Heme oxygenase mutant and application thereof in preparation of bilirubin by multi-enzyme coupling

Abstract

The application discloses a kind of heme oxygenase mutant and its multienzyme coupling preparation of application of bilirubin, belong to biological catalysis engineering field.The application obtains the positive mutant of heme oxygenase by directed evolution technology, can efficiently catalyze heme to synthesize chlorophyllin;Further introduce chlorophyllin reductase, coupling cytochrome P450 reductase, formic acid dehydrogenase, and two kinds of core enzyme are coupled with exogenous auxiliary enzyme, constructs recombinant escherichia coli, catalyzes heme to synthesize bilirubin.In the system, without exogenous addition NADPH, reaction time is about 4h, and bilirubin yield is as high as 2.10g / L, and no intermediate accumulation is observed in catalytic process, reaches the highest yield of the synthesis of bilirubin reported at home and abroad, and the shortest reaction time level.Compared with traditional pig bile extraction, the method of the application breaks through the resource limit, and the operation is simple, the yield is high, realizes the efficient, green synthesis of bilirubin.

Description

Technical Field

[0001] This invention relates to a heme oxygenase mutant and its application in the preparation of bilirubin via multi-enzyme coupling, belonging to the field of biocatalysis engineering. Background Technology

[0002] Bilirubin (CAS No. 635-65-4) is the main pigment in bile. Excessive accumulation of bilirubin in the body (especially in newborns) can easily lead to various diseases. For example, bilirubin deposition in the central nervous system can cause bilirubin encephalopathy in newborns, impairing hearing, motor function, and mental function; and high concentrations of free bilirubin in plasma can easily cause jaundice. Therefore, for a long time, bilirubin was considered a metabolic waste product.

[0003] However, in recent years, with in-depth research on bile pigments, their beneficial physiological effects have been gradually discovered and reported. Bilirubin has antioxidant properties, physiologically preventing the oxidation of biomolecules such as fats and proteins and scavenging oxygen free radicals; appropriate concentrations of bilirubin in serum can prevent cardiovascular and metabolic diseases; and it plays a key role in reducing neuronal damage in the central nervous system.

[0004] Given the important role and potential market value of bilirubin, its production methods have been developed and reported. Traditional preparation methods involve extraction from the bile of livestock such as pigs and cattle. Recent research progress has mainly focused on the development of extraction and purification processes. However, this method is limited by the availability of pig bile resources, resulting in low extraction rates and yields that cannot meet market demand. With the development of synthetic biology, biocatalysis, as a green manufacturing method, has attracted widespread attention in the synthesis of high-value-added bio-organic molecules in recent years. Biocatalysis has natural advantages, such as mild reaction conditions, environmental friendliness, almost no byproduct formation, and high stereoselectivity.

[0005] Eukaryotes typically possess a complete bilirubin synthesis pathway. A key metabolic intermediate in this pathway is heme. Heme undergoes ring-opening oxidation catalyzed by heme oxygenase (HO, EC 1.14.14.18) to form the intermediate biliverdin. Biliverdin then undergoes hydrogenation reduction catalyzed by biliverdin reductase (BVR, EC 1.3.1.24) to revert to bilirubin. The reaction equation is as follows: Figure 5 As shown.

[0006] To date, there are limited reports on the biosynthesis of bilirubin and its key intermediates. For example, Yan Sihan et al. constructed a heme oxygenase and glutamate dehydrogenase coenzyme regeneration system derived from Clostridium tetani and displayed it on a membrane surface, achieving a biliverdin yield of 76.3 mg / L using whole-cell E. coli as a catalyst. Jianfeng Mei et al. used biliverdin as a substrate and E. coli as a host to express biliverdin reductase derived from cyanobacteria as a whole-cell catalyst, achieving a bilirubin yield of 325 mg / L. Chinese invention patent CN119331839A constructed recombinant E. coli with a bilirubin yield of 1611 mg / L, but the yield is still insufficient. How to further increase bilirubin yield and achieve efficient bilirubin preparation under higher substrate concentrations has become a research hotspot and challenge.

[0007] Research reports indicate that the catalytic synthesis of bilirubin using heme as a substrate involves two steps: the first step involves heme oxygenase catalyzing the synthesis of biliverdin from heme, and the second step involves biliverdin reductase catalyzing the synthesis of bilirubin from biliverdin. The reaction formulas are as follows: Figure 5 As shown, the rate of heme conversion catalyzed by heme oxygenase in the first step of the reaction is much lower than that catalyzed by biliverdin reductase in the second step. This severe imbalance in the catalytic functions of the two core enzymes results in low efficiency in the synthesis of bilirubin from the substrate heme using the multi-enzyme coupled system. Although preliminary enzyme engineering modifications have been performed on heme oxygenase, further increasing the substrate concentration remains a significant challenge. Therefore, further targeted evolution of heme oxygenase is needed to enhance its industrial properties. Furthermore, since the reaction requires expensive coenzymes, coupling a coenzyme regeneration system to the reaction system of the two core enzymes to achieve a one-pot, highly efficient catalytic synthesis of bilirubin from heme is of great significance. Summary of the Invention

[0008] This invention synthesizes heme oxygenase (ScHO), cytochrome P450 reductase (ScCPR), and biliverdin reductase (MmBVR) based on the bilirubin biosynthesis pathway. Using the model strain *Escherichia coli* BL21(DE3) as the expression host, all protein sequences were codon-optimized. To improve the soluble expression levels of the proteins, the transmembrane regions of ScHO and ScCPR were truncated during gene synthesis. The truncated sequences are the C-terminal amino acid sequences 289-318 of ScHO and the N-terminal amino acid sequences 1-24 of ScCPR (hereinafter, ScHO and ScCPR refer to the truncated proteins). Additionally, NADP was expressed in the engineered strain. + Formate-dependent dehydrogenases are used for the regeneration of the coenzyme NADPH.

[0009] Because the rate of heme conversion catalyzed by heme oxygenase is much lower than that catalyzed by biliverdin reductase, ScHO underwent enzymatic modification. Specifically, through random full-sequence mutation, three single-point mutants with enhanced activity were obtained, and combined mutants with further enhanced activity were obtained by combining mutations of the single-point mutants.

[0010] This invention utilizes directed evolution technology to obtain a positive mutant of heme oxygenase, which can efficiently catalyze the synthesis of biliverdin from heme. It further introduces biliverdin reductase to catalyze the conversion of biliverdin to bilirubin; simultaneously, it couples cytochrome P450 reductase to provide sufficient electrons for heme oxygenase; and by using formate dehydrogenase for in-situ regeneration of the coenzyme, and coupling the two core enzymes with exogenous coenzymes, it constructs recombinant *E. coli*, enabling the recombinant bacteria to catalyze the synthesis of bilirubin from heme substrates. In this system, a one-pot reverse reaction can be completed without the need for exogenous NADPH, with a reaction time of approximately 4 hours and a bilirubin yield as high as 2.10 g / L. No intermediate product accumulation was observed during the catalytic process, achieving the highest yield and shortest reaction time reported in bilirubin synthesis both domestically and internationally to date.

[0011] This invention provides a heme oxygenase mutant, which is obtained by mutating alanine at position 29 of the heme oxygenase amino acid sequence as shown in SEQ ID NO.1 to lysine, and is named A29K;

[0012] Alternatively, it can be obtained by mutating the asparagine at position 31 of the heme oxygenase, as shown in SEQ ID NO.1, to histidine, and named N31H;

[0013] Alternatively, it can be obtained by mutating asparagine at position 258 of heme oxygenase, as shown in SEQ ID NO.1, to methionine, and named N258M;

[0014] Alternatively, the alanine at position 29 of the heme oxygenase shown in SEQ ID NO.1 can be mutated to lysine, and the asparagine at position 31 of the heme oxygenase shown in SEQ ID NO.1 can be mutated to histidine, and named A29K / N31H.

[0015] Alternatively, the amino acid sequence of heme oxygenase, as shown in SEQ ID NO.1, can be modified by mutating alanine at position 29 to lysine, as well as asparagine at position 31 to histidine, and asparagine at position 258 to methionine, and named A29K / N31H / N258M.

[0016] The amino acid sequence of the parental enzyme heme oxygenase (WT) is shown in SEQ ID NO.1:

[0017] MEDSSNTIIPSPTDVGALANRINFQTRDAHNKINTFMGIKMAIAMRHGFIYRQGILAYYYVFDAIEQEIDRLLNDPVTEEELQTSTILKQFWLEDFRRSTQIYKDLKLLYSNTFKSTESLNEFLATFQKPPLLQQFINNIHENI HKEPCTILSYCHVLYLALFAGGKLIRSNLYRRLGLFPNFEKLSQKELVKKGTNFFTFSDLGPTEETRLKWEYKKNYELATRTELTEAQKLQIISVAEGIFDWNFNIVAEIGELNRRELMGKFSFKCITYLYEEWMFNKDSATRR

[0018] The nucleotide sequence encoding the parental enzyme heme oxygenase (WT) is shown in SEQ ID NO.2:

[0019] atggaagatagcagcaacaccatcatcccgtccccgaccgatgttggcgccctggcgaaccgtatcaacttccagacccgcgatgcgcacaacaaaatcaacaccttcatgggtatcaaaatggcgatcgcaatgcgtcacggcttcatctaccgccagggcatcctggcgtactactacgttttcgacgcgatcgaacaggaaatcgatcgtctgctgaacgatccggtgaccgaagaagaattgcagacctccaccatcctgaaacagttctggctggaggacttccgccgtagcacccagatctacaaagatctgaaactgctgtacagcaacaccttcaaatccaccgaaagcctgaacgagttcctggcgaccttccagaaaccgccgctgctgcaacagttcatcaacaacatccatgaaaacatccacaaagaaccgtgcaccatcctgtcctactgccacgttctgtacctggccctgttcgcgggcggcaaactgatccgttccaacctgtaccgtcgcctgggtctgttcccgaacttcgaaaaactgtctcagaaagaactggttaaaaaaggcaccaacttcttcaccttctccgacctgggcccgaccgaagaaacccgcctgaaatgggaatataaaaagaactacgaactggcaacccgtaccgaactgaccgaagcgcagaaactgcaaatcatctccgttgcggaaggtatcttcgactggaactttaacatcgtggcggaaatcggcgaactgaaccgtcgtgaactgatgggcaaattctctttcaaatgcatcacctacctgtacgaagaatggatgttcaacaaagatagcgcgacccgccgttaa

[0020] The nucleotide sequence encoding the mutant enzyme A29K / N31H / N258M is shown in SEQ ID NO.6:

[0021] atggaagatagcagcaacaccatcatcccgtccccgaccgatgttggcgccctggcgaaccgtatcaacttccagacccgcgataaacaccataaaatcaacaccttc atgggtatcaaaatggcgatcgcaatgcgtcacggcttcatctaccgccagggcatcctggcgtactactacgttttcgacgcgatcgaacaggaaatcgatcgtctg ctgaacgatccggtgaccgaagaagaattgcagacctccaccatcctgaaacagttctggctggaggacttccgccgtagcacccagatctacaaagatctgaaactg ctgtacagcaacaccttcaaatccaccgaaagcctgaacgagttcctggcgaccttccagaaaccgccgctgctgcaacagttcatcaacaacatccatgaaaacatcc acaaagaaccgtgcaccatcctgtcctactgccacgttctgtacctggccctgttcgcgggcggcaaactgatccgttccaacctgtaccgtcgcctgggtctgttcc cgaacttcgaaaaactgtctcagaaagaactggttaaaaaaggcaccaacttcttcaccttctccgacctgggcccgaccgaagaaacccgcctgaaatgggaatataa aaagaactacgaactggcaacccgtaccgaactgaccgaagcgcagaaactgcaaatcatctccgttgcggaaggtatcttcgactggaactttaacatcgtggcgga aatcggcgaactgatgcgtcgtgaactgatgggcaaattctctttcaaatgcatcacctacctgtacgaagaatggatgttcaacaaagatagcgcgacccgccgttaa

[0022] The present invention also provides a gene encoding the above-mentioned heme oxygenase mutant.

[0023] The present invention also provides a recombinant vector carrying the above-mentioned genes.

[0024] The present invention also provides recombinant cells expressing the above-mentioned mutants or carrying the above-mentioned genes or recombinant vectors.

[0025] In one embodiment of the present invention, the recombinant cells are bacteria or fungi as host cells.

[0026] The present invention also provides a recombinant enzyme catalyst containing the above-mentioned heme oxygenase mutant, which is any one of the following forms:

[0027] (1) Culture recombinant expression transformants containing the heme oxygenase mutant and isolate transformant cells containing the recombinant heme oxygenase mutant enzyme;

[0028] (2) Culture recombinant expression transformants containing the heme oxygenase mutant, isolate transformant cells containing the recombinant heme oxygenase mutant enzyme, and break the transformant cells containing the recombinant heme oxygenase mutant enzyme to obtain cell lysate;

[0029] (3) Cultivate recombinant expression transformants containing the heme oxygenase mutant, isolate transformant cells containing the recombinant heme oxygenase mutant enzyme, break the transformant cells containing the recombinant heme oxygenase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate of the recombinant heme oxygenase mutant enzyme to obtain lyophilized enzyme powder.

[0030] The present invention also provides a method for improving the conversion rate of heme oxygenase to substrate and / or improving its enzyme activity, wherein the method comprises mutating alanine at position 29 of the heme oxygenase as shown in SEQ ID NO.1 to lysine;

[0031] Alternatively, the asparagine at position 31 of the heme oxygenase amino acid sequence shown in SEQ ID NO.1 may be mutated to histidine;

[0032] Alternatively, the asparagine at position 258 of the heme oxygenase amino acid sequence shown in SEQ ID NO.1 may be mutated to methionine;

[0033] Alternatively, the amino acid sequence of heme oxygenase, as shown in SEQ ID NO.1, may be modified by mutating alanine at position 29 to lysine, as well as asparagine at position 31 to histidine, and asparagine at position 258 to methionine.

[0034] The present invention also provides the use of the above-mentioned mutants, recombinant cells, or recombinase catalysts in the preparation of biliverdin or bilirubin.

[0035] In one embodiment of the present invention, the application in the preparation of biliverdin includes a reaction system comprising a whole-cell reaction system and an enzyme reaction system, wherein:

[0036] The whole-cell reaction was performed using heme chloride as a substrate, and the recombinant cells also expressed NADP derived from Azospirillum palustre. + Formate-dependent dehydrogenase, cytochrome P450 reductase derived from Saccharomyces cerevisiae;

[0037] The enzyme reaction system is as follows: using heme chloride as the substrate, and simultaneously adding NADP derived from Azospirillum palustre. + Formate-dependent dehydrogenase, cytochrome P450 reductase derived from Saccharomyces cerevisiae;

[0038] In one embodiment of the present invention, the application in the preparation of bilirubin includes a reaction system comprising a whole-cell reaction system and an enzyme reaction system, wherein:

[0039] The whole-cell reaction was performed using heme chloride as a substrate. The recombinant cells also expressed biliverdin reductase from Musmusculus and NADP from Azospirillum palustre. + Formate-dependent dehydrogenase, cytochrome P450 reductase derived from Saccharomyces cerevisiae;

[0040] The enzyme reaction system is as follows: using heme chloride as the substrate, and simultaneously adding biliverdin reductase from Mus musculus and NADP from Azospirillum palustre. + Formate-dependent dehydrogenase, cytochrome P450 reductase derived from Saccharomyces cerevisiae.

[0041] This invention provides a genetically engineered bacterium that expresses heme oxygenase or a mutant derived from *Saccharomyces cerevisiae*, the wild-type nucleotide sequence of which is shown in SEQ ID NO. 2; cytochrome P450 reductase derived from *Saccharomyces cerevisiae*, the nucleotide sequence of which is shown in SEQ ID NO. 3; biliverdin reductase derived from *Mus musculus*, the nucleotide sequence of which is shown in SEQ ID NO. 4; and NADP derived from *Azospirillum palustre*. +The nucleotide sequence of the formate-dependent dehydrogenase is shown in SEQ ID NO.5.

[0042] SEQ ID NO.3:

[0043]

[0044] SEQ ID NO.4:

[0045] Atgagcaccgaaccgaaacgtaaattcggcgttgttgttgttggtgttggtcgcgcgggcagcgttcgtatccgtgatctgaaagatccgcactctagcgcgttcctgaacctgatcggttacgtttctcgtcgtgaactgggcagcctggataacgttcgtcagatctctctggaagatgcgctgcgtagccaggaagttgatgttgcatacatctgcactgaatcctcttcccacgaggactacatccgtcagttcctccaggcgggtaaacacgttctggttgaatacccgatggcgctgagcttcgcggcggcgcaggaactgtgggaactggcggcgcagaaaggtcgtgttctgcacgaagaacacatcgaactgctgatggaagaatttgagttcctgaaacgtgaagttgcaggcaaagaactgctgaaaggcagcctgcgtttcaccgcgagcccgctggaagaagaaaaattcggcttcccggcgttcagcggcatctctcgtctgacctggctggtgtctctgttcggtgaactgtctctgatcagcgcgactatggaaaaccgtaaagaagatcagtatatgaaaatgaccgttcagctggaaacccagaacaaatctccgctgagctggattgaagaaaaaggtccgggcctgaaacgtaaccgtcacatcagcatccacttcaaatccggcagcctggaagaagttccgaacgttggtgttaacaaaaacatcttcctgaaagatcaggatattttcatccagaaactgctgggccaggttagcgcggaagatctggcggcggaaaagaaacgtatcctgcactgcctggaactggcgagcgatattcagcgtctgtgccaccgtaaacagtaa

[0046] SEQ ID NO.5:

[0047]

[0048] In one embodiment of the present invention, the genetically engineered bacteria include, but are not limited to, Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and yeast.

[0049] In one embodiment of the present invention, the genetically engineered bacteria is recombinant Escherichia coli.

[0050] In one embodiment of the present invention, the recombinant Escherichia coli is expressed as E. coli BL21(DE3).

[0051] In one embodiment of the present invention, the recombinant Escherichia coli is expressed using pET28a(+), pET21a(+), pET32a(+), pETDuet-1, pRSFDuet-1, or pCDFDuet-1 as an expression vector.

[0052] In one embodiment of the present invention, the high-performance liquid chromatography detection method refers to detecting the content of biliverdin, a product catalyzed by heme oxygenase, or the content of bilirubin, a product of a two-step reaction.

[0053] In one embodiment of the present invention, the enzyme activity of the heme oxygenase is measured by high-performance liquid chromatography (HPLC) analysis of the peak area of ​​the biliverdin product, which is obtained by reacting with heme as a substrate. Screening for heme oxygenase mutants is performed by comparing enzyme activities.

[0054] The present invention also provides base sequences encoding the above-mentioned heme oxygenase, biliverdin reductase, formate dehydrogenase and chaperone protein.

[0055] The present invention also provides one or more recombinant vectors carrying the above-mentioned genes.

[0056] In one embodiment of the present invention, the recombinant vector is pET28a(+), pET21a(+), pET32a(+), pETDuet-1, pRSFDuet-1 or pCDFDuet-1 as the expression vector.

[0057] The present invention also provides one or more recombinant cells expressing the above-mentioned genes or carrying the above-mentioned recombinant vectors.

[0058] In one embodiment of the present invention, the recombinant cells use Escherichia coli as the expression host.

[0059] In one embodiment of the present invention, the recombinant bacteria are constructed by PCR amplification of heme oxygenase (ScHO) or its mutant (ScHOmut), cytochrome P450 reductase (ScCPR) or formate dehydrogenase (ApFDH), cloned into plasmid pETDuet-1, constructing recombinant plasmid pETDuet-1-ScHO-ScCPR-ApFDH, or pETDuet-1-ScHOmut-ScCPR-ApFDH, and constructing recombinant Escherichia coli catalyst.

[0060] In one embodiment of the present invention, the recombinant bacteria are constructed by PCR amplification of biliverdin reductase (MmBVR) and cloning it into plasmid pCDFDuet-1 to construct recombinant plasmid pCDFDuet-1-MmBVR, thereby constructing a recombinant Escherichia coli whole-cell catalyst.

[0061] In one embodiment of the present invention, the recombinant bacteria are constructed by co-transforming recombinant plasmid pETDuet-1-ScHO-ScCPR-ApFDH or pETDuet-1-ScHOmut-ScCPR-ApFDH with recombinant plasmid pCDFDuet-1-MmBVR into E. coli BL21(DE3) to construct a recombinant Escherichia coli whole-cell catalyst.

[0062] This invention provides a method for improving the bilirubin synthesis capacity of *Escherichia coli*. The method involves using *Escherichia coli* BL21(DE3) as the host cell and expressing heme oxygenase and cytochrome P450 reductase from *Saccharomyces cerevisiae*, biliverdin reductase from *Mus musculus*, and NADP from *Azospirillum lumpus*. + Formate-dependent dehydrogenase.

[0063] In one embodiment of the present invention, the heme oxygenase is a heme oxygenase obtained by deleting amino acids 289-318 from the C-terminus of the heme oxygenase with NCBI accession number GHM92019.1, or a mutant of the above-mentioned heme oxygenase; the biliverdin reductase has NCBI accession number XP_030102511.1, and the cytochrome P450 reductase is a cytochrome P450 reductase obtained by deleting amino acids 1-24 from the N-terminus of the cytochrome P450 reductase with NCBI accession number NP_011908.1; the NADP + The NCBI accession number for formate-dependent dehydrogenase is WP_098736599.1.

[0064] The present invention also provides a method for whole-cell catalytic synthesis of bilirubin, wherein the method comprises using heme as a substrate and, under aeration conditions, using the above-mentioned recombinant cell lysate or the above-mentioned recombinant Escherichia coli whole-cell catalytic conversion to synthesize bilirubin.

[0065] In one embodiment of the present invention, the whole-cell catalytic system includes, but is not limited to, 4 mM heme (equivalent to 2.604 g / L), an aeration rate of 8 vvm, and heme pre-dissolved in 50% of the total reaction volume of NaOH solution (100 mM). After complete dissolution, the pH is adjusted to 6.5 with HCl, 100 mM ammonium formate is added, and the reaction volume is made up with 100 mM citric acid-sodium citrate solution (pH 6.5). The reaction time is 4 h, and the reaction temperature is 35 °C.

[0066] In one embodiment of the present invention, the whole-cell catalyst is obtained by culturing recombinant bacteria and inducing them to express heme oxygenase, biliverdin reductase, cytochrome P450 reductase, and formate dehydrogenase, and then collecting the recombinant bacterial cells.

[0067] In one embodiment of the present invention, the whole-cell catalyst is obtained by inoculating recombinant *E. coli* co-expressing heme oxygenase, biliverdin reductase, cytochrome P450 reductase, and formate dehydrogenase into 100 mL LB medium at a 1% inoculation rate and culturing at 37°C. When OD... 600 When the pH reaches 0.4–0.6, add 0.1 mM IPTG to induce protein expression. After induction at 20°C for 16 h, centrifuge at 8,000 × g for 10 min to collect the bacterial cells. Wash the bacterial cells twice with 20 mM Tris-HCl (pH 7.5) buffer to obtain the final product.

[0068] In one embodiment of the present invention, the reaction conditions for whole-cell catalysis are: stirring speed 200 r / min, temperature 35℃, pH 6.5, aeration rate 8 vvm, and reaction time 1-4 h.

[0069] In one embodiment of the present invention, the amount of heme added is 1-4 mM (equivalent to 651-2604 mg / L), and the amount of catalyst added is 20 g / L (cell dry weight).

[0070] This invention provides the application of the above-described recombinant Escherichia coli or the above-described method in the preparation of bilirubin or products containing bilirubin.

[0071] Beneficial effects

[0072] (1) This invention successfully obtained a high-activity mutant of heme oxygenase, improving the catalytic efficiency of heme oxygenase. The heme oxygenase mutant, cytochrome P450 reductase, and biliverdin reductase were co-expressed in *E. coli*, and coupled with a high-activity formate dehydrogenase. This successfully reconstructed a complete heme degradation cascade reaction in a single cell, achieving high catalytic activity without the need for exogenous NADPH addition, utilizing endogenous coenzymes. No intermediate product, biliverdin, accumulated during the catalytic process. Using heme as a substrate, through a one-pot cascade catalysis of multiple enzymes, the final bilirubin yield reached 2.10 g / L, with a conversion rate >90%. The multi-enzyme coupling system constructed in this invention solves the problem of low conversion rates in the catalytic conversion of heme to biliverdin and in bilirubin research.

[0073] (2) The establishment of this whole-cell transformation system solves the problems of cumbersome steps, low yield and large wastewater discharge in the traditional biological tissue extraction method for producing bilirubin. It has good atom economy and realizes efficient and green synthesis of bilirubin using heme as substrate. According to all current data, the bilirubin yield in this invention is the highest level in biological synthesis starting from heme substrate. Attached Figure Description

[0074] Figure 1 Protein expression of strains containing recombinant plasmid pETDuet-1-ScHO-ScCPR-ApFDH. Lane 1 is cell lysate supernatant.

[0075] Figure 2 Protein expression of strains containing recombinant plasmids pETDuet-1-ScHO-ScCPR-ApFDH and pCDFDuet-1-MmBVR. Lane 1 is the cell lysate supernatant.

[0076] Figure 3 The standard curve of biliverdin was determined using an enzyme-linked immunosorbent assay (ELISA) reader.

[0077] Figure 4 : HPLC chromatogram and standard curve of bilirubin; where, a, chromatogram of bilirubin; b, standard curve of bilirubin.

[0078] Figure 5 : Bilirubin reaction formula. Detailed Implementation

[0079] The preparation of nucleic acid gels involved in the following examples:

[0080] 50×TAE buffer: Accurately weigh 242g of Tris and 37.2g of Na2EDTA·2H2O into a 1L volumetric flask, add 800mL of ultrapure water to dissolve thoroughly and shake well. Then add 57.1mL of glacial acetic acid, shake well, and add deionized water to bring the volume to 1L. Prepare the 50×TAE buffer, then dilute it 50 times to make 1×TAE buffer. Using the 1×TAE buffer as a solvent, prepare a 0.9% agarose solution (dissolved by heating). After the agarose is completely dissolved, add the nucleic acid dye (1.5μL / 20mL), gently shake to mix evenly, avoiding the formation of bubbles, pour into a nucleic acid gel mold and cool to obtain an agarose gel.

[0081] The gel electrophoresis involved in the following examples:

[0082] Place the gel in an electrophoresis tank containing 1×TAE buffer. Set the electrophoresis conditions to 160V for 15 min. After electrophoresis, observe the bands under UV light. Template digestion: Take 50 μL of the PCR product that tested positive on electrophoresis, add 1 μL of the restriction enzyme DpnⅠ and 5 μL of the corresponding buffer to remove the methylated template DNA. The reaction conditions are 37℃ for 3 h. After the reaction, incubate at 65℃ for 10 min to inactivate DpnⅠ.

[0083] The product purification involved in the following examples:

[0084] according to The product instructions for the FastPure Gel DNA Extraction Mini Kit specify the purification of digestion products.

[0085] Homologous recombination involved in the following embodiments:

[0086] according to The ClonExpress II One Step Cloning Kit product instructions describe using homologous recombination to ligate a truncated, mutated DNA fragment into a linearized vector to obtain a recombinant plasmid.

[0087] The transformation of recombinant plasmids involved in the following examples:

[0088] All the homologous recombination reaction solution was added to E. coli BL21(DE3) competent cells, mixed well, and placed on ice for 30 min; then heat-shocked in a 42℃ water bath for 90 s, quickly transferred to an ice bath for 5 min, added 600 μL of LB medium, and cultured at 37℃ and 200 rpm for 1 h; 50 μL was plated on LB plates containing the corresponding antibiotic and incubated upside down at 37℃ for 12 h; single colonies were picked for sequencing verification, and strains that had been correctly introduced with the recombinant plasmid were preserved.

[0089] The detection methods involved in the following embodiments are as follows:

[0090] SDS-PAGE gel electrophoresis:

[0091] Prepare the BBI blocking-free SDS-PAGE denaturing acrylamide color gel rapid preparation kit and prepare the protein gel according to the instructions. Sample preparation: Take wet bacterial cells containing the target protein, prepare a 20 g / L cell suspension with Tris-HCl buffer (pH 7.5, 100 mM), and sonicate 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, mix with 8 μL of loading buffer, heat in a boiling water bath for 10 min, centrifuge at 12,000 × g for 5 min, and set aside.

[0092] Electrophoresis. Protein gel loading volume: 10 μL sample, 3 μL marker; Electrophoresis conditions: 160 V, 60 min; Electrophoresis buffer (g / L): glycine 14.4, SDS 1, Tris 3.

[0093] Staining: Immerse the protein gel in staining solution (Coomassie Brilliant Blue R250 1g, methanol 450mL, acetic acid 100mL, water 450mL) for 15 minutes, then wash twice with water. Destaining: Immerse in destaining solution (ethanol 10%, acetic acid 10%, water 80%) and gently shake, replacing with fresh destaining solution until the bands are clearly visible.

[0094] bilirubin testing

[0095] Mobile phase: A (25 mM ammonium acetate, pH adjusted to 3.5 with acetic acid); B (acetonitrile:methanol:isopropanol = 8:1:1); A:B = 5:95; Column temperature: 35℃; Column: C18; Injection volume: 5 μL; Run time: 10 min; Detection wavelength: 400 nm. The chromatogram of bilirubin and the corresponding standard curve are shown in the instruction manual. Figure 4 As shown.

[0096] Conversion rate = substrate of the reaction / total substrate.

[0097] Yield = Product generated / Total substrate.

[0098] Detection of heme oxygenase activity

[0099] In a 1 mL reaction system, 100 mg of purified heme oxygenase or its mutant, 100 mg of purified cytochrome P450 reductase, 30 mM NADPH, and 1 mM heme chloride were added. Using Tris-HCl (100 mM, pH 7.5) as the solvent, the mixture was placed in a 35°C constant-temperature shaker and reacted at 500 rpm for 20 min. Subsequently, 50 μL of the mixture was taken and mixed thoroughly with 950 μL of methanol. After centrifugation at 12,000 × g for 1 min, 200 μL of the supernatant was collected and the absorbance at 690 nm was measured in a 96-well plate. The concentration of biliverdin was calculated based on the standard curve, and the activity units (U) and specific activity (U / g) were further calculated.

[0100] The activity unit U is defined as the amount of enzyme required to catalyze the synthesis of 1 μmol of biliverdin per minute under conditions of 35°C and pH 7.5.

[0101] Specific activity is the number of activity units per gram of heme oxygenase or its mutant, expressed in U / g.

[0102] The protein sequence information involved in the following examples is shown in Table 1.

[0103] Table 1: Protein Information

[0104] protein name source NCBI reference seq. / GenBank Heme oxygenase (ScHO) Saccharomyces cerevisiae GHM92019.1 Biliverdin reductase (MmBVR) Mus musculus XP_030102511.1 Cytochrome P450 reductase (ScCPR) Saccharomyces cerevisiae NP_011908.1 Formate dehydrogenase (ApFDH) Azospirillum palustre WP_098736599.1

[0105] In this invention, the heme oxygenase obtained by deleting amino acids 289-318 from the C-terminus of the heme oxygenase with NCBI accession number GHM92019.1 is used.

[0106] The present invention uses ScCPR obtained by deleting the amino acid sequence from the N-terminus 1 to 24 of ScCPR with NCBI accession number NP_011908.1.

[0107] Example 1: Gene Synthesis and Expression

[0108] The specific steps are as follows:

[0109] 1. Construction of recombinant vectors

[0110] (1) Gene Acquisition

[0111] Heme oxygenase (ScHO) with nucleotide sequences as shown in SEQ ID NO.2, biliverdin reductase (MmBVR) with nucleotide sequences as shown in SEQ ID NO.4, cytochrome P450 reductase (ScCPR) with nucleotide sequences as shown in SEQ ID NO.3, and formate dehydrogenase (ApFDH) with nucleotide sequences as shown in SEQ ID NO.5 were synthesized respectively. These were submitted to Sangon Biotech (Shanghai) Co., Ltd. for codon optimization, gene synthesis, and construction of recombinant vectors.

[0112] (2) Construction of recombinant vector

[0113] The vectors used were the commonly used expression plasmids pETDuet-1 and pCDFDuet-1 for Escherichia coli.

[0114] The synthesized ScHO, ScCPR, and ApFDH genes were sequentially inserted into the pETDuet-1 vector. The nucleotide sequence of ScHO was inserted between the BamHI and SacI restriction sites of pETDuet-1 in a 5'→3' order. The nucleotide sequence of ScCPR was added to the 5' end with a ribosome binding sequence (5'-tttgtttaactttaagaaggagatatacc-3') and then inserted between the SalI and NotI restriction sites of pETDuet-1 in a 5'→3' order. The nucleotide sequence of ApFDH was inserted between the NdeI and XhoI restriction sites of pETDuet-1 in a 5'→3' order, resulting in the recombinant plasmid pETDuet-1-ScHO-ScCPR-ApFDH.

[0115] The synthesized MmBVR gene was inserted into the pCDFDuet-1 vector, with the nucleotide sequence of MmBVR inserted in a 5'→3' order between the restriction enzyme sites BamHI and SacI of pCDFDuet-1 to obtain the recombinant plasmid pCDFDuet-1-MmBVR.

[0116] 2. Construction of recombinant strains

[0117] E. coli BL21(DE3) was selected as the expression host. The recombinant plasmids obtained in step 1 were introduced into competent E. coli BL21(DE3) cells and plated onto LB agar plates containing ampicillin or streptomycin, or both of the above antibiotics. Positive transformants were picked and preserved to obtain the corresponding engineered strains.

[0118] E.coli BL21(DE3) / pETDuet-1-ScHO-ScCPR-ApFDH, E.coli BL21(DE3) / pCDFDuet-1-MmBVR.

[0119] 3. Protein expression

[0120] The engineered strains obtained in step 2 were inoculated into test tubes containing ampicillin or streptomycin (both at a concentration of 50 mg / L), or either of the above two antibiotics, 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 ampicillin or streptomycin (both at a concentration of 50 mg / L), or either of the above two antibiotics, and cultured at 37°C and 200 rpm until OD (dose retardation). 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 16 h to induce protein expression; the fermentation broth is then obtained.

[0121] The obtained fermentation broth was centrifuged at low temperature under the following conditions: 8,000×g, 10 min, 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 ultrasonically disrupted under the following conditions: 400W, 0℃, working for 2 s, intermittent for 4 s, for a total time of 10 min, to obtain cell lysate, which is the catalyst to be added in the embodiments of the present invention.

[0122] The obtained cell lysates were analyzed by SDS-PAGE gel electrophoresis to verify the soluble expression of proteins. Protein expression was as follows: Figure 1 and Figure 2 As shown in the figure, the results indicate that all four proteins were successfully expressed.

[0123] Example 2: Obtaining the mutant sequence of heme oxygenase

[0124] The specific steps are as follows:

[0125] Using pETDuet-1-ScHO-ScCPR-ApFDH as a template for PCR amplification, error-prone PCR was performed on the ScHO-encoding gene. Primers were designed based on the gene sequence for amplification. During amplification, low-fidelity Taq DNA polymerase and exogenous modifications to the PCR reaction conditions were employed, and Mn was added to the PCR system. 2+ (0.15mM) is used to reduce the fidelity of DNA replication, mainly due to Mn. 2+This can reduce the specificity of polymerase for the template, increasing the probability of base mismatches and deletions during the synthesis of new DNA strands, thus leading to multi-point mutations or deletions in the amplification products. Therefore, a ScHO random mutant library can be obtained.

[0126] Using pETDuet-1-ScHO-ScCPR-ApFDH as a template for PCR amplification, reverse PCR was performed using high-fidelity DNA polymerase to obtain a vector fragment homologous to ScHO.

[0127] The primer sequences involved in this embodiment are shown in Table 2.

[0128] Table 2: Mutation sites and primers

[0129]

[0130] The PCR reaction conditions were as follows: (1) 95℃ pre-denaturation for 3 min; (2) 95℃ denaturation for 15 s; (3) 55℃ annealing for 15 s; (4) 72℃ extension for 2 min; (2) to (4) were repeated 30 times; and finally, the reaction was extended at 72℃ for 5 min and then cooled to 16℃ to end.

[0131] High-fidelity PCR system: plasmid template pETDuet-1-ScHO-ScCPR-ApFDH 1μL; forward and reverse primers 1μL each; Takara primeSTAR Max high-fidelity enzyme 25μL; ddH2O 22μL.

[0132] Error-prone PCR reaction system: plasmid template pETDuet-1-ScHO-ScCPR-ApFDH 1μL; error-prone PCR 10×mix 3μL, error-prone PCR MgCl2 3μL, error-prone PCR 10×dNTP 3μL, forward and reverse primers 1μL each; Taq DNA polymerase 1μL, ddH2O 18μL.

[0133] The PCR product was treated with Dpn I to eliminate the template plasmid. The system and reaction conditions were as follows: 50 μL of PCR product, 1 μL of Takaraquickcut Dpn I, and 5 μL of 10×quickcut buffer. After mixing, the mixture was placed in a 37°C water bath for 2 hours.

[0134] After the reaction was completed, the reaction solution was purified using the Vazyme FastPure Gel DNA Extraction Mini Kit and in accordance with its instructions to obtain high-purity ScHO fragments and vector fragments containing random mutations.

[0135] according to Following homologous recombination as per the ClonExpress II One Step Cloning Kit's product instructions, a recombinant plasmid pETDuet-1-ScHO containing a random ScHO mutant sequence was obtained. mut -ScCPR-ApFDH.

[0136] Following the recombinant strain construction method described in Example 1, the purified product was introduced into E. coli BL21(DE3) competent cells and plated onto LB plates containing ampicillin resistance. After incubation at 37°C for 12 h, a single clone containing the mutant was obtained.

[0137] Example 3: High-throughput screening of positive mutants

[0138] Establishment of high-throughput detection methods:

[0139] Biliverdin standard was dissolved in methanol, and biliverdin standard solutions of 0–200 mg / L were prepared in a gradient manner. 200 μL of each concentration was placed in a 96-well plate, and the absorbance of each well at 690 nm was read using a microplate reader. A well containing 200 μL of methanol was used as a blank control. The results were as follows: Figure 3 The standard curve shown.

[0140] High-throughput screening process:

[0141] The monoclonal strains obtained in Example 2 were inoculated into 48-well plates containing 1 mL of LB medium per well, and ampicillin at a concentration of 50 mg / L was added. After incubation at 37°C and 200 rpm for 8 h, a seed culture was obtained. The seed culture was then transferred to another 48-well plate containing fresh LB medium at an inoculation rate of 1% (v / v), and ampicillin at a concentration of 50 mg / L was added. After incubation at 37°C and 200 rpm for 2 h, IPTG was added to each well to a final concentration of 0.1 mM, and the plates were transferred to a shaker at 20°C and 200 rpm for 16 h. After incubation, the plates were centrifuged at 8,000 × g at 4°C for 10 min using a matching rotor. The supernatant was discarded, and the plates containing the bacterial pellet were frozen at -20°C for 24 h to increase cell membrane permeability.

[0142] The following reaction mixture (total volume 0.5 mL) was then added to each well of the plate: 1 mM heme chloride, 30 mM ammonium formate, and Tris-HCl (100 mM, pH 7.5) as solvent. The mixture was shaken to resuspend the bacterial precipitate. The plate was incubated at 35°C and 200 rpm for 1 hour. After the reaction, 0.5 mL of methanol was added to each well to terminate the reaction. The plate was then centrifuged at 8,000 × g at 4°C for 10 min using a centrifuge with a matching rotor. 200 μL of the supernatant was added to a 96-well plate, and the absorbance of biliverdin, the product of heme oxygenase, was measured at 690 nm using a microplate reader. The absorbance value was then substituted into the... Figure 4 The standard curve is shown, and the concentration is calculated.

[0143] Compared with the wild-type enzyme, single clones with increased product concentration were selected and sent to Qingke Biotechnology (Suzhou) for sequencing to obtain the corresponding mutant sequences. The corresponding single clones were then preserved in bacterial strains. The amino acid changes before and after mutation are shown in Table 3.

[0144] Table 3: Sequencing results of mutants and concentrations of biliverdin catalyzed synthesis

[0145] mutation site Product biliverdin concentration (mg / L) A29K 40.32 N31H 38.76 N258M 31.47 WT 21.78

[0146] Example 4: Combinatorial Mutation of ScHO

[0147] The three positive mutations A29K, N31H, and N258M obtained in Example 3 were combined with mutations to further obtain highly active mutants. The specific steps are as follows:

[0148] The recombinant strain containing three positive mutants obtained in Example 3 was inoculated into test tubes containing 5 mL of LB medium, ampicillin was added, and the tubes were incubated at 37°C and 200 rpm for 8 h. Plasmid extraction was then performed using the Vazyme FastPurePlasmid Mini Kit-BOX 2, following the instructions provided in the kit. Three recombinant plasmids, pETDuet-1-ScHO, were obtained. A29K -ScCPR-ApFDH、pETDuet-1-ScHO N31H -ScCPR-ApFDH and pETDuet-1-ScHO N258M -ScCPR-ApFDH. Subsequently, using the mutation primers shown in Table 4 and the above three plasmids as templates, a PCR reaction was performed to introduce the second / third mutation site.

[0149] Table 4: Site-directed mutagenesis primers

[0150]

[0151] The PCR, template digestion, and product purification procedures were as described in Example 2. The double-mutant recombinant plasmid pETDuet-1-ScHO was obtained. A29K / N31H -ScCPR-ApFDH、pETDuet-1-ScHO A29K / N258M -ScCPR-ApFDH、pETDuet-1-ScHO N31H / N258M -ScCPR-ApFDH; and the triple mutant recombinant plasmid pETDuet-1-ScHO A29K / N31H / N258M -ScCPR-ApFDH. Subsequently, the corresponding recombinant strains were obtained according to the recombinant strain construction method described in Example 1, and the two-point combination mutants and three-point combination mutants were screened according to the screening method described in Example 3. The results are shown in Table 5.

[0152] Table 5: Concentration of biliverdin synthesized by the combined mutant

[0153] mutation site Product biliverdin concentration (mg / L) A29K / N31H 46.99 A29K / N258M 39.72 N31H / N258M 46.01 A29K / N31H / N258M 63.76 WT 22.32

[0154] The results showed that the combined mutants of A29K, N31H and N258M with positive effects, after two-point or three-point combination mutations, had higher catalytic efficiency than wild-type enzymes; among them, the three-point combination mutants showed a further improvement in the ability to catalyze the synthesis of biliverdin, which was 2.8 times higher than that of wild-type.

[0155] Example 5: Enzymatic Properties Analysis of Mutant and Wild-Type ScHO

[0156] The specific steps are as follows:

[0157] For wild-type heme oxygenase ScHO (SEQ ID NO.1) and cytochrome P450 reductase ScCPR, as well as the recombinant plasmid containing mutants screened in Example 3, the mutant sequences were amplified using PCR primers designed according to Table 6, and then ligated to pET28a(+) using homologous recombination technology.

[0158] Table 6: Homologous recombination primers

[0159]

[0160] Recombinant plasmids pET28a(+)-ScHO, pET28a(+)-A29K, pET28a(+)-N31H, pET28a(+)-N258M, pET28a(+)-A29K / N31H, pET28a(+)-A29K / N258M, pET28a(+)-N31H / N258M, pET28a(+)-A29K / N31H / N258M, and pET28a(+)-ScCPR were constructed. Recombinant strains were constructed and proteins were expressed according to Example 1, and proteins were purified according to the following methods.

[0161] (1) Preparation of protein purification reagents

[0162] Binging Buffer: Tris-HCl buffer (100mM, pH 7.5). Washing Buffer: Take 1L of Tris-HCl buffer (100mM, pH 7.5), add 3.4g of imidazole (final concentration 50mM), and adjust the pH of the buffer to 7.5 with hydrochloric acid or sodium hydroxide. Elution Buffer: Take 1L of Tris-HCl buffer (100mM, pH 7.5), add 34g of imidazole (final concentration 500mM), and adjust the pH of the buffer to 7.5 with hydrochloric acid or sodium hydroxide.

[0163] (2) Protein purification process

[0164] ScHO and its mutants, as well as ScCPR, all carry histidine tags. The supernatant of the crude enzyme solution obtained after disruption was purified using a Ni affinity column (40×12.6 mm, Bio-Rad, USA). The specific steps were as follows: a. Wash the Ni column with Binging Buffer at a flow rate of 0.4 mL / min until UV baseline equilibrium is reached, approximately 15 times the Ni column volume. b. After leveling the baseline with Binging Buffer, load the sample at a flow rate of 0.2 mL / min to ensure sufficient binding of the target protein to the Ni column. c. Wash away contaminating proteins with Washing Buffer at a flow rate of 0.3 mL / min until UV baseline equilibrium is reached, approximately 10 times the Ni column volume. d. Elute the target protein with Elution Buffer at a flow rate of 0.25 mL / min. Start collecting when the absorbance reaches 0.1 and stop collecting when the absorbance drops to 0.1. e. Continue rinsing with Elution Buffer at a flow rate of 0.3 mL / min until UV baseline equilibration. f. Replace Elution Buffer with Binging Buffer and continue rinsing. Equilibrate the Ni column with Binging Buffer at a flow rate of 0.35 mL / min until UV baseline equilibration, approximately 10 column volumes of Ni column. g. Preserve the Ni column with 8 column volumes of 20% ethanol and store it at 4°C. g. Salt ion removal: Dialyze the collected target protein eluent in Tris-HCl buffer (100 mM, pH 7.5) overnight to obtain purified enzyme solution.

[0165] (3) After obtaining purified ScHO wild-type enzyme, A29K, N31H, N258M, A29K / N31H, A29K / N258M, N31H / N258M, A29K / N31H / N258M, and ScCPR, the protein concentration was determined using an ultra-micro spectrophotometer, and the reaction system was prepared according to the following:

[0166] ScHO wild-type or mutant enzyme 20 μg, ScCPR 20 μg, NADPH final concentration 15 mM, heme chloride final concentration in the reaction system (0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mM, respectively), and Tris-HCl buffer (100 mM, pH 7.5) to control the total volume to 200 μL, and place in a 96-well plate to measure the reaction progress.

[0167] Reaction progress measurement procedure: Place the 96-well plate in a microplate reader, set the temperature to 35℃, shake for 2 seconds every 5 seconds, scan the absorbance value of each well at 690nm at 30-second intervals, and then proceed as follows. Figure 4The standard curves shown were converted to biliverdin concentrations. The reaction rate was calculated for the first 10% of each reaction process, and the reaction rates at different substrate concentrations were fitted using the Michaelis-Menten equation. The kinetic parameters of wild-type and mutant ScHO calculated based on the fitting results are shown in Table 7. Enzyme activity data were also detected, and the results are shown in Table 7.

[0168] Table 7: Kinetic parameters of SchO wild-type and mutant strains

[0169] mutation site <![CDATA[K m (mM)]]> <![CDATA[k cat (×10 -3 s -1 )]]> <![CDATA[k cat / K m (×10 -2 (s·mM) -1 )]]> Specific activity (U / g) A29K 0.102 55.87 54.77 110.10 N31H 0.099 49.09 49.58 102.77 N258M 0.123 43.23 35.15 86.42 A29K / N31H 0.097 72.33 74.57 139.84 A29K / N258M 0.109 50.68 46.50 98.79 N31H / N258M 0.110 63.77 57.97 129.65 A29K / N31H / N258M 0.098 96.79 98.76 200.43 WT 0.093 31.62 34.00 64.98

[0170] Kinetic parameter determination experiments showed that the SchO combined mutant A29K / N31H / N258M had the highest catalytic efficiency and specific activity.

[0171] Example 6: Construction of multi-enzyme co-expression engineered strains

[0172] Preferred ScHO mutant ScHO A29K / N31H / N258M The specific steps for integrating the remaining proteins into the same strain of E. coli are as follows:

[0173] (1) Plasmid preparation

[0174] The preferred ScHO mutant obtained in Example 4 A29K / N31H / N258M Plasmid extraction was performed on the strain using the Vazyme FastPure Plasmid Mini Kit-BOX 2, following the instructions provided in the kit. The recombinant plasmid pETDuet-1-ScHO was obtained. A29K / N31H / N258M -ScCPR-ApFDH;

[0175] The recombinant plasmid pCDFDuet-1-MmBVR was constructed according to the method in Example 1;

[0176] (2) Construction of recombinant strains

[0177] Following the recombinant strain construction method described in Example 1, the two plasmids were simultaneously transformed into the same E. coli BL21(DE3) competent cells to obtain cells containing ScHO. A29K / N31H / N258M Four-enzyme co-expression strain: E. coli BL21(DE3) / pETDuet-1-ScHO A29K / N31H / N258M -ScCPR-ApFDH / pCDFDuet-1-MmBVR, named E. coli BL21(DE3). Mut .

[0178] Example 7: Establishing an efficient reaction system

[0179] The recombinant Escherichia coli E. coli BL21(DE3) obtained in Example 6 was used. Mut As a preferred catalyst, the reaction system was established. Following step 3 of Example 1, a fermentation broth was prepared. The fermentation broth was centrifuged at low temperature under the following conditions: 8,000 × g, 10 min, 4 °C. After centrifugation, the supernatant was discarded, and the precipitate was washed twice with physiological saline to obtain whole cells. A total dry weight of 20 g / L of whole cells was weighed and ultrasonically disrupted at 400 W, 0 °C, with a 2-second working time followed by a 4-second pause, for a total time of 10 min, to obtain cell lysate. Catalytic reactions under different concentrations of heme chloride were also investigated.

[0180] (1) The reaction conditions are as follows: 100 mL reaction system, 20 g / L of recombinant Escherichia coli cell lysis buffer (the amount of lysis buffer added is: first weigh 20 g / L of whole cell dry weight, and after rupture, it is the corresponding lysis buffer to be added), the solvent is citrate-sodium citrate buffer (pH 6.5, 100 mM), EDTA-Na2 5 mM, 35℃, 200 rpm.

[0181] Under the above reaction conditions, 1 mM heme chloride was added. Samples were continuously taken during the reaction process. For each sample, 50 μL of the reaction solution was added to a mixture of 950 μL of DMSO and dichloromethane (volume ratio 5:1), centrifuged at 12,000 × g for 1 min, and the supernatant was collected. The bilirubin concentration was determined by HPLC. After 4 hours of reaction, the conversion rate was 96.7%, and the concentration of bilirubin produced was 0.563 g / L.

[0182] (2) The reaction conditions are as follows: 100 mL reaction system, 20 g / L of recombinant Escherichia coli cell lysis buffer (the amount of lysis buffer added is: first weigh 20 g / L of whole cell dry weight, and after rupture, it is the corresponding lysis buffer to be added), the solvent is citrate-sodium citrate buffer (pH 6.5, 100 mM), EDTA-Na2 5 mM, 35℃, 200 rpm.

[0183] Under the above reaction conditions, 2 mM heme chloride was added. Samples were continuously taken during the reaction process. For each sample, 50 μL of the reaction solution was added to a mixture of 950 μL of DMSO and dichloromethane (volume ratio 5:1), centrifuged at 12,000 × g for 1 min, and the supernatant was collected. The bilirubin concentration was determined by HPLC. After 4 hours of reaction, the conversion rate was 96.2%, and the concentration of bilirubin produced was 1.120 g / L.

[0184] (3) The reaction conditions are as follows: 100 mL reaction system, 20 g / L of recombinant Escherichia coli cell lysis buffer (the amount of lysis buffer added is: first weigh 20 g / L of whole cell dry weight, and after rupture, it is the corresponding lysis buffer to be added), the solvent is citrate-sodium citrate buffer (pH 6.5, 100 mM), EDTA-Na2 5 mM, 35℃, 200 rpm.

[0185] Under the above reaction conditions, 3 mM heme chloride was added. Samples were continuously taken during the reaction process. For each sample, 50 μL of the reaction solution was added to a mixture of 950 μL of DMSO and dichloromethane (volume ratio 5:1), centrifuged at 12,000 × g for 1 min, and the supernatant was collected. The bilirubin concentration was determined by HPLC. After 4 hours of reaction, the conversion rate was 95.6%, and the concentration of bilirubin produced was 1.675 g / L.

[0186] (4) The reaction conditions are as follows: 100 mL reaction system, 20 g / L of recombinant Escherichia coli cell lysis buffer (the amount of lysis buffer added is: first weigh 20 g / L of whole cell dry weight, and after rupture, it is the corresponding lysis buffer to be added), the solvent is citrate-sodium citrate buffer (pH 6.5, 100 mM), EDTA-Na2 5 mM, 35℃, 200 rpm.

[0187] Under the above reaction conditions, 4 mM heme chloride was added. Samples were continuously taken during the reaction process. For each sample, 50 μL of the reaction solution was added to a mixture of 950 μL of DMSO and dichloromethane (volume ratio 5:1), centrifuged at 12,000 × g for 1 min, and the supernatant was collected. The bilirubin concentration was determined by HPLC. After 4 hours of reaction, the conversion rate was 92.1%, and the concentration of bilirubin produced was 2.10 g / L.

[0188] (5) The E. coli BL21(DE3) / pETDuet-1-ScHO described in the text of Chinese invention patent application with publication number CN119331839A H156N / N247H -ScCPR-ApFDH / pCDFDuet-1-MmBVR-AcCODH was used as the control strain; following the method in step (4) above, after 4 hours of reaction with the addition of 4mM heme chloride, the conversion rate was 74.2% and the concentration of bilirubin produced was 1.71g / L.

[0189] The present invention relates to E. coli BL21(DE3). Mut It can achieve reactions with high substrate concentrations. When the substrate concentration is 4 mM, the product bilirubin reaches 2.10 g / L after 4 h, with a yield of 90.02%.

[0190] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A heme oxygenase mutant, characterized in that, The heme oxygenase mutant is... The amino acid sequence of heme oxygenase, as shown in SEQ ID NO.1, is obtained by mutating alanine at position 29 to lysine, asparagine at position 31 to histidine, and asparagine at position 258 to methionine.

2. The gene encoding the heme oxygenase mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. Recombinant cells expressing the heme oxygenase mutant of claim 1, or carrying the gene of claim 2, or carrying the recombinant vector of claim 3.

5. The recombinant cell according to claim 4, characterized in that, The recombinant cells use bacteria or fungi as host cells.

6. A recombinant enzyme catalyst containing the heme oxygenase mutant of claim 1, characterized in that, It is any of the following forms: (1) Culture recombinant expression transformants containing the heme oxygenase mutant and isolate transformant cells containing the recombinant heme oxygenase mutant enzyme; (2) Culture recombinant expression transformants containing the heme oxygenase mutant, isolate transformant cells containing the recombinant heme oxygenase mutant enzyme, and break the transformant cells containing the recombinant heme oxygenase mutant enzyme to obtain cell lysate; (3) Cultivate recombinant expression transformants containing the heme oxygenase mutant, isolate transformant cells containing the recombinant heme oxygenase mutant enzyme, break the transformant cells containing the recombinant heme oxygenase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate of the recombinant heme oxygenase mutant enzyme to obtain lyophilized enzyme powder.

7. A method for improving the conversion rate of heme oxygenase to substrates and / or increasing the activity of heme oxygenase, characterized in that, The method involves mutating alanine at position 29 of the heme oxygenase amino acid sequence as shown in SEQ ID NO.1 to lysine, mutating asparagine at position 31 to histidine, and mutating asparagine at position 258 to methionine.

8. The use of the heme oxygenase mutant of claim 1, the recombinant cell of claim 4 or 5, or the recombinant enzyme catalyst of claim 6 in the preparation of biliverdin or bilirubin, characterized in that, The application in the preparation of biliverdin involves a reaction system comprising a whole-cell reaction system and an enzyme reaction system, wherein: The whole-cell reaction system consisted of whole-cell transformation using heme chloride as a substrate to prepare biliverdin. The recombinant cells also expressed a gene derived from... Azospirillum palustre NADP + Formate-dependent dehydrogenase, derived from Saccharomyces cerevisiae Cytochrome P450 reductase; The enzyme reaction system is as follows: using heme chloride as a substrate, biliverdin is prepared by catalysis, while adding ingredients derived from... Azospirillum palustre NADP + Formate-dependent dehydrogenase, derived from Saccharomyces cerevisiae Cytochrome P450 reductase; The application described in the preparation of bilirubin involves a reaction system comprising a whole-cell reaction system and an enzyme reaction system, wherein: The whole-cell reaction system was as follows: bilirubin was prepared by whole-cell transformation using heme chloride as a substrate. The recombinant cells also expressed [a specific gene / product] derived from [a specific source]. Mus musculus biliverdin reductase, derived from Azospirillum palustre NADP + Formate-dependent dehydrogenase, derived from Saccharomyces cerevisiae Cytochrome P450 reductase; The enzyme reaction system is as follows: using heme chloride as a substrate, bilirubin is prepared by catalysis, while adding substances derived from... Mus musculus biliverdin reductase, derived from Azospirillum palustre NADP + Formate-dependent dehydrogenase, derived from Saccharomyces cerevisiae Cytochrome P450 reductase.

9. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria expressed the heme oxygenase mutant of claim 1, derived from... Mus musculus biliverdin reductase, derived from Azospirillum palustre NADP + Formate-dependent dehydrogenase, derived from Saccharomyces cerevisiae Cytochrome P450 reductase.

10. The genetically engineered bacterium according to claim 9, characterized in that, The nucleotide sequence of the cytochrome P450 reductase is shown in SEQ ID NO. 3; the nucleotide sequence of the biliverdin reductase is shown in SEQ ID NO. 4; the NADP... + The nucleotide sequence of the formate-dependent dehydrogenase is shown in SEQ ID NO.

5.

11. The genetically engineered bacterium according to claim 10, characterized in that, The genetically engineered bacteria include Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and yeast.

12. A method for synthesizing bilirubin, characterized in that, The method involves using heme as a substrate and employing the genetically engineered bacteria or their fermentation broth as catalysts according to any one of claims 9-11 to catalyze the conversion and synthesis of bilirubin.

13. The method according to claim 12, characterized in that, The amount of heme added is 1~4 mM, the stirring speed is 100~500 r / min, the temperature is 25~40 ℃, the pH is 6.0~8.0, and the reaction time is 1~4 h.

14. The use of the genetically engineered bacteria according to any one of claims 9 to 11 in the preparation of bilirubin or products containing bilirubin.

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