Enzyme for synthesizing bilirubin, gene combination, recombinant plasmid for high-yield bilirubin, engineering bacterium, method and application
By designing multi-enzyme complexes and optimizing codons, we constructed highly efficient recombinant plasmids and engineered bacteria, solving the problems of low enzyme catalytic efficiency and viral contamination risk in bilirubin production, and achieving high-yield, low-cost bilirubin production.
Patent Information
- Application Number
- CN202511120131.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-11
- Publication Date
- 2025-11-14
AI Technical Summary
Existing methods for producing bilirubin rely on animal sources and suffer from low enzyme catalytic efficiency, low conversion rate, high cost, difficulty in scaling up, and risk of viral contamination. Current multi-enzyme complex designs have failed to effectively address issues such as low enzyme catalytic efficiency, competitive consumption of precursors, and exogenous expression barriers.
A multi-enzyme complex design was adopted, including the heme precursor synthesis fusion multi-enzyme complex ALAS-hemB, the bilirubin synthesis multi-enzyme complex GST-hemH-HO1-BVR, and glucose dehydrogenase. Through codon optimization and host metabolic engineering, efficient recombinant plasmids and engineered bacteria were constructed to achieve substrate channelization transport and in-situ regeneration of the coenzyme NADPH.
It has nearly doubled bilirubin production, reduced raw material costs by more than 60%, completely avoided the risk of animal-derived viruses, and provided an efficient, safe, and low-cost bilirubin production solution.
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Figure CN120944842A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of synthetic biology technology, specifically relating to enzymes, gene combinations, recombinant plasmids for high bilirubin production, engineered bacteria, methods, and applications for bilirubin synthesis. Background Technology
[0002] Bilirubin (CAS No. 635-65-4), the main pigment in bile, was once considered a harmful metabolic waste, but recent studies have revealed its significant physiological functions. As a potent antioxidant, bilirubin protects biomolecules from oxidative damage, scavenge oxygen free radicals, and plays an important role in reducing cardiovascular disease risk, neuroprotection, and anti-inflammatory and antiviral effects. Furthermore, bilirubin is a core component of the precious traditional Chinese medicine bezoar (niuhuang), widely used in prepared Chinese medicines such as Angong Niuhuang Wan. However, natural bezoar resources are scarce, and the bilirubin content in artificial bezoar (only 0.7%) is far lower than that in natural bezoar (35%), necessitating an increase in its purity and yield to meet medical needs.
[0003] Currently, bilirubin production mainly relies on extraction from the bile of animals such as pigs and cattle. This method is limited by unstable raw material supply, low extraction efficiency, high cost, and difficulty in large-scale production. In recent years, the development of synthetic biology has provided new ideas for bilirubin biosynthesis. Existing biological methods mainly employ a two-step enzymatic catalytic pathway: heme is catalyzed by heme oxygenase (HO) to produce biliverdin, which is then reduced to bilirubin by biliverdin reductase (BVR). However, this pathway has significant bottlenecks: the catalytic efficiency of HO is much lower than that of BVR, resulting in a low overall conversion rate; and heme still depends on animal sources, posing a risk of mammalian viral contamination. In addition, the bilirubin yield of existing engineered bacteria is generally below 500 mg / L, which is insufficient to meet industrial-scale demands.
[0004] Multi-enzyme complex technology, with its advantages such as efficient substrate channeling and low byproduct generation, has become a potential solution to overcome the aforementioned bottlenecks. However, current technologies for multi-enzyme complex design have not effectively addressed issues such as low enzyme catalytic efficiency, precursor competition and consumption, and exogenous expression barriers in bilirubin synthesis. Therefore, there is an urgent need to develop an efficient, safe, and economical strategy for bilirubin biosynthesis to achieve large-scale green production of bilirubin. Summary of the Invention
[0005] The purpose of this invention is to provide enzymes, gene combinations, high-bilirubin-producing recombinant plasmids, engineered bacteria, methods, and applications for bilirubin synthesis. This invention provides enzyme combinations for bilirubin synthesis, developing an efficient, economical, and safe strategy for bilirubin biosynthesis. Through multi-enzyme complex design, codon optimization, and host metabolic engineering, bilirubin production is synergistically increased, which is of great significance for promoting its industrial application.
[0006] This invention provides an enzyme combination for bilirubin synthesis, comprising a heme precursor synthesis fusion multi-enzyme complex ALAS-hemB, a bilirubin synthesis multi-enzyme complex GST-hemH-HO1-BVR, and glucose dehydrogenase; the amino acid sequence of the heme precursor synthesis fusion multi-enzyme complex ALAS-hemB is shown in SEQ ID NO. 8; the amino acid sequence of the bilirubin synthesis multi-enzyme complex GST-hemH-HO1-BVR is shown in SEQ ID NO. 14; and the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO. 1.
[0007] The present invention also provides a gene combination for bilirubin synthesis, comprising a gene encoding a heme precursor synthesis fusion multienzyme complex ALAS-hemB, a gene encoding a bilirubin synthesis multienzyme complex GST-hemH-HO1-BVR, and a gene encoding glucose dehydrogenase; the nucleotide sequence of the gene encoding the heme precursor synthesis fusion multienzyme complex ALAS-hemB is shown in SEQ ID NO. 9; the nucleotide sequence of the gene encoding the bilirubin synthesis multienzyme complex GST-hemH-HO1-BVR is shown in SEQ ID NO. 15; and the amino acid sequence of the gene encoding glucose dehydrogenase is shown in SEQ ID NO. 2.
[0008] The present invention also provides a recombinant plasmid for high bilirubin production, wherein the recombinant plasmid contains the gene combination for bilirubin synthesis described in the above technical solution.
[0009] Preferably, the backbone vector of the recombinant plasmid includes pET24a.
[0010] Preferably, the full-length nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO.16.
[0011] The present invention also provides an engineered bacterium that produces high levels of bilirubin, wherein the engineered bacterium contains the recombinant plasmid described in the above technical solution.
[0012] Preferably, the host cell of the engineered bacteria includes Escherichia coli; the Escherichia coli includes E. coli Nissle 1917 (DE3).
[0013] This invention also provides a method for constructing engineered bacteria that produce high levels of bilirubin, comprising the following steps:
[0014] The recombinant plasmid described in the above technical solution is transformed into a host cell to obtain an engineered bacterium that produces high levels of bilirubin.
[0015] The present invention also provides the application of the enzyme combination, gene combination, recombinant plasmid, or engineered bacteria described in the above-mentioned technical solutions in the preparation of bilirubin.
[0016] This invention also provides a method for preparing bilirubin, comprising the following steps:
[0017] Using glucose or glycerol as a carbon source, the engineered bacteria described in the above technical solution are fermented, induced, and extracted to obtain bilirubin.
[0018] This invention provides an enzyme assemblages for bilirubin synthesis, comprising the heme precursor synthesis fusion multi-enzyme complex ALAS-hemB, the bilirubin synthesis multi-enzyme complex GST-hemH-HO1-BVR, and glucose dehydrogenase. This invention achieves substrate channelization transport through the design of the multi-enzyme complexes ALAS-hemB and GST-hemH-HO1-BVR, solving the problem of enzyme catalytic efficiency mismatch; and achieves in-situ regeneration of the coenzyme NADPH by coupling with glucose dehydrogenase, eliminating the need for exogenous addition. The enzyme assemblages described in this invention can be used for the efficient, safe, and economical synthesis of bilirubin.
[0019] This invention also provides a gene encoding the enzyme combination used for bilirubin synthesis, which, through codon optimization, significantly increases the expression levels of key enzymes. Based on this gene, this invention further constructs recombinant plasmids and engineered bacteria. Experimental results show that the engineered bacteria of this invention, fermenting with glucose / glycerol as a substrate for 72 hours, achieves a bilirubin yield as high as 786 mg / L, nearly double that of existing engineered bacteria. The bilirubin preparation method of this invention utilizes entirely microbial synthesis, completely avoiding the risk of animal-derived viruses, reducing raw material costs by more than 60%, and providing efficient, safe, and low-cost bilirubin raw materials for artificial bezoar and traditional Chinese medicine. Attached Figure Description
[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0021] Figure 1 This is a gel image showing the protein expression of pET-BS01 in engineered Escherichia coli provided in Comparative Example 1 of this invention;
[0022] Figure 2 This is a diagram of the bilirubin product solution extracted with dichloromethane provided in Comparative Example 1 of the present invention.
[0023] Figure 3The HPLC detection results of the bilirubin product provided in Comparative Example 1 of this invention are shown in the figure.
[0024] Figure 4 This is a standard curve for spectrophotometric quantification of biosynthesized bilirubin provided in Comparative Example 1 of the present invention. Detailed Implementation
[0025] This invention provides an enzyme combination for bilirubin synthesis, comprising a heme precursor synthesis fusion multi-enzyme complex ALAS-hemB, a bilirubin synthesis multi-enzyme complex GST-hemH-HO1-BVR, and glucose dehydrogenase; the amino acid sequence of the heme precursor synthesis fusion multi-enzyme complex ALAS-hemB is shown in SEQ ID NO. 8; the amino acid sequence of the bilirubin synthesis multi-enzyme complex GST-hemH-HO1-BVR is shown in SEQ ID NO. 14; and the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO. 1. This invention designs and constructs a fusion multi-enzyme complex that can improve catalytic efficiency. Specifically, this invention selects 5-aminolevulinic acid synthase (ALAS) from Komagataeibacter diospyri and hemB from Escherichia coli, and connects them to form the fusion multi-enzyme complex ALAS-hemB, which is used to increase the host cell's ability to synthesize heme and solve the problem of precursor competitive consumption. In a specific embodiment, the connection is performed using a linker. In specific embodiments, the protein sequence of the linker can be selected from various flexible and rigid linker sequences. In a specific embodiment, the amino acid sequence of the selected linker is shown in SEQ ID NO. 17. In a specific embodiment, the nucleotide sequence of the codon-optimized heme precursor synthesis fusion multienzyme complex ALAS-hemB is shown in SEQ ID NO. 9. This invention selects the GST tag, heme oxygenase (HO1) from *E. coli*, and biliverdin reductase (BVR) from *Thermosynechococcus elongatus*, and links them to form the fusion multienzyme complex GST-hemH-HO1-BVR, which converts heme synthesized by the host cell into the final product bilirubin. In a specific embodiment, the amino acid sequence of the GST is shown in SEQ ID NO. 10; the amino acid sequence of the heme oxygenase is shown in SEQ ID NO. 11; the amino acid sequence of the heme oxygenase is shown in SEQ ID NO. 12; and the amino acid sequence of the biliverdin reductase is shown in SEQ ID NO. 13. In a specific embodiment, the linking is performed using a linker. In a specific embodiment, the protein sequence of the linker can be selected from a variety of flexible and rigid linker sequences. In a specific embodiment, the amino acid sequence of the selected linker is shown in SEQ ID NO. 17. In a specific embodiment, the nucleotide sequence of the codon-optimized bilirubin synthesis multienzyme complex GST-hemH-HO1-BVR is shown in SEQ ID NO. 15. This invention achieves in-situ regeneration of the coenzyme NADPH by coupling glucose dehydrogenase (PpGDH).In a specific embodiment, the nucleotide sequence of glucose dehydrogenase after codon optimization is shown in SEQ ID NO.2.
[0026] This invention also provides a gene combination for bilirubin synthesis, including a gene encoding the heme precursor synthesis fusion multienzyme complex ALAS-hemB, a gene encoding the bilirubin synthesis multienzyme complex GST-hemH-HO1-BVR, and a gene encoding glucose dehydrogenase; the nucleotide sequence of the gene encoding the heme precursor synthesis fusion multienzyme complex ALAS-hemB is shown in SEQ ID NO. 9; the nucleotide sequence of the gene encoding the bilirubin synthesis multienzyme complex GST-hemH-HO1-BVR is shown in SEQ ID NO. 15; and the amino acid sequence of the gene encoding glucose dehydrogenase is shown in SEQ ID NO. 2. This invention has optimized the E. coli-preferred codons of the genes encoding the heme precursor synthesis fusion multienzyme complex ALAS-hemB, the bilirubin synthesis multienzyme complex GST-hemH-HO1-BVR, and the glucose dehydrogenase, thus overcoming the obstacle of exogenous expression.
[0027] This invention also provides a recombinant plasmid for high bilirubin production, wherein the recombinant plasmid contains the gene combination for bilirubin synthesis described in the above-mentioned technical solution. In a specific embodiment, the recombinant plasmid further includes essential gene expression elements; the essential gene expression elements include a T7 promoter, RBS, a stop codon, and a transcription termination signal. In a specific embodiment, the backbone vector of the recombinant plasmid includes pET24a. In a specific embodiment, the full-length nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO.16. This invention does not impose any special limitations on the preparation method of the recombinant plasmid; conventional synthesis methods can be used.
[0028] This invention also provides a high-bilirubin-producing engineered bacterium, wherein the engineered bacterium contains the recombinant plasmid described in the above-mentioned technical solution. In a specific embodiment, the host cell of the engineered bacterium includes Escherichia coli; the Escherichia coli includes E. coli Nissle 1917(DE3).
[0029] This invention also provides a method for constructing engineered bacteria that produce high levels of bilirubin, comprising the following steps:
[0030] The recombinant plasmid described in the above technical solution is transformed into host cells to obtain engineered bacteria that produce high levels of bilirubin. In a specific embodiment, the host cells include *Escherichia coli*, specifically *E. coli* Nissle 1917(DE3). Specifically, this invention transforms the recombinant plasmid described in the above technical solution into competent *E. coli* Nissle 1917(DE3) cells to obtain engineered bacteria. This invention does not specifically limit the transformation method; conventional transformation methods are acceptable. The engineered bacteria of this invention ferment using glucose / glycerol as a substrate, achieving high bilirubin production.
[0031] This invention also provides the application of the enzyme combination, gene combination, recombinant plasmid, or engineered bacteria described in the above-mentioned technical solutions in the preparation of bilirubin. The bilirubin preparation process of this invention utilizes a microbial synthesis method throughout, avoiding the risk of animal-derived contamination, eliminating the need for exogenous heme and NADPH addition, and reducing raw material costs by more than 60%. This invention provides an efficient, safe, and low-cost bilirubin production solution for artificial bezoar and traditional Chinese medicine.
[0032] This invention also provides a method for preparing bilirubin, comprising the following steps:
[0033] Using glucose or glycerol as a carbon source, the engineered bacteria described in the above technical solution are fermented, induced, and extracted to obtain bilirubin. In this invention, the induction includes induction using IPTG. In a specific embodiment, the induction culture temperature is 20°C, and the culture time after induction is 48–72 h. After 48–72 h of culture after induction, the wet bacterial cells are collected by centrifugation, reselected, and ultrasonically disrupted to obtain cell lysate. In a specific embodiment, the extraction solvent used includes dichloromethane. This invention mixes the cell lysate with dichloromethane to extract bilirubin. In this invention, the culture medium using glucose or glycerol as a carbon source includes TB liquid medium.
[0034] To further illustrate the present invention, the enzymes, gene combinations, high-bilirubin recombinant plasmids, engineered bacteria, methods, and applications for bilirubin synthesis provided by the present invention are described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of the present invention.
[0035] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.
[0036] The following are the materials and reagents used in the examples:
[0037] E. coli Nissle 1917(DE3) competent cells were purchased from Shanghai Weidi.
[0038] Transformation of recombinant plasmids:
[0039] Add 2 μL (100 μg / mL) of recombinant plasmid to a centrifuge tube containing competent *E. coli* cells that has been thawed in an ice bath. Gently tap the tube wall to mix, and incubate on ice for 30 min. Heat shock at 42°C for 90 s, then immediately incubate on ice for 5 min. Under aseptic conditions, add 786 μL of LB medium to the centrifuge tube, mix by pipetting, and incubate at 37°C and 180 rpm for 60 min with shaking. Centrifuge the tube at 6000 rpm for 5 min, remove the 786 μL supernatant, mix the remaining liquid by pipetting, and spread it onto an LB agar plate containing 50 μg / mL kanamycin. Incubate the LB plate upside down at 37°C overnight until single colonies are clearly visible. Pick positive transformants for colony PCR verification to obtain the genetically engineered *E. coli* strain.
[0040] SDS-PAGE gel electrophoresis:
[0041] Sample preparation: Take wet bacterial cells containing the target protein and prepare a 20 g / L cell suspension using Tris-HCl buffer (pH 7.5, 100 mM). Sonicate the cells on ice (450 W, 1 s on, 4 s off, 10 min total). Centrifuge at 12,000 × g, 4 °C for 1 min to separate the supernatant and precipitate. Take 20 μL of the supernatant and mix it with 4 μL of 5 × SDS PAGE loading buffer. Heat in a boiling water bath for 10 min, then centrifuge at 12,000 × g for 5 min. Electrophoresis: Protein gel loading volume: 10 μL sample, 3 μL marker; Electrophoresis conditions: 160 V, 60 min. Staining: Immerse the protein gel in staining solution for 15 min, then wash twice with water. Destaining: Immerse in destaining solution and gently shake, replacing with fresh destaining solution until the bands are clearly visible.
[0042] 1M DTT: Weigh 1.543g of DTT, dissolve it in 6ml of ddH2O, and bring the volume to 10mL. Store at -20℃. DTT was purchased from BBI Corporation.
[0043] 5×SDS-PAGE loading buffer: Measure 12.5 mL of 1M Tris-HCl buffer (pH 6.8) and 25 mL of glycerol. Weigh 5 g of SDS and 0.25 g of bromophenol blue, dissolve in ddH2O, and bring the volume to 80 mL. Store at 4°C. For each use, mix 2 mL of the above solution with 0.5 mL of 1M DTT at a 4:1 ratio, mix thoroughly, and store at 4°C.
[0044] 5×Tris-Gly SDS PAGE electrophoresis buffer: Weigh 15.1g Tris base, 94g glycine, and 5g SDS, dissolve in approximately 800mL ddH2O by stirring, then bring the volume up to 1L and store at room temperature.
[0045] SDS-PAGE staining solution: Prepare the solution according to the ratio of anhydrous ethanol:ddH2O:glacial acetic acid = 4.5:4.5:1 (volume ratio), add 1g of Coomassie Brilliant Blue R250, dissolve thoroughly, and then make up to 1L. Store at room temperature.
[0046] SDS-PAGE decolorization solution: Prepare the solution according to the ratio of anhydrous ethanol:ddH2O:glacial acetic acid = 25:65:8 (volume ratio) and store at room temperature.
[0047] Bilirubin test:
[0048] The bilirubin detection method in the detection methods for bezoar in Part I of the Pharmacopoeia was adopted.
[0049] Comparative Example 1
[0050] Selection of glucose dehydrogenase, heme oxygenase and biliverdin reductase, and construction and expression of recombinant plasmids.
[0051] Glucose dehydrogenase (PpGDH) from Paenibacilluspini was selected as the catalytic enzyme for in-situ regeneration of the coenzyme, and its amino acid sequence is shown in SEQ ID NO.1. The nucleotide sequence encoding the glucose dehydrogenase, optimized by Hongxun Bio's codon optimization software, is shown in SEQ ID NO.2.
[0052] The amino acid sequence of heme oxygenase, which catalyzes the conversion of heme to biliverdin, with the addition of the GST fusion tag is shown in SEQ ID NO.3. The nucleotide sequence of the heme oxygenase fused with the GST tag, optimized by Hongxun Bio's codon optimization software, is shown in SEQ ID NO.4.
[0053] The biliverdin reductase, which catalyzes the conversion of biliverdin to bilirubin, was selected, and its amino acid sequence after adding the GST fusion tag is shown in SEQ ID NO. 5. The nucleotide sequence encoding the biliverdin reductase fused with the GST tag, optimized by Hongxun Bio's codon optimization software, is shown in SEQ ID NO. 6.
[0054] Using pET24a as the backbone vector, the nucleotide sequences of the above three enzymes were modified by adding essential gene expression elements such as the T7 promoter, RBS, stop codon, and transcription termination signal. The full sequence of the recombinant plasmid designed is shown in SEQ ID NO.7. The recombinant plasmid is named pET-BS01.
[0055] The recombinant plasmid pET-BS01 sequence was synthesized artificially using the Hongxun Biotechnology gene synthesis platform to obtain the recombinant plasmid. This plasmid was then transformed into E. coli Nissle 1917(DE3) competent cells to obtain the engineered strain.
[0056] The pET-BS01 engineered strain was inoculated into test tubes containing LB liquid medium (concentration: 50 mg / L) of kanamycin and cultured at 37°C and 200 rpm for 8–10 h to obtain seed culture. The seed culture was then inoculated at a 1% (v / v) inoculation rate into shake flasks containing TB liquid medium (50 mL / 500 mL volume) of kanamycin and cultured at 37°C and 200 rpm until OD... 600 Once the concentration reaches 0.4–0.6, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM, adjust the temperature to 20 °C, and continue culturing for 72 h to induce protein expression; the fermentation broth is then obtained.
[0057] The obtained fermentation broth was centrifuged at low temperature at 8,000 rpm for 10 min at 4℃. After centrifugation, the supernatant was discarded, and the precipitate was washed twice with physiological saline to obtain wet bacterial cells containing the target protein. The obtained wet bacterial cells were resuspended in 50 mL of physiological saline and ultrasonically disrupted under the conditions of 400 W, 0℃, working for 2 s, intermittent for 4 s, and a total time of 20 min to obtain cell lysate.
[0058] 20 μL of the obtained cell lysis buffer was used to verify the soluble expression of proteins by SDS-PAGE gel electrophoresis. Protein expression results are shown below. Figure 1 As shown in the figure, the results indicate that all three proteins were successfully expressed.
[0059] The remaining cell lysate was extracted three times with 10 mL of dichloromethane to obtain bilirubin. The extracts were combined, diluted 10-fold with dichloromethane, and the content was determined spectrophotometrically. The extracted bilirubin was as follows: Figure 2 As shown. HPLC detection results are as follows. Figure 3 As shown, the standard curve for spectrophotometric quantification of biosynthesized bilirubin is as follows: Figure 4 As shown in the figure. The results showed that biosynthesized bilirubin was obtained, with a yield of 90 mg / L.
[0060] Example 2
[0061] Design of a fusion multienzyme complex for heme precursor synthesis.
[0062] 5-Aminolevulinic acid synthase (ALAS) from *Komagataeibacter diospyri* and hemB from *E. coli* were linked by a linker to form a fusion multi-enzyme complex, ALAS-hemB, to increase the host cell's ability to synthesize heme. The protein sequence of the linker can be selected from various flexible and rigid linker sequences. In this embodiment, LEGKSSGSGSESKST (SEQ ID NO. 17) was selected as the linker. The amino acid sequence of the designed fusion multi-enzyme complex ALAS-hemB is shown in SEQ ID NO. 8.
[0063] The nucleotide sequence of the fusion multienzyme complex ALAS-hemB, optimized by Hongxun Bio's codon optimization software, is shown in SEQ ID NO.9.
[0064] Example 3
[0065] Design of a multi-enzyme complex for bilirubin synthesis.
[0066] The following amino acid sequences were selected: GST tag (amino acid sequence shown in SEQ ID NO. 10), hemH from *E. coli* (amino acid sequence shown in SEQ ID NO. 11), heme oxygenase from *Thermosynechococcus elongatus* (amino acid sequence shown in SEQ ID NO. 12), and biliverdin reductase from rat (amino acid sequence shown in SEQ ID NO. 13). These were linked by a linker to form a fusion multi-enzyme complex, GST-hemH-HO1-BVR, which converts heme synthesized by the host cell into the final product bilirubin. The linker protein sequence can be selected from various flexible and rigid linker sequences. In this embodiment, LEGKSSGSGSESKST (SEQ ID NO. 17) was selected as the linker. The amino acid sequence of the designed fusion multi-enzyme complex GST-hemH-HO1-BVR is shown in SEQ ID NO. 14, and the codon-optimized nucleotide sequence after optimization using Hongxun Biotechnology's codon optimization software is shown in SEQ ID NO. 15.
[0067] Example 4
[0068] Design and synthesis of expression plasmids for glucose dehydrogenase, heme precursor synthesis multienzyme complex ALAS-hemB, and bilirubin synthesis multienzyme complex GST-hemH-HO1-BVR.
[0069] Using pET24a as the backbone vector, the nucleotide sequences of the above three enzymes and multi-enzyme complex proteins were supplemented with essential gene expression elements such as the T7 promoter, RBS, stop codon, and transcription termination signal. The full sequence of the recombinant plasmid designed is shown in SEQ ID NO.16. This recombinant plasmid is named pET-BS02.
[0070] The recombinant plasmid pET-BS02 (SEQ ID NO.16) sequence was synthesized artificially using the Hongxun Biotechnology gene synthesis platform to obtain the recombinant plasmid. This plasmid was then transformed into E. coli Nissle 1917(DE3) competent cells to obtain the engineered strain.
[0071] Example 5
[0072] Bilirubin synthesis and yield identification by multi-enzyme complex expression strains.
[0073] The pET-BS02 engineered strain was inoculated into test tubes containing LB liquid medium (concentration: 50 mg / L) of kanamycin and cultured at 37°C and 200 rpm for 8–10 h to obtain seed culture. The seed culture was then inoculated at a 1% (v / v) inoculation rate into shake flasks containing TB liquid medium (50 mL / 500 mL volume) of kanamycin and cultured at 37°C and 200 rpm until OD... 600 When the concentration 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 72 h to induce protein expression and product synthesis; thus obtaining the fermentation broth.
[0074] The obtained fermentation broth was centrifuged at low temperature at 8,000 rpm for 10 min at 4℃. After centrifugation, the supernatant was discarded, and the precipitate was washed twice with physiological saline to obtain wet bacterial cells containing the target protein. The obtained wet bacterial cells were resuspended in 50 mL of physiological saline and ultrasonically disrupted under the conditions of 400 W, 0℃, working for 2 s, intermittent for 4 s, and a total time of 20 min to obtain cell lysate.
[0075] The cell lysate was extracted three times with 10 mL of dichloromethane to extract the bilirubin product. The extracts were combined, diluted 10 times with dichloromethane, and the content was determined by spectrophotometry to obtain biosynthesized bilirubin with a yield of 786 mg / L.
[0076] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. An enzyme assembly for the synthesis of bilirubin, characterized in that, The invention comprises a heme precursor synthesis fusion multienzyme complex ALAS-hemB, a bilirubin synthesis multienzyme complex GST-hemH-HO1-BVR, and glucose dehydrogenase; the amino acid sequence of the heme precursor synthesis fusion multienzyme complex ALAS-hemB is shown in SEQ ID NO.8; the amino acid sequence of the bilirubin synthesis multienzyme complex GST-hemH-HO1-BVR is shown in SEQ ID NO.14; and the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO.
1.
2. A gene combination for synthesizing bilirubin, characterized in that, The invention includes genes encoding the heme precursor synthesis fusion multienzyme complex ALAS-hemB, the bilirubin synthesis multienzyme complex GST-hemH-HO1-BVR, and glucose dehydrogenase; the nucleotide sequence of the gene encoding the heme precursor synthesis fusion multienzyme complex ALAS-hemB is shown in SEQ ID NO. 9; the nucleotide sequence of the gene encoding the bilirubin synthesis multienzyme complex GST-hemH-HO1-BVR is shown in SEQ ID NO. 15; and the amino acid sequence of the gene encoding glucose dehydrogenase is shown in SEQ ID NO.
2.
3. A recombinant plasmid that produces high levels of bilirubin, characterized in that, The recombinant plasmid contains the gene combination for bilirubin synthesis as described in claim 2.
4. The recombinant plasmid according to claim 3, characterized in that, The backbone vector of the recombinant plasmid includes pET24a.
5. The recombinant plasmid according to claim 3 or 4, characterized in that, The full-length nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO.
16.
6. A high-bilirubin-producing engineered bacterium, characterized in that, The engineered bacteria contain the recombinant plasmid as described in any one of claims 3 to 5.
7. The engineered bacteria according to claim 6, characterized in that, The host cells of the engineered bacteria include Escherichia coli; the Escherichia coli includes E. coli Nissle 1917(DE3).
8. A method for constructing an engineered bacterium that produces high levels of bilirubin, characterized in that, Includes the following steps: Transform the recombinant plasmid according to any one of claims 3 to 5 into a host cell to obtain an engineered bacterium that produces high levels of bilirubin.
9. The application of the enzyme combination of claim 1, the gene combination of claim 2, the recombinant plasmid of any one of claims 3 to 5, or the engineered bacteria of claim 6 or 7 in the preparation of bilirubin.
10. A method for preparing bilirubin, characterized in that, Includes the following steps: Using glucose or glycerol as a carbon source, the engineered bacteria described in claim 6 or 7 are fermented, induced, and extracted to obtain bilirubin.