Method for synthesizing bilirubin from heme through whole enzyme catalysis and application

Abstract

The invention belongs to the technical field of bilirubin synthesis, and particularly relates to a method for synthesizing bilirubin through whole enzyme catalysis of heme and application. The method comprises the following steps: fermenting and centrifuging a pichia pastoris engineering strain for expressing heme oxygenase to obtain a thallus precipitate, and crushing to obtain a pichia pastoris cell lysis solution; the method comprises the following steps: fermenting and centrifuging an escherichia coli strain for expressing biliverdin reductase to obtain a thallus precipitate, and crushing to obtain an escherichia coli cell lysis solution; the method comprises the following steps: fermenting and centrifuging an escherichia coli engineering strain for expressing glucose dehydrogenase to obtain a thallus precipitate, and crushing to obtain an escherichia coli cell lysis solution; mixing the pichia pastoris cell lysis buffer with the escherichia coli cell lysis buffer, adding a glucose solution and heme, and reacting to obtain the bilirubin. The whole-cell transformation system established by the invention has good atom economy, and realizes efficient and green synthesis of bilirubin by taking heme as a substrate.

Description

Technical Field

[0001] The invention belongs to the technical field of bilirubin synthesis, and specifically relates to a method and application of synthesizing bilirubin from heme catalyzed by a holoenzyme. Background Art

[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Bilirubin is the main pigment in human bile, has multiple pharmacological effects, and is the main raw material for making artificial bezoar.

[0004] Conventional methods for producing bilirubin mainly use animal bile, such as pig bile, as materials and are extracted by chemical methods. Due to the low technical threshold and extremely low gross profit margin, there are many domestic manufacturers, and the production process uses alkaline hydrolysis and acid to adjust the pH, resulting in a large amount of wastewater. At present, bilirubin comes from bile, and one kilogram of bilirubin requires about 50,000 pig biles, so the cost of bilirubin is relatively high, and therefore the selling price is also high. The comprehensive cost of enzyme-catalyzed production of high-purity bilirubin is relatively low, so the economic benefits are excellent.

[0005] Eukaryotes usually have a complete bilirubin synthesis pathway, in which the key metabolic intermediate is heme. Heme undergoes ring-opening oxidation catalyzed by heme oxygenase (HO, EC 1.14.14.18) to form the intermediate product biliverdin, which is then hydrogenated and reduced by biliverdin reductase (BVR, EC 1.3.1.24) to form bilirubin. Clostridium tetani The heme oxygenase and glutamate dehydrogenase coenzyme regeneration system from the source was displayed on the membrane surface. E. coli The whole cell was used as catalyst, and the biliverdin production reached 76.3 mg / L; Jianfeng Mei et al. used biliverdin as substrate and E. coli Biliverdin reductase from cyanobacteria was expressed as a whole-cell catalyst for the host, and the bilirubin production reached 325 mg / L. Current research reports show that the heme conversion rate catalyzed by heme oxygenase is much lower than the biliverdin conversion rate catalyzed by biliverdin reductase, making it difficult to use low-cost heme as a substrate for two-step biocatalysis. Summary of the invention

[0006] In view of the shortcomings of the prior art, the first object of the present invention is to provide a method for synthesizing bilirubin by catalyzing heme with a holoenzyme. Heme is converted into biliverdin under the action of heme oxygenase, and biliverdin is converted into bilirubin under the action of biliverdin reductase.

[0007] The second object of the present invention is to provide the application of bilirubin synthesized by the above method.

[0008] To achieve the above object, the present invention adopts the following technical solution: A method for synthesizing bilirubin by using heme to catalyze the synthesis of bilirubin by a holoenzyme, the method comprising the following steps: (1) fermenting an engineered strain of Pichia pastoris expressing heme oxygenase, centrifuging the fermentation broth of Pichia pastoris to obtain a bacterial precipitate, and then crushing the precipitate to obtain a lysate of Pichia pastoris cells expressing heme oxygenase; (2) fermenting an engineered strain of Escherichia coli expressing biliverdin reductase, centrifuging the fermented Escherichia coli liquid to obtain a bacterial cell precipitate, and then crushing the cell precipitate to obtain a cell lysate of Escherichia coli expressing biliverdin reductase; (3) fermenting the engineered strain of Escherichia coli expressing glucose dehydrogenase, centrifuging the fermented Escherichia coli liquid to obtain a bacterial cell precipitate, and crushing the cell precipitate to obtain a cell lysate of Escherichia coli expressing glucose dehydrogenase; (4) Mix the Pichia pastoris cell lysate expressing heme oxygenase obtained in step (1), the Escherichia coli cell lysate expressing biliverdin reductase obtained in step (2), and the Escherichia coli cell lysate expressing glucose dehydrogenase obtained in step (3), add glucose solution and heme, and react to obtain bilirubin.

[0009] The present invention uses the model strain Pichia pastoris GS115 is the expression host, and the heme oxygenase protein sequence has been codon-optimized. Heme oxygenase lacks the coenzyme binding domain and cannot directly obtain electrons and reducing power from NADPH. The process of electron transfer to the substrate heme requires the participation of redox chaperone proteins, which contain FAD, FMN or Fe-S clusters and transfer electrons from NADPH to the heme molecule through conformational rearrangement. Pichia pastoris synthesizes heme oxygenase by fermentation, and Pichia pastoris can produce the above coenzyme by itself, which can complete the reaction without the need to synthesize the above coenzyme separately.

[0010] The present invention uses the model strain Escherichia coli BL21 (DE3) was used as the expression host, and the biliverdin reductase protein sequence was codon optimized. Both heme oxygenase and biliverdin reductase require NADPH. In order to further reduce production costs, we Escherichia coli NADP was expressed in the BL21 (DE3) engineered strain + The dependent glucose dehydrogenase was used for the regeneration of the coenzyme NADPH, achieving efficient catalysis with the addition of very small amounts of coenzymes. Finally, the Pichia pastoris and Escherichia coli cell disrupted fluids were mixed to achieve an efficient one-step synthesis of bilirubin, laying a solid foundation for its industrial production.

[0011] Furthermore, in step (1), the Pichia pastoris engineered strain is a recombinant Pichia pastoris GS115 strain, and the recombinant Pichia pastoris expresses Corynebacterium diphtheriae The heme oxygenase of the present invention has an amino acid sequence as shown in SEQ ID NO.1: SEQ ID NO. 1: MTTATAGLAVELKQSTAQAHEKAEHSTFMSDLLEGRLGVAEFTRLQEQAWLFYTALEQAADAVRASGFAESLLDPALNRAEVLARDLDKLNDGSEWRSRITASPAVID YVNRLEEIRDNVDGPALVAHHYVRYLGDLSGGQVIARMMQRHYGVDPEALGFYHFEGIAKLKVYKDEYREKLNNLELSDEQRENLLKEATDAFVFNHQVFADLGKGL.

[0012] Furthermore, in step (1), the OD of the Pichia fermentation broth is 600 =120-150.

[0013] The centrifugation speed is 8000×g, the time is 10 min, and the temperature is 4°C.

[0014] The construction method of the Pichia pastoris engineered strain expressing heme oxygenase is as follows: The heme oxygenase gene sequence is synthesized according to the amino acid sequence of heme oxygenase; the amino acid sequence of the heme oxygenase is shown in SEQ ID NO. 1; the heme oxygenase gene sequence is inserted into pPIC9K to construct a recombinant plasmid pPIC9K-CD, and then transformed into Pichia pastoris Pichia pastori In GS115, the Pichia pastoris engineered strain expressing heme oxygenase was obtained.

[0015] Furthermore, in step (1), the fermentation is carried out by the following steps: The Pichia yeast engineered strain was inoculated into YPD liquid medium, cultured at 30°C and 220 rpm for 16-24 h, then inoculated into BMGY liquid medium at a 1% inoculum amount, and cultured at 30°C and 220 rpm until OD 600 When the reaction mixture reaches 1-3, the mixture is transferred to BMMY liquid culture medium, methanol is added, and the mixture is cultured at 30° C. for 120 h to obtain the fermentation broth.

[0016] Furthermore, in step (2), the engineered Escherichia coli strain is E. coliBL21 (DE3) is the host for the expression of biliverdin reductase. The E. coli engineered strain expresses Castor canadensis Biliverdin reductase, the amino acid sequence is shown in SEQ ID NO. 2: SEQ ID NO. 2: MNTEPGRKFGVVVVGVGRAGSVRIRDLRNPHASSAFLNLIGFVSRRELGSVDEVQQISLENALSSQEIDVAYICSESSSHEDYIRQFLHAGKHVLVEYPMTLSLAAAEELWELAEEKGKVLHEEHVELLMEEFTFLKKEVVGKELLKG SLLFTAGPLEEEHFGFPAFSGISRLTWLVSLFGELSLVSATLEEKKEDQYMKMTVHLETKNKCPLSWIEEKGPGLKRNRYLSFHFKSGSLENVPNVGVNKNIFLKDQNIFVQKLLGQVPEKELAAERKRILHCLALAEEIQKHCRPTK.

[0017] Furthermore, in step (2), the OD of the E. coli fermentation broth is 600 =80-100.

[0018] The centrifugation speed is 8000×g, the time is 10 min, and the temperature is 4°C.

[0019] The Escherichia coli engineering strain expressing biliverdin reductase is constructed as follows: The biliverdin reductase gene sequence was synthesized according to the amino acid sequence of biliverdin reductase; the amino acid sequence of the biliverdin reductase was shown in SEQ ID NO. 2; the biliverdin reductase gene sequence was inserted into pET28a(+) to construct a recombinant plasmid BVR-CC, and then transformed into Escherichia coli E. coli In BL21, the Escherichia coli engineered strain expressing biliverdin reductase was obtained.

[0020] Furthermore, in step (2), the fermentation is carried out by the following steps: The E. coli strain expressing biliverdin reductase was inoculated into LB liquid medium containing Kan, and cultured at 37°C and 200 rpm for 8-10 h. The seed liquid was inoculated into LB liquid medium containing Kan at an inoculum of 1%, and cultured at 37°C and 200 rpm until OD 600 When the pH value reaches 0.4-0.6, isopropyl-β-D-thiogalactoside is added, the temperature is adjusted to 20° C., and the culture is continued for 16 hours to obtain the fermentation broth.

[0021] Furthermore, in step (3), the Escherichia coli engineered strain is E. coli BL21 (DE3) is the host for the expression of biliverdin reductase. The E. coli engineered strain expresses Bacillus megaterium NADP + Dependent formate dehydrogenase, the amino acid sequence is shown in SEQ ID NO.3: SEQ ID NO. 3: MYTDLKDKVVVVTGGSKGLGRAMAVRFGQEQSKVVVNYRSNEEEALEVKKEIEQAGGQAIIVRGDVTKEEDVVNLVETAVKEFGTLDVMINNAGVENPVPSHELSLENWNQVIDTNLTGAFLGSREAIKYF VENDIKGNVINMSSVHEMIPWPLFVHYAASKGGMKLMTETLALEYAPKGIRVNNIGPGAIDTPINAEKFADPEQRADVESMIPMGYIGNPEEIASVAAFLASSQASYVTGITLFADGGMTKYPSFQAGRG.

[0022] Furthermore, in step (3), the OD of the E. coli fermentation broth is 600 =80-100.

[0023] The centrifugation speed is 8000×g, the time is 10 min, and the temperature is 4°C.

[0024] The construction method of the Escherichia coli engineered strain expressing glucose dehydrogenase is as follows: The glucose dehydrogenase gene sequence was synthesized according to the amino acid sequence of glucose dehydrogenase; the amino acid sequence of glucose dehydrogenase is shown in SEQ ID NO.3, and the glucose dehydrogenase gene sequence was inserted into pET28a(+) to construct a recombinant plasmid GDH-BM, which was then transformed into Escherichia coli E. coli In BL21, the Escherichia coli engineered strain expressing glucose dehydrogenase was obtained.

[0025] Furthermore, in step (3), the fermentation is carried out by the following steps: The engineered strain of Escherichia coli expressing glucose dehydrogenase was inoculated into LB liquid medium containing Kan, and cultured at 37°C and 200 rpm for 8-10 h. The seed liquid was inoculated into LB liquid medium containing Kan at an inoculum of 1%, and cultured at 37°C and 200 rpm until OD 600When the pH value reaches 0.4-0.6, isopropyl-β-D-thiogalactoside is added, the temperature is adjusted to 20° C., and the culture is continued for 16 hours to obtain the fermentation broth.

[0026] The high performance liquid phase detection method of the present invention refers to detecting the content of biliverdin, a product catalyzed by heme oxygenase, or detecting the content of bilirubin, a product catalyzed by biliverdin reductase.

[0027] The enzyme activity of the heme oxygenase of the present invention is measured by the peak area of ​​the product biliverdin detected by HPLC using heme as substrate for reaction. The screening of the heme oxygenase is carried out by comparing the enzyme activity.

[0028] The enzymatic activity of the biliverdin reductase of the present invention is measured by using biliverdin as substrate to react and detecting the peak area of ​​the product bilirubin by HPLC. The screening of the biliverdin reductase is carried out by comparing the enzyme activity.

[0029] In one embodiment of the present invention, the whole enzyme catalytic system includes but is not limited to 300 mg / L hemoglobin, aeration volume 1 vvm, hemoglobin is pre-dissolved in 10% NaOH solution (0.05M) of the total reaction volume, the reaction volume is made up with 10 g / L glucose solution (pH 7.5), the reaction time is 1 h, and the reaction temperature is 37°C.

[0030] In one embodiment of the present invention, the whole enzyme catalyst is obtained by culturing recombinant yeast and inducing it to express heme oxygenase, culturing recombinant Escherichia coli and inducing it to express biliverdin reductase or glucose dehydrogenase, and further collecting the recombinant bacterial cells and crushing them.

[0031] In one embodiment of the present invention, the holoenzyme catalyst is obtained by inoculating 1% of the recombinant Pichia pastoris expressing heme oxygenase into 100 mL YPD medium and culturing at 30°C. 600 When the OD reaches 1-3, transfer to BMGY medium. 600 After reaching 1-3, the cells were collected by centrifugation and transferred into BMMY medium. 0.5% methanol was added every day to induce protein expression. After induction at 30℃ for 120h, the cells were collected by low-temperature centrifugation at 8,000×g for 10min and washed twice with 20 mM Tris-HCl (pH 7.5) buffer.

[0032] In one embodiment of the present invention, the holoenzyme catalyst is obtained by inoculating 1% of the recombinant Escherichia coli expressing biliverdin reductase and glucose dehydrogenase into 100 mL LB medium, culturing at 37°C, and culturing until the OD 600When the p - value reached 0.4-0.6, 0.5 mM IPTG was added to induce protein expression. After induction at 20°C for 16 h, the cells were collected by low-temperature centrifugation at 8,000 × g for 10 min, and the cells were washed twice with 20 mM Tris-HCl (pH 7.5) buffer.

[0033] In one embodiment of the present invention, the reaction conditions of the whole-cell catalysis are: stirring speed 220 r / min, temperature 37° C., pH 7.5, ventilation volume 10 vvm, and reaction time 1 to 3 h.

[0034] In one embodiment of the present invention, the amount of heme added is: 0.5-4 mM (325-2604 mg / L), and the amount of the catalyst added is: 10-40 g / L.

[0035] The application of bilirubin synthesized by the above method in the medical field.

[0036] Beneficial effects: (1) The present invention successfully screened highly active heme oxygenase and biliverdin reductase, which were expressed in Pichia pastoris and Escherichia coli, respectively. After the yeast expressed heme oxygenase, it utilized the redox chaperone proteins FAD, FMN or Fe-S cluster produced by itself; Escherichia coli expressed biliverdin reductase and glucose dehydrogenase, respectively. After the three enzymes were mixed, the heme degradation cascade reaction of the whole enzyme was successfully realized. At the same time, only a small amount of NADP and glucose were needed to achieve high catalytic activity, and there was no accumulation of the intermediate product biliverdin during the catalytic process. With heme as the substrate, after multi-enzyme one-pot cascade catalysis, the final bilirubin production could reach 300 mg / L.

[0037] (2) The whole-cell conversion system established in the present invention solves the problems of complicated steps, low yield, and large wastewater discharge in the process of producing bilirubin by traditional biological tissue extraction method. It has good atom economy and realizes efficient and green synthesis of bilirubin using heme as substrate. According to all current data, the bilirubin production in the present invention is the highest level of biological synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the exemplary embodiments of the present invention and their description are used to explain the present invention, and do not constitute an improper limitation of the present invention.

[0039] Figure 1 This is a quantitative analysis diagram of protein expression based on protein gel; Figure 2 is the chromatogram of biliverdin; Figure 3 is the standard curve of biliverdin; Figure 4 is the chromatogram of bilirubin; Figure 5 is the standard curve of bilirubin; Figure 6 are the reaction results under different substrate concentrations; Figure 7 is the standard curve of hemoglobin; Figure 8 The reaction progress curves of heme oxygenase, biliverdin reductase and glucose dehydrogenase at 2 L scale; Fig. 9 This is the bilirubin reaction formula. DETAILED DESCRIPTION

[0040] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0041] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents or raw materials used in the present invention can be purchased through conventional channels. If not otherwise specified, the reagents or raw materials used in the present invention are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar to or equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials described in the present invention are for demonstration purposes only. The present invention is further described according to the accompanying drawings and specific embodiments of the specification.

[0042] Example 1 Gene Synthesis and Expression 1. Construction of recombinant vector The amino acid sequences of heme oxygenase, biliverdin reductase and glucose dehydrogenase selected through experiments were submitted to GenScript (Nanjing) Co., Ltd. for codon optimization, gene synthesis and construction of recombinant vectors.

[0043] (1) The heme oxygenase vector used was the commonly used expression plasmid pPIC9K of Pichia pastoris. The synthetic heme oxygenase (HO) gene sequence was shown in SEQ ID NO. 4: SEQ ID NO. 4: .

[0044] Insert into pPIC9K in the 5'- to 3'-direction Bm I and Not I endonuclease recognition site to obtain the corresponding recombinant plasmid: pPIC9K-CD.

[0045] (2) The biliverdin reductase and glucose dehydrogenase vectors used were pET28a(+), a commonly used expression vector of Escherichia coli. The synthetic biliverdin reductase (BVR) gene sequence is shown in SEQ ID NO. 5: SEQ ID NO. 5: ATGAACACCGAACCGGGCCGCAAATTTGGCGTGGTGGTGGTGGGCGTGGGCCGCGCGGGCAGCGTGCGCATTCGCGATCTGCGCAACCCGCATGCGAGCAGCGCGTTTCTGAACCTGATTGGCTTTGTGAGCCGCCGCGAACTGGGCAGCGTGGATGAAGTGCAGCAGATTAGCCTGGAAAACGCGCTGAGCAGCCAGGAAATTGATGTGGCGTATATTTGCAGCGAAAGCAGCAGCCATGAAGATTATATTCGCCAGTTTCTGCATGCGGGCAAACATGTGCTGGTGGAATATCCGATGACCCTGAGCCTGGCGGCGGCGGAAGAACTGTGGGAACTGGCGGAAGAAAAAGGCAAAGTGCTGCATGAAGAACATGTGGAACTGCTGATGGAAGAATTTACCTTTCTGAAAAAAGAAGTGGTGGGCAAAGAACTGCTGAAAGGCAGCCTGCTGTTTACCGCGGGCCCGCTGGAAGAAGAACATTTTGGCTTTCCGGCGTTTAGCGGCATTAGCCGCCTGACCTGGCTGGTGAGCCTGTTTGGCGAACTGAGCCTGGTGAGCGCGACCCTGGAAGAAAAAAAAGAAGATCAGTATATGAAAATGACCGTGCATCTGGAAACCAAAAACAAATGCCCGCTGAGCTGGATTGAAGAAAAAGGCCCGGGCCTGAAACGCAACCGCTATCTGAGCTTTCATTTTAAAAGCGGCAGCCTGGAAAACGTGCCGAACGTGGGCGTGAACAAAAACATTTTTCTGAAAGATCAGAACATTTTTGTGCAGAAACTGCTGGGCCAGGTGCCGGAAAAAGAACTGGCGGCGGAACGCAAACGCATTCTGCATTGCCTGGCGCTGGCGGAAGAAATTCAGAAACATTGCCGCCCGACCAAATAA。

[0046] The gene sequence of glucose dehydrogenase (GDH) is shown in SEQ ID NO. 6: SEQ ID NO. 6: .

[0047] Inserted into pET28a(+) in the 5'- to 3'-direction Nco I and XOt I endonuclease recognition sites, and the corresponding recombinant plasmids were obtained: BVR-CC, GDH-BM.

[0048] 2. Construction of recombinant strains (1) Construction of recombinant Pichia pastoris strain: Selection of Pichia pastoris Pichia pastoriGS115 was used as the expression host, and the recombinant plasmid pPIC9K-CD obtained in step (1) was linearized with Sac I, Sal After linearization, the electroconversion Pichia pastoris GS115 competent cells (purchased from Invitrogen, Shanghai) were plated on MD plates, and high copies were screened using YPD solid medium plates containing 0.5 mg / L, 1.0 mg / L, 1.5 mg / L, and 2 mg / L G418 sulfate (McLean), and finally, strains were preserved to obtain the corresponding Pichia engineering strains; (2) Construction of recombinant Escherichia coli strains: Selection of Escherichia coli Escherichia coli BL21 (DE3) (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) was used as the expression host, and the recombinant plasmids BVR-CC and GDH-BM obtained in step (1) were respectively introduced into E. coli The transformed cells were cultured in competent cells of BL21 (DE3) and spread onto LB solid culture medium plates containing kanamycin sulfate (Kan). Positive transformants were selected for strain preservation to obtain the corresponding Escherichia coli engineered strains.

[0049] 3. Protein expression (1) Expression of heme oxygenase protein: ① The obtained Pichia yeast engineered strains were inoculated into YPD liquid medium test tubes, cultured at 30°C and 220 rpm for 16-24 h, and then the seed liquid was inoculated into BMGY liquid medium shake flasks (100 mL liquid / 500 mL volume) at a 1% inoculum rate, and cultured at 30°C and 220 rpm until OD 600 When the culture medium reaches 1-3, transfer to BMMY liquid medium, add methanol at a final concentration of 0.5%, and continue culturing for 120 h to induce protein expression. During the induction process, add 0.5% methanol every day to finally obtain the fermentation broth; ② The fermentation broth obtained in step ① was centrifuged at low temperature at 8000×g for 10 min at 4°C. The supernatant was discarded after centrifugation, and the precipitate was washed twice with physiological saline to obtain wet cells containing the target protein; the obtained wet cells were diluted 5 times with 50 mM PBS buffer, and then crushed with a high-pressure homogenizer at 1500 MP to obtain a cell lysate, which is a yeast whole-cell catalyst containing bilirubin oxygenase.

[0050] (2) Expression of biliverdin reductase and glucose dehydrogenase proteins: ① Inoculate the obtained E. coli engineered strain into a test tube of LB liquid culture medium containing Kan, and culture at 37°C and 200 rpm for 8-10 h to obtain seed solution; inoculate the seed solution into a shake flask of LB liquid culture medium containing Kan (100 mL liquid / 500 mL volume) at a 1% inoculum amount, and culture at 37°C and 200 rpm until the OD 600 When the pH value reaches 0.4-0.6, add isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.5 mM, adjust the temperature to 20°C, and continue culturing for 16 h to induce protein expression; obtain fermentation broth respectively; ② The fermentation broth obtained in step ① was centrifuged at low temperature at 8000×g, 10 min, 4°C. The supernatant was discarded after centrifugation, and the precipitate was washed twice with physiological saline to obtain wet bacteria containing the target protein; the obtained wet bacteria were diluted 5 times with 50 mM PBS buffer, and then ultrasonically disrupted with a high-pressure homogenizer at 1200 MP, and the obtained cell lysate was the required catalyst.

[0051] The lysates of yeast cells and E. coli cells obtained above were subjected to SDS-PAGE gel electrophoresis to verify the soluble expression of the protein. Figure 1 As shown, Figure 1 The protein expression diagram of heme oxygenase (HO), biliverdin reductase (BVR) and glucose dehydrogenase (GDH) shows that all the synthesized genes were successfully heterologously expressed in Escherichia coli and showed different expression levels.

[0052] 4. Synthesis of bilirubin (1) Heme conversion to biliverdin: 100 mL reaction system, crude heme oxygenase solution plus 50 g / L of cell lysate, 100 mg / L hemin chloride (dissolved in 0.05 M NaOH), NADP + 0.001 g / L, glucose dehydrogenase cell lysate 20 g / L, Tris-HCl buffer (pH 7.5, 50 mM) was added to 1 mL, placed in a 2 mL EP tube, reacted at 35°C, 200 rpm for 10 min, after the reaction, 50 μL of the reaction solution was taken, 950 μL of methanol was added, centrifuged at 12000×g for 1 min, the supernatant was taken, and the biliverdin concentration was detected by HPLC.

[0053] (2) Conditions for conversion of biliverdin to bilirubin: 100 mL reaction system, 20 g / L of biliverdin reductase cell lysate (the amount of lysate added is: first weigh 1 g / L of whole cells, and then break them to obtain the corresponding lysate to be added), 600 mg / L of biliverdin hydrochloride, 0.001 g / L of NADP, 20 g / L of glucose dehydrogenase cell lysate, and Tris-HCl buffer (pH 7.5, 50 mM) to 1 mL, placed in a 2 mL EP tube, reacted at 35°C, 200 rpm for 10 min, after the reaction was completed, 50 μL of the reaction solution was taken, 950 μL of a mixture of DMSO and dichloromethane (volume ratio 5:1) was added, and the mixture was centrifuged at 12,000 × g for 1 min. The supernatant was taken and the bilirubin concentration was detected according to the HPLC method described in the second example.

[0054] Figure 2-5 HPLC spectra and standard curves of biliverdin and bilirubin. Figure 2 is the chromatogram of biliverdin, Figure 3 is the standard curve of biliverdin, Figure 4 is the chromatogram of bilirubin, Figure 5 The standard curve of bilirubin.

[0055] Second Example: Recombinant Pichia pastoris and Escherichia coli catalyze the synthesis of bilirubin Pichia pastoris lysate and Escherichia coli lysate were used as preferred catalysts for amplified transformation experiments.

[0056] 1. Shake flask experiment (1) Catalyst substrate loading ① In a 100 mL reaction system, add 50 g / L recombinant Pichia pastoris cell lysate, 20 g / L Escherichia coli biliverdin reductase cell lysate and 20 g / L glucose dehydrogenase cell lysate, the solvent is glucose solution (pH 7.5, 50 mM, after adding other substances, use glucose solution to replenish to 100 mL), EDTA-Na2 2 mM, 35℃, 200 rpm.

[0057] Under the above reaction conditions, 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, and 300 mg / L of hemoglobin were added respectively. Sampling was continuously taken during the reaction. 950 μL of DMSO and dichloromethane mixture (volume ratio 5:1) was added to 50 μL of the reaction solution sample, and the mixture was centrifuged at 12000×g for 1 min. The supernatant was taken and the bilirubin concentration was detected by HPLC.

[0058] The reaction results at different substrate concentrations are shown in the instruction manual. Figure 6As shown, the test results show that the substrate heme has a certain inhibitory effect on the reaction process, and the heme concentration should not be too high. At a substrate loading of 1 mM, a good bilirubin yield of 200 mg / L was obtained, with a yield of 99%.

[0059] The final reaction conditions were determined as follows: 50 g / L Pichia pastoris cell lysate, 20 g / L Escherichia coli biliverdin reductase cell lysate and 20 g / L glucose dehydrogenase cell lysate, 200 mg / L heme, and the solvent was glucose solution (pH 7.5, 50 mM), 35°C, and 200 rpm.

[0060] (2) Scale-up culture The reaction conditions are as follows: 5 L bioreactor, 2 L liquid volume, 20 g / L of recombinant Pichia pastoris and Escherichia coli cell lysates respectively (the amount of lysate added is: first weigh 20 g / L of whole cells, and then break them to obtain the corresponding lysate to be added), 200 mg / L of heme, the solvent is glucose solution (pH 7.5, 50 mM, and the glucose solution is used to supplement to 2 L after adding other substances), aeration volume of 10 vvm, 37°C, and 400 rpm.

[0061] During the reaction, samples were continuously taken and the concentrations of bilirubin, biliverdin and heme were detected by HPLC.

[0062] The method for detecting bilirubin concentration is as follows: add 950 μL of DMSO and dichloromethane mixture (volume ratio 5:1) to 50 μL of reaction solution sample, centrifuge at 12000×g for 1 min, take the supernatant, and detect the bilirubin concentration by HPLC.

[0063] The method for detecting the biliverdin concentration is as follows: take 50 μL of the reaction solution sample, add 950 μL of methanol, centrifuge at 12000×g for 1 min, take the supernatant, and detect the biliverdin concentration by HPLC.

[0064] The method for detecting heme concentration is as follows: take 50 μL of reaction solution sample, add 950 μL NaOH (100 mM), centrifuge at 12000×g for 1 min, take the supernatant, and detect the heme concentration by HPLC; the detection conditions of heme are as follows: mobile phase: A (0.5% trifluoroacetic acid aqueous solution); B (acetonitrile: methanol = 9:1); injection program: 0 min, 20% A, 0-30 min, 20%→90% A, 30-31 min, 90%→20% A, 31-35 min, 20% A; column temperature: 40°C, chromatographic column: C18, injection volume: 5 μL, running time 35 min, detection wavelength 400 nm.

[0065] The standard curve of hemoglobin is as attached in the instruction manual. Figure 7 shown.

[0066] According to the HPLC test results, the change curves of the substrate heme, the intermediate product biliverdin and the final product bilirubin in the 2 L system as the reaction proceeds are as follows: Figure 8 As shown, the specific data are shown in Table 1. Table 1 shows the changes in the concentrations of heme, biliverdin and bilirubin over time during the reaction process.

[0067] Table 1 Changes of heme, biliverdin and bilirubin concentrations over time Fig. 9 This is the reaction equation for bilirubin.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for synthesizing bilirubin by using heme to catalyze the whole enzyme, characterized in that: The method comprises the following steps: (1) fermenting an engineered strain of Pichia pastoris expressing heme oxygenase, centrifuging the fermentation broth of Pichia pastoris to obtain a bacterial precipitate, and then crushing the precipitate to obtain a lysate of Pichia pastoris cells expressing heme oxygenase; (2) fermenting the Escherichia coli strain expressing biliverdin reductase, centrifuging the Escherichia coli fermentation broth to obtain a bacterial precipitate, and then crushing the bacterial precipitate to obtain an Escherichia coli cell lysate expressing biliverdin reductase; (3) fermenting the engineered strain of Escherichia coli expressing glucose dehydrogenase, centrifuging the fermented Escherichia coli liquid to obtain a bacterial cell precipitate, and crushing the cell precipitate to obtain a cell lysate of Escherichia coli expressing glucose dehydrogenase; (4) Mix the Pichia pastoris cell lysate expressing heme oxygenase obtained in step (1), the Escherichia coli cell lysate expressing biliverdin reductase obtained in step (2), and the Escherichia coli cell lysate expressing glucose dehydrogenase obtained in step (3), add glucose solution and heme, and react to obtain bilirubin.

2. The method according to claim 1, characterized in that In step (4), the concentration of the Pichia pastoris cell lysate expressing heme oxygenase in the reaction system is 10 to 40 g / L; The concentration of the Escherichia coli cell lysate expressing biliverdin reductase in the reaction system is 10-40 g / L; The concentration of the Escherichia coli cell lysate expressing glucose dehydrogenase in the reaction system is 10 to 40 g / L; The concentration of the glucose solution is 50 mM; The concentration of heme in the reaction system is 0.5-4 mM; The reaction conditions are: ventilation volume of 1-10 vvm, temperature of 30-40°C, time of 1-3 h, and rotation speed of 200-400 rpm.

3. The method according to claim 1, characterized in that In step (1), the OD of the Pichia fermentation broth 600 =120-150; The centrifugation speed is 8000×g, the time is 10 min, and the temperature is 4°C; The construction method of the Pichia pastoris engineered strain expressing heme oxygenase is as follows: The heme oxygenase gene sequence is synthesized according to the amino acid sequence of the heme oxygenase; the amino acid sequence of the heme oxygenase is shown in SEQ ID NO. 1; the heme oxygenase gene sequence is inserted into pPIC9K to construct a recombinant plasmid pPIC9K-CD, and then transformed into Pichia pastoris Pichia pastori In GS115, the Pichia pastoris engineered strain expressing heme oxygenase was obtained.

4. The method according to claim 1, characterized in that: In step (1), the fermentation is carried out by the following steps: The Pichia yeast engineered strain was inoculated into YPD liquid medium, cultured at 30°C and 220 rpm for 16-24 h, then inoculated into BMGY liquid medium at a 1% inoculum amount, and cultured at 30°C and 220 rpm until OD 600 When the reaction mixture reaches 1-3, the mixture is transferred to BMMY liquid culture medium, methanol is added, and the mixture is cultured at 30° C. for 120 h to obtain the fermentation broth.

5. The method according to claim 1, characterized in that: In step (2), the OD of the E. coli fermentation broth is 600 =80-100; The centrifugation speed is 8000×g, the time is 10 min, and the temperature is 4°C; The Escherichia coli engineering strain expressing biliverdin reductase is constructed as follows: The biliverdin reductase gene sequence is synthesized according to the amino acid sequence of biliverdin reductase; the amino acid sequence of the biliverdin reductase is shown in SEQ ID NO. 2; The biliverdin reductase gene sequence was inserted into pET28a(+) to construct a recombinant plasmid BVR-CC, which was then transformed into Escherichia coli E. coli In BL21, the Escherichia coli engineered strain expressing biliverdin reductase was obtained.

6. The method according to claim 1, characterized in that In step (2), the fermentation is carried out by the following steps: The E. coli strain expressing biliverdin reductase was inoculated into LB liquid medium containing Kan, and cultured at 37°C and 200 rpm for 8-10 h. The seed liquid was inoculated into LB liquid medium containing Kan at an inoculum of 1%, and cultured at 37°C and 200 rpm until OD 600 When the pH value reaches 0.4-0.6, isopropyl-β-D-thiogalactoside is added, the temperature is adjusted to 20° C., and the culture is continued for 16 hours to obtain the fermentation broth.

7. The method according to claim 1, characterized in that In step (3), the OD of the E. coli fermentation broth is 600 =80-100; The centrifugation speed is 8000×g, the time is 10 min, and the temperature is 4°C; The construction method of the Escherichia coli engineered strain expressing glucose dehydrogenase is as follows: The glucose dehydrogenase gene sequence was synthesized according to the amino acid sequence of glucose dehydrogenase; the amino acid sequence of glucose dehydrogenase is shown in SEQ ID NO.3, and the glucose dehydrogenase gene sequence was inserted into pET28a(+) to construct a recombinant plasmid GDH-BM, which was then transformed into Escherichia coli E. coli In BL21, the Escherichia coli engineered strain expressing glucose dehydrogenase was obtained.

8. The method according to claim 1, characterized in that In step (3), the fermentation is carried out by the following steps: The engineered strain of Escherichia coli expressing glucose dehydrogenase was inoculated into LB liquid medium containing Kan, and cultured at 37°C and 200 rpm for 8-10 h. The seed liquid was inoculated into LB liquid medium containing Kan at an inoculum of 1%, and cultured at 37°C and 200 rpm until OD 600 When the pH value reaches 0.4-0.6, isopropyl-β-D-thiogalactoside is added, the temperature is adjusted to 20° C., and the culture is continued for 16 hours to obtain the fermentation broth.

9. Use of bilirubin synthesized by the method according to any one of claims 1 to 8 in the medical field.

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