanamycin. Transformants were grown, and single colonies were transferred into LB liquid medium containing kanamycin. After overnight incubation at 37°C, the recombinant plasmid pCOLADuet-TeCphA was extracted and sent to a commercial biotechnology company for sequencing to confirm that it contained the correct gene fragment.

[0052] The DNA fragment of the polyphosphate kinase gene RsPPK and the pACYCDuet-1 vector were digested with restriction endonucleases NdeI and XhoI, respectively. The gene DNA fragment and the vector fragment were ligated using T4 ligase. The ligation product was transformed into Escherichia coli host strain DH5α by heat shock. The transformed cells were plated on LB solid medium containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 1.5% agar powder and 0.03 g / L chloramphenicol. Transformants were grown, and single colonies were transferred into LB liquid medium containing chloramphenicol. After incubation at 37°C overnight, the recombinant plasmid pACYCDuet-RsPPK was extracted and sent to a commercial biotechnology company for sequencing to confirm that it contained the correct gene fragment.

[0053] The recombinant plasmids pCOLADuet-TeCphA and pACYCDuet-RsPPK were co-transformed into the Escherichia coli host strain BL21(DE3) using the heat shock method. The transformed strains were then spread onto LB solid medium containing kanamycin and chloramphenicol and grown. Single colonies were picked to obtain the engineered strain (TeCphA-RsPPK).

[0054] Example 2: Whole-cell preparation of engineered bacteria (TeCphA-RsPPK)

[0055] Prepare 100 mL of seed culture medium containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 0.05 g / L kanamycin, 0.03 g / L chloramphenicol, and the remainder being purified water. Adjust the pH to 7.0 with ammonia. Sterilize the medium in a 250 mL Erlenmeyer flask, inoculate with a single colony of engineered bacteria (TeCphA-RsPPK), and incubate at 37°C and 200 rpm for 8 hours. Then, inoculate into a 1000 mL Erlenmeyer flask containing 200 mL of induction expression medium containing 12 g / L peptone, 24 g / L yeast extract, 4 g / L glycerol, 2.3 g / L potassium dihydrogen phosphate, 12.5 g / L dipotassium hydrogen phosphate, 0.05 g / L kanamycin, and 0.03 g / L chloramphenicol. The solution contained g / L chloramphenicol, with the remainder being purified water; the shake flasks were incubated at 37℃ and 200 rpm for 3 hours, then cooled to 25℃, and 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added, followed by incubation for another 12 hours; after incubation, the culture medium was centrifuged at 4000 rpm for 20 minutes at 4℃, and the supernatant was discarded to obtain whole cells containing phycocyanin synthase and polyphosphate kinase. The expression levels of the two enzymes in the whole cells were detected by SDS-PAGE electrophoresis. Figure 2 As shown, lanes 1 and 3 represent uninduced whole-cell proteins, while lanes 2 and 4 represent induced whole-cell proteins. The molecular weight of TeCphA is approximately 98 kDa, and the molecular weight of RsPPK is approximately 38 kDa.

[0056] Example 3: Preparation of amino acid concentration standard curve

[0057] Preparation of OPA derivatization reagent: Weigh 0.0343 g of o-phthalaldehyde and 0.1472 g of acetylcysteine, add 5 mL of anhydrous ethanol, and dilute to 25 mL with 0.1 M sodium borate solution (pH 9.5).

[0058] Standard solutions of aspartic acid and arginine with concentrations of 200 µM, 400 µM, 600 µM, 800 µM, and 1000 µM were prepared respectively.

[0059] Take 0.25 mL of amino acid standard solution, mix it with 0.3 mL of 0.1 M sodium borate solution (pH 9.5) and 0.2 mL of OPA derivatizing agent, and let it stand in the dark for 2 min. Filter the mixture through a 0.45 µm filter membrane and transfer it into a sample vial. Detect the peak areas of the two amino acids in the sample vial by high performance liquid chromatography. Plot a standard curve with amino acid concentration as the x-axis and peak area as the y-axis.

[0060] The operating conditions for high performance liquid chromatography were as follows: C-18 column, column temperature 35℃, flow rate 0.8 mL / min, mobile phase A (pH 7.0 0.05 M sodium acetate solution): mobile phase B (chromatographic grade methanol) = 70%: 30%, detection wavelength 334 nm, and injection volume 5 µL.

[0061] like Figure 3 As shown, the retention time for aspartic acid is 2.8 min, and the retention time for arginine is 5.7 min. The standard curves for the two standard amino acids are shown below. Figure 4 As shown, the regression equation for the aspartic acid standard curve is y = 852.4x - 20511, R0 2 =0.9939; the regression equation for the arginine standard curve is y=822.89x-558.3, R0 2 =0.9995.

[0062] Example 4: Detection of amino acid concentration in reaction solution by high performance liquid chromatography

[0063] Preparation of OPA derivatization reagent: Weigh 0.0343 g of o-phthalaldehyde and 0.1472 g of acetylcysteine, add 5 mL of anhydrous ethanol, and dilute to 25 mL with 0.1 M sodium borate solution (pH 9.5).

[0064] Centrifuge 0.5 mL of the reaction solution at 10,000 rpm for 10 min, and dilute the supernatant 100 times with pure water. Mix 0.25 mL of the diluted solution with 0.3 mL of 0.1 M sodium borate solution (pH 9.5) and 0.2 mL of OPA derivatizing agent, and let stand in the dark for 2 min. Filter the mixture through a 0.45 µm filter membrane and transfer it into a sample vial. Detect the peak areas of the two amino acids in the sample vial using high performance liquid chromatography (HPLC). Calculate the concentrations of the two amino acids in the reaction solution based on the amino acid standard curve. The HPLC operating conditions are: C-18 column, column temperature 35℃, flow rate 0.8 mL / min, mobile phase A (pH 7.0 0.05 M sodium acetate solution): mobile phase B (chromatographic grade methanol) = 70%: 30%, detection wavelength 334 nm, and injection volume 5 µL.

[0065] Example 5: Extraction and purification of phycocyanin

[0066] Collect the phycocyanin synthesis reaction solution, add 3 M HCl to adjust the pH of the reaction solution to 1.5, and place it in a shaker at 25 ℃ and 250 rpm for 20 h to dissolve. Centrifuge the reaction solution at 9000 rpm and 4 ℃ for 20 min. Take the supernatant from the first centrifugation, add 5 M NaOH to adjust the pH to 7.0, and centrifuge at 9000 rpm and 4 ℃ for 20 min. Collect the precipitate from the second centrifugation, which is the insoluble phycocyanin. Collect the supernatant from the second centrifugation, add twice the volume of anhydrous ethanol, mix well, and centrifuge at 9000 rpm and 4 ℃ for 20 min. Collect the precipitate from the third centrifugation, which is the soluble phycocyanin. Wash each of the two phycocyanin precipitates with twice the volume of acetone, centrifuge at 9000 rpm and 4 ℃ for 20 min, discard the supernatant, and collect the two phycocyanin precipitates. Place the two phycocyanin precipitates in a pre-cooled vacuum freeze dryer and freeze-dry for 24 hours. h, two pure phycocyanin products were obtained, weighed, and the results were recorded.

[0067] Example 6: Effect of reaction conditions on phycocyanin synthesis efficiency

[0068] (1) Prepare whole cells of engineered bacteria (TeCphA-RsPPK) according to Example 2;

[0069] (2) Effect of temperature on phycocyanin synthesis efficiency

[0070] 1 mL of phycocyanin synthesis reaction solution was prepared using a pH 7 buffer solution containing 80 mM aspartic acid (Asp), 80 mM arginine (Arg), 16 mM sodium hexametaphosphate (polyP), 8 mM adenosine triphosphate (ATP), 30 mM MgCl2, 50 mM KCl, and 20 OD engineered bacteria (TeCphA-RsPPK) whole cells. The reaction solution was placed in a water bath at different temperature gradients from 20 to 80°C for 1 hour. After the reaction, samples were taken and analyzed by high-performance liquid chromatography (HPLC). The consumption of amino acids was calculated according to Example 4 (the maximum consumption was counted as 100%), and the relative enzyme activity at each temperature was calculated. The data were imported into Prism 8.0 software for plotting, and the results are shown below. Figure 5 As shown, the optimal synthesis temperature for phycocyanin is 40℃.

[0071] (3) Effect of pH on phycocyanin synthesis efficiency

[0072] 1 mL of phycocyanin synthesis reaction solution was prepared using buffers with different pH gradients from 4 to 11 (Tris-HCl buffer was used for reaction systems with pH 4 to 6, and acetate-sodium acetate buffer was used for reaction systems with pH 6 to 10). The solution contained 80 mM aspartic acid (Asp), 80 mM arginine (Arg), 16 mM sodium hexametaphosphate (polyP), 8 mM adenosine triphosphate (ATP), 30 mM MgCl2, 50 mM KCl, and 20 OD engineered bacteria (TeCphA-RsPPK) whole cells. The reaction solution was placed in a 40°C water bath for 1 hour. After the reaction, samples were taken and analyzed by high-performance liquid chromatography (HPLC). The amino acid consumption was calculated according to Example 4 (maximum consumption was counted as 100%), and the relative enzyme activity at each temperature was calculated. The data were imported into Prism 8.0 software for plotting, and the results are shown below. Figure 5 As shown, the optimal pH for phycocyanin synthesis is 7.0.

[0073] (4) Effect of amino acid substrate concentration on phycocyanin synthesis efficiency

[0074] 1 mL of phycocyanin synthesis reaction solution was prepared using a pH 7 buffer solution containing amino acid substrates (Asp and Arg) at different concentration gradients ranging from 60 to 100 mM, 16 mM sodium hexametaphosphate (polyP), 8 mM adenosine triphosphate (ATP), 30 mM MgCl2, 50 mM KCl, and 20 OD engineered bacteria (TeCphA-RsPPK) whole cells. The reaction solution was placed in a 40°C water bath for 1 hour. After the reaction, samples were taken and analyzed by high-performance liquid chromatography (HPLC). The consumption of amino acids was calculated according to Example 4 (the maximum consumption was counted as 100%), and the relative enzyme activity at each temperature was calculated. The data were imported into Prism 8.0 software for plotting, and the results are shown below. Figure 5 As shown, the optimal amino acid substrate concentration for phycocyanin synthesis is 80 mM.

[0075] (5) Effect of polyP concentration on phycocyanin synthesis efficiency

[0076] 1 mL of phycocyanin synthesis reaction solution was prepared using a pH 7 buffer solution, containing 80 mM aspartic acid (Asp), 80 mM arginine (Arg), sodium hexametaphosphate (polyP) at different concentration gradients from 4 to 28 mM, 8 mM adenosine triphosphate (ATP), 30 mM MgCl2, 50 mM KCl, and 20 OD engineered bacteria (TeCphA-RsPPK) whole cells. The reaction solution was placed in a 40°C water bath for 1 hour. After the reaction, samples were taken and analyzed by high-performance liquid chromatography (HPLC). The amino acid consumption was calculated according to Example 4 (the maximum consumption was counted as 100%), and the relative enzyme activity at each temperature was calculated. The data were imported into Prism 8.0 software for plotting, and the results are shown below. Figure 5 As shown, the optimal polyP concentration for phycocyanin synthesis is 16 mM.

[0077] Example 7: Biosynthesis of phycocyanin (65 mM substrate)

[0078] (1) Preparation of whole cells

[0079] The method is the same as in Example 2;

[0080] (2) Preparation of reaction solution

[0081] Prepare 1.5 L of phycocyanin synthesis reaction solution using pure water, which contains 65 mM aspartic acid (Asp), 65 mM arginine (Arg), 16 mM sodium hexametaphosphate (polyP), 8 mM adenosine triphosphate (ATP), 30 mM MgCl2, 50 mM KCl, and 20 OD engineered bacteria (TeCphA-RsPPK) whole cells, and adjust the pH to 7.0;

[0082] (3) Control of the synthesis reaction

[0083] The reaction solution was poured into a bioreactor to synthesize phycocyanin. The temperature was controlled at 40℃, the rotation speed at 500 rpm, and the reaction was continued for 24 hours.

[0084] (4) Detection of amino acid substrate concentration

[0085] The method is the same as in Example 4; samples are taken every 2 hours, and the peak areas of the two amino acids in the reaction solution are detected by high performance liquid chromatography (e.g., Figure 6 As shown in the figure, the concentrations of the two amino acids in the reaction solution were calculated based on the amino acid standard curve;

[0086] (6) Supplementation of amino acid substrates

[0087] Aspartic acid and arginine substrates were added separately according to the amount of substrate consumed in the reaction solution, so that the concentrations of the two amino acids in the reaction solution were maintained at 65 mM.

[0088] (7) Extraction and purification of phycocyanin

[0089] The method was the same as in Example 5; the weighing results showed that the yield of insoluble phycocyanin was 7.19 g / L, the yield of soluble phycocyanin was 12.50 g / L, and the total yield was 19.69 g / L.

[0090] Example 8: Biosynthesis of phycocyanin (100 mM substrate)

[0091] (1) Preparation of whole cells

[0092] The method is the same as in Example 2;

[0093] (3) Preparation of reaction solution

[0094] Prepare 1.5 L of phycocyanin synthesis reaction solution using pure water, which contains 100 mM aspartic acid (Asp), 100 mM arginine (Arg), 16 mM sodium hexametaphosphate (polyP), 8 mM adenosine triphosphate (ATP), 30 mM MgCl2, 50 mM KCl, and 20 OD engineered bacteria (TeCphA-RsPPK) whole cells, and adjust the pH to 7.0;

[0095] (3) Control of the synthesis reaction

[0096] The reaction solution was poured into a bioreactor to synthesize phycocyanin. The temperature was controlled at 40℃, the rotation speed at 500 rpm, and the reaction was continued for 24 hours.

[0097] (4) Detection of amino acid substrate concentration

[0098] The method is the same as in Example 4; samples are taken every 2 hours, and the peak areas of the two amino acids in the reaction solution are detected by high performance liquid chromatography. The concentrations of the two amino acids in the reaction solution are calculated according to the amino acid standard curve.

[0099] (8) Supplementation of amino acid substrates

[0100] Aspartic acid and arginine substrates were added separately according to the amount of substrate consumed in the reaction solution, so that the concentrations of the two amino acids in the reaction solution were maintained at 100 mM respectively.

[0101] (9) Extraction and purification of phycocyanin

[0102] The method was the same as in Example 5; the weighing results showed that the yield of insoluble phycocyanin was 9.33 g / L, the yield of soluble phycocyanin was 14.10 g / L, and the total yield was 23.43 g / L.

[0103] Example 9: Biosynthesis of phycocyanin (80 mM substrate)

[0104] (1) Preparation of whole cells

[0105] The method is the same as in Example 2;

[0106] (2) Preparation of reaction solution

[0107] Prepare 1.5 L of phycocyanin synthesis reaction solution using pure water, which contains 80 mM aspartic acid (Asp), 80 mM arginine (Arg), 16 mM sodium hexametaphosphate (polyP), 8 mM adenosine triphosphate (ATP), 30 mM MgCl2, 50 mM KCl, and 20 OD engineered bacteria (TeCphA-RsPPK) whole cells, and adjust the pH to 7.0;

[0108] (3) Control of the synthesis reaction

[0109] The reaction solution was poured into a bioreactor to synthesize phycocyanin. The temperature was controlled at 40℃, the rotation speed at 500 rpm, and the reaction was continued for 24 hours.

[0110] (4) Detection of amino acid substrate concentration

[0111] The method is the same as in Example 4; samples are taken every 2 hours, and the peak areas of the two amino acids in the reaction solution are detected by high performance liquid chromatography. The concentrations of the two amino acids in the reaction solution are calculated according to the amino acid standard curve.

[0112] (5) Supplementation of amino acid substrates

[0113] Aspartic acid and arginine substrates were added separately according to the amount of substrate consumed in the reaction solution, so that the concentrations of the two amino acids in the reaction solution were maintained at 80 mM.

[0114] (6) Extraction and purification of phycocyanin

[0115] The method was the same as in Example 5; the weighing results are shown in Table 1. The average yield of insoluble phycocyanin was 10.16 g / L, the average yield of soluble phycocyanin was 20.26 g / L, and the average total yield was 30.42 g / L.

[0116] (7) Purity detection of the two phycocyanin

[0117] 5 mg of each of the two phycocyanin compounds were weighed and dissolved in 500 µL of 0.1 M HCl. The purity and size of the two phycocyanin compounds were determined by polyacrylamide gel electrophoresis. The results are as follows: Figure 7 As shown, odd-numbered lanes contain insoluble phycocyanin with a molecular weight of approximately 20-33 kDa, while even-numbered lanes contain soluble phycocyanin with a molecular weight of approximately 14-20 kDa.

[0118] Table 1. Yield of biosynthesized phycocyanin

[0119]

Claims

1. A method for the biosynthesis of phycocyanin, characterized in that, The synthesis method includes the following steps: (1) Prepare the phycocyanin synthesis reaction solution, which contains amino acid substrate, polyP, ATP, MgCl2, KCl and whole cells of engineered bacteria (TeCphA-RsPPK); (2) Pour the reaction solution into the bioreactor to continuously synthesize phycocyanin, detect the concentration of amino acid substrate in the reaction solution, and add substrate in a timely manner according to the reaction requirements; (3) Phycocyanin was extracted and purified by acid treatment-ethanol precipitation method; The method for constructing the engineered bacterium (TeCphA-RsPPK) is as follows: DNA fragments encoding the phycocyanin synthase gene TeCphA and the polyphosphate kinase gene RsPPK are artificially synthesized and cloned into the pCOLADuet-1 and pACYCDuet-1 vectors, respectively, to obtain recombinant plasmids pCOLADuet-TeCphA and pACYCDuet-RsPPK; the two recombinant plasmids are co-transformed into the Escherichia coli host bacterium BL21(DE3) to obtain the engineered bacterium (TeCphA-RsPPK); the nucleotide sequences of the phycocyanin synthase gene TeCphA and the polyphosphate kinase gene RsPPK are shown in SEQ ID NO: 1 and SEQ ID NO:

2.

2. The method for biosynthesizing phycocyanin according to claim 1, characterized in that, Includes the following steps: (1) Prepare the phycocyanin synthesis reaction solution using pure water, which contains 65~100 mM aspartic acid (Asp), 65~100 mM arginine (Arg), 16 mM sodium hexametaphosphate (polyP), 7~8 mM adenosine triphosphate (ATP), 30 mM MgCl2, 50 mM MKCl and 20 OD engineered bacteria (TeCphA-RsPPK) whole cells, and adjust the pH to 7.0; (2) Pour the reaction solution into the bioreactor to synthesize phycocyanin. Control the temperature at 40℃ and the rotation speed at 500 rpm. Take samples every 2 hours to detect the concentration of the two amino acid substrates. Replenish to an initial concentration of 65~80 mM according to the amount of substrate consumed. (3) After the reaction is completed, soluble phycocyanin and insoluble phycocyanin are extracted by acid treatment-ethanol precipitation method, and then pure phycocyanin is obtained by vacuum freeze drying.

3. The method for biosynthesizing phycocyanin according to claim 1, characterized in that, Includes the following steps: (1) Prepare the phycocyanin synthesis reaction solution using pure water, which contains 80 mM aspartic acid (Asp), 80 mM arginine (Arg), 16 mM sodium hexametaphosphate (polyP), 8 mM adenosine triphosphate (ATP), 30 mM MgCl2, 50 mM KCl and 20 OD engineered bacteria (TeCphA-RsPPK) whole cells, and adjust the pH to 7.0; (2) Pour the reaction solution into the bioreactor to synthesize phycocyanin. Control the temperature at 40℃ and the rotation speed at 500 rpm. Take samples every 2 hours to detect the concentration of the two amino acid substrates. Replenish to the initial concentration of 80 mM according to the amount of substrate consumed. (3) After the reaction was completed, soluble phycocyanin and insoluble phycocyanin were extracted by acid treatment-ethanol precipitation method, and then pure phycocyanin was obtained by vacuum freeze drying. The average yield of insoluble phycocyanin was 10.16 g / L, the average yield of soluble phycocyanin was 20.26 g / L, and the average total yield was 30.42 g / L.

4. The method for biosynthesizing phycocyanin according to claim 1, characterized in that, The whole-cell preparation method described in step (1) includes the following steps: Prepare 100 mL of seed culture medium containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 0.05 g / L kanamycin, 0.03 g / L chloramphenicol, with the remainder being purified water. Adjust the pH to 7.0-7.2 with ammonia. After sterilizing the medium in a 250 mL Erlenmeyer flask, inoculate it with a single colony of engineered bacteria (TeCphA-RsPPK) and culture it in a shaker at 37°C and 200 rpm for 8-12 hours. Then, inoculate it into a 1000 mL Erlenmeyer flask containing 200 mL of induction expression medium containing 12 g / L peptone, 24 g / L yeast extract, 4 g / L glycerol, 2.3 g / L potassium dihydrogen phosphate, 12.5 g / L dipotassium hydrogen phosphate, 0.05 g / L kanamycin, 0.03 g / L chloramphenicol, and the remainder being purified water. The solution was prepared with g / L chloramphenicol and the remainder was purified water. The shake flask was placed in a shaker at 37℃ and 200 rpm for 2-4 hours, then cooled to 25-28℃. 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) was added, and the culture was continued for 12 hours. After the culture was completed, the culture medium was centrifuged at 4000 rpm and 4℃ for 20 minutes. The supernatant was discarded to obtain whole cells containing phycocyanin synthase and polyphosphate kinase.

5. The method for biosynthesizing phycocyanin according to claim 1, characterized in that, The method for constructing the engineered bacteria (TeCphA-RsPPK) includes the following steps: (1) DNA fragments of the phycocyanin synthase gene TeCphA and the polyphosphate kinase gene RsPPK, which were commercially synthesized artificially. (2) The DNA fragment of the phycocyanin synthase gene TeCphA and the pCOLADuet-1 vector were digested with SalI and NotI respectively. The gene DNA fragment and the vector fragment were ligated with T4 ligase. The ligation product was transformed into the host strain of Escherichia coli DH5α by heat shock method. The transformed cells were plated on LB solid medium containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder and 0.05 g / L kanamycin. Transformants were grown. Single colonies were picked and transformed into LB liquid medium containing kanamycin. After being cultured at 37°C overnight, the recombinant plasmid pCOLADuet-TeCphA was extracted and sent to a commercial biotechnology company for sequencing to confirm that it contained the correct gene fragment. (3) The DNA fragment of the polyphosphate kinase gene RsPPK and the pACYCDuet-1 vector were digested with NdeI and XhoI, respectively. The gene DNA fragment and the vector fragment were ligated with T4 ligase. The ligation product was transformed into the host strain of Escherichia coli DH5α by heat shock method. The transformed cells were plated on LB solid medium containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 1.5% agar powder and 0.03 g / L chloramphenicol. Transformants were grown. Single colonies were picked and transformed into LB liquid medium containing chloramphenicol. After being cultured at 37°C overnight, the recombinant plasmid pACYCDuet-RsPPK was extracted and sent to a commercial biotechnology company for sequencing to confirm that it contained the correct gene fragment. (4) The recombinant plasmids pCOLADuet-TeCphA and pACYCDuet-RsPPK were co-transformed into the Escherichia coli host strain BL21(DE3) by heat shock method. The transformed strains were spread on LB solid medium containing kanamycin and chloramphenicol and transformed. Single colonies were picked to obtain the engineered strain (TeCphA-RsPPK).

6. The method for biosynthesizing phycocyanin according to claim 1, characterized in that, The method for detecting the concentration of the two amino acid substrates in step (2) includes the following steps: take 0.5 mL of the reaction solution and centrifuge at 10,000 rpm for 10 min, and dilute the supernatant 100 times with pure water; take 0.25 mL of the diluted solution and mix it with 0.3 mL of 0.1 M sodium borate solution (pH 9.5) and 0.2 mL of OPA derivatizing agent, and let it stand in the dark for 2 min; filter the mixture with a 0.45 µm filter membrane and transfer it into a sample vial, detect the peak area of ​​the two amino acids in the sample vial by high performance liquid chromatography, and calculate the concentration of the two amino acids in the reaction solution according to the amino acid standard curve; the working conditions of high performance liquid chromatography are: C-18 column, column temperature 35℃, flow rate 0.8 mL / min, mobile phase A (pH 7.0 0.05 M sodium acetate solution): mobile phase B (chromatographic grade methanol) = 70%: 30%, detection wavelength 334 nm, and injection volume 5 µL.

7. The method for biosynthesizing phycocyanin according to claim 1, characterized in that, The extraction and purification method of phycocyanin described in step (3) includes the following steps: collecting the phycocyanin synthesis reaction solution, adding 3 M HCl to adjust the pH of the reaction solution to 1.5, and dissolving it in a shaker at 25 ℃ and 250 rpm for 20 h; centrifuging the reaction solution at 9000 rpm and 4℃ for the first time for 20 min; taking the supernatant from the first centrifugation, adding 5 M NaOH to adjust the pH to 7.0; centrifuging the solution at 9000 rpm and 4℃ for the second time for 20 min, and collecting the precipitate from the second centrifugation, which is the insoluble phycocyanin; collecting the supernatant from the second centrifugation, adding two volumes of anhydrous ethanol and mixing evenly, centrifuging the solution at 9000 rpm and 4℃ for the third time for 20 min, and collecting the precipitate from the third centrifugation, which is the soluble phycocyanin; washing the two phycocyanin solutions with acetone equal to the volume of the precipitate, centrifuging the solutions at 9000 rpm and 4℃ for 20 min, collecting the two phycocyanin precipitates, and freeze-drying them in a pre-cooled vacuum freeze dryer for 24 hours. Two pure phycocyanin products were obtained; the purity and size of the two phycocyanin products were determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

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