Efficient synthesis of bilirubin by heme oxygenase mutants and their multi-enzyme coupling systems

Abstract

The present invention discloses a heme oxygenase mutant and its multi-enzyme coupling system for the efficient synthesis of bilirubin, belonging to the field of biocatalytic engineering. The present invention provides a heme oxygenase mutant that catalyzes the synthesis of biliverdin from heme; couples biliverdin reductase to catalyze the synthesis of bilirubin from biliverdin; introduces cytochrome P450 reductase to provide electrons; carbon monoxide dehydrogenase removes the competitive binding of by-products to the substrate heme; formate dehydrogenase is used for in-situ regeneration of coenzymes, and bilirubin is efficiently synthesized by a one-pot method. The heme oxygenase mutant of the present invention couples the above two core enzymes with three foreign auxiliary enzymes to construct recombinant Escherichia coli, which catalyzes heme without adding exogenous NADPH, and the bilirubin yield is as high as 1611 mg / L within 4 h, which is the highest reported bilirubin synthesis level with the shortest time. This technology not only removes resource limitations, but also has a simple operation method and a high yield, realizing the efficient and green synthesis of bilirubin.

Description

Technical Field

[0001] The present invention relates to the efficient synthesis of bilirubin by heme oxygenase mutants and their multi-enzyme coupling systems, belonging to the field of biocatalytic engineering. Background Art

[0002] Bilirubin (CAS No. 635-65-4) is the main pigment in bile. Since the excessive accumulation of bilirubin in the body (especially in newborns) is likely to cause various diseases, such as the deposition of bilirubin in the central nervous system of the brain leading to newborns suffering from bilirubin encephalopathy, which damages auditory, motor and mental functions; and the excessive concentration of free bilirubin in plasma is likely to cause jaundice, etc. Therefore, bilirubin was considered a metabolic waste in the early days.

[0003] However, in recent years, with the in-depth study of bile pigments, their beneficial physiological effects have been gradually discovered and reported. Bilirubin has antioxidant effects and can prevent the oxidation of biological macromolecules such as fats and proteins and scavenge oxygen free radicals physiologically; an appropriate concentration of bilirubin in serum can prevent cardiovascular diseases and metabolic diseases; it plays a key role in reducing neuronal damage in the central nervous system; in addition, due to its significant anti-inflammatory, antioxidant and antiviral effects, bilirubin has also been applied to the treatment of systemic complications of COVID-19 infection.

[0004] Based on the important role and potential market value of bilirubin, the development of its production methods and corresponding reports have been made. The traditional preparation method is to extract from the bile of livestock such as pigs and cows. In recent years, the research progress has mainly focused on the development of extraction and purification processes. This method is limited by the resources of pig bile, with a low extraction rate and the output being difficult to meet the market demand. With the development of synthetic biology, biocatalysis, as a green manufacturing method, has attracted extensive 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 by-products generated in the reaction, and high stereoselectivity.

[0005] Eukaryotes usually have a complete bilirubin synthesis pathway. The key metabolic intermediate of this pathway is heme. Heme is catalytically oxidized by heme oxygenase (HO, EC 1.14.14.18) to form the intermediate biliverdin, and then undergoes hydrogenation reduction catalyzed by biliverdin reductase (BVR, E.C. 1.3.1.24) to form bilirubin. The reaction formula is as Figure 12 shown.

[0006] So far, there have been few reports on the biosynthesis of bilirubin and its key intermediates. For example, SiHan Yan et al. constructed a heme oxygenase and coenzyme regeneration system of glutamate dehydrogenase from Clostridium tetani and carried out membrane surface display. Using E. coli whole cells as catalysts, the biliverdin production reached 76.3 mg / L; Jianfeng Mei et al. used biliverdin as a substrate and expressed biliverdin reductase from cyanobacteria as a whole cell catalyst in E. coli host, and the bilirubin production reached 325 mg / L. In addition, in previous work, our technical team constructed recombinant E. coli containing heme oxygenase, biliverdin reductase, formate dehydrogenase and cytochrome P450 reductase, and the bilirubin production reached 415.5 mg / L (Patent Publication No. CN117487733A).

[0007] Research reports show that in the process of catalytic synthesis of bilirubin with heme as a substrate, it is divided into two-step reactions: the first reaction is catalyzed by heme oxygenase to synthesize biliverdin from heme, and the second reaction is catalyzed by biliverdin reductase to synthesize bilirubin from biliverdin. The reaction formula is as Figure 12 shown. The conversion rate of heme catalyzed by heme oxygenase in the first reaction of the reaction process is much lower than the conversion rate of biliverdin catalyzed by biliverdin reductase in the second reaction. The catalytic functions of the two core enzymes are seriously unbalanced, resulting in a low efficiency of the multi-enzyme coupling system constructed by the two to catalyze the substrate heme to synthesize bilirubin. Therefore, there is an urgent need to develop a method for directed evolution of heme oxygenase to improve its industrial properties. In addition, since expensive coenzymes are required in the reaction process and by-product carbon monoxide is produced, it is of great significance to couple the coenzyme regeneration system and carbon monoxide by-product elimination system in the reaction system of the two core enzymes to achieve one-pot high-efficiency catalysis of heme to synthesize bilirubin. Summary of the Invention

[0008] In the present invention, heme oxygenase (ScHO), cytochrome P450 reductase (ScCPR), and biliverdin reductase (MmBVR) were synthesized based on the bilirubin synthesis and metabolism pathway. Using the model strain Escherichia coli BL21(DE3) as the expression host, all protein sequences were codon-optimized; in order to improve the soluble expression level of the proteins, the transmembrane regions of ScHO and ScCPR were truncated during gene synthesis. The truncated sequences were the C-terminal 289-318 amino acid sequence of ScHO and the N-terminal 1-24 amino acid sequence of ScCPR (ScHO and ScCPR mentioned below both represent the truncated proteins). In addition, NADP + -dependent formate dehydrogenase was additionally expressed in the engineered strain for the regeneration of coenzyme NADPH.

[0009] Since the conversion rate of heme catalyzed by heme oxygenase is much lower than that of biliverdin catalyzed by biliverdin reductase, enzyme modification of ScHO was thus carried out. Specifically, amino acid residues within the range of the substrate in the active center were obtained through crystal structure analysis and alanine scanning and site-directed saturation mutagenesis were performed, resulting in three single mutants with increased activity, and combined mutants with further increased activity were obtained after combining the single mutations. The present invention provides a heme oxygenase mutant, which is obtained by mutating glutamine at position 25 of the heme oxygenase with the amino acid sequence shown in SEQ ID NO.1 to valine, named Q25V; or by mutating histidine at position 156 of the heme oxygenase with the amino acid sequence shown in SEQ ID NO.1 to asparagine, named H156N; or by mutating asparagine at position 247 of the heme oxygenase with the amino acid sequence shown in SEQ ID NO.1 to histidine, named N247H; or by mutating histidine at position 156 of the heme oxygenase with the amino acid sequence shown in SEQ ID NO.1 to asparagine and simultaneously mutating asparagine at position 247 to histidine, named H156N / N247H; or by mutating glutamine at position 25 of the heme oxygenase with the amino acid sequence shown in SEQ ID NO.1 to valine and simultaneously mutating histidine at position 156 to asparagine, named Q25V / H156N; or by mutating glutamine at position 25 of the heme oxygenase with the amino acid sequence shown in SEQ ID NO.1 to valine and simultaneously mutating asparagine at position 247 to histidine, named Q25V / N247H.

[0010] The amino acid sequence of the heme oxygenase is shown in SEQ ID NO.1:

[0011]

[0012] ​MEDSSNTIIPSPTDVGALANRINFQTRDAHNKINTFMGIKMAIAMRHGFIYRQGILAYYYVFDAIEQEIDRLLNDPVTEEELQTSTILKQFWLEDFRRSTQIYKDLKLLYSNTFKSTESLNEFLATFQKPPLLQQFINNIHENIHKEPCTILSYCHVLYLALFAGGKLIRSNLYRRLGLFPNFEKLSQKELVKKGTNFFTFSDLGPTEETRLKWEYKKNYELATRTELTEAQKLQIISVAEGIFDWNFNIVAEIGELNRRELMGKFSFKCITYLYEEWMFNKDSATRR

[0013] The nucleotide sequence encoding the heme oxygenase is shown in SEQ ID NO.2:

[0014] atggaagatagcagcaacaccatcatcccgtccccgaccgatgttggcgccctggcgaaccgtatcaacttccagacccgcgatgcgcacaacaaaatcaacaccttcatgggtatcaaaatggcgatcgcaatgcgtcacggcttcatctaccgccagggcatcctggcgtactactacgttttcgacgcgatcgaacaggaaatcgatcgtctgctgaacgatccggtgaccgaagaagaattgcagacctccaccatcctgaaacagttctggctggaggacttccgccgtagcacccagatctacaaagatctgaaactgctgtacagcaacaccttcaaatccaccgaaagcctgaacgagttcctggcgaccttccagaaaccgccgctgctgcaacagttcatcaacaacatccatgaaaacatccacaaagaaccgtgcaccatcctgtcctactgccacgttctgtacctggccctgttcgcgggcggcaaactgatccgttccaacctgtaccgtcgcctgggtctgttcccgaacttcgaaaaactgtctcagaaagaactggttaaaaaaggcaccaacttcttcaccttctccgacctgggcccgaccgaagaaacccgcctgaaatgggaatataaaaagaactacgaactggcaacccgtaccgaactgaccgaagcgcagaaactgcaaatcatctccgttgcggaaggtatcttcgactggaactttaacatcgtggcggaaatcggcgaactgaaccgtcgtgaactgatgggcaaattctctttcaaatgcatcacctacctgtacgaagaatggatgttcaacaaagatagcgcgacccgccgttaa

[0015] SEQ ID NO.7 (Nucleotide sequence of Q25V):

[0016] atggaagatagcagcaacaccatcatcccgtccccgaccgatgttggcgccctggcgaaccgtatcaacttcgttacccgcgatgcgcacaacaaaatcaacaccttcatgggtatcaaaatggcgatcgcaatgcgtcacggcttcatctaccgccagggcatcctggcgtactactacgttttcgacgcgatcgaacaggaaatcgatcgtctgctgaacgatccggtgaccgaagaagaattgcagacctccaccatcctgaaacagttctggctggaggacttccgccgtagcacccagatctacaaagatctgaaactgctgtacagcaacaccttcaaatccaccgaaagcctgaacgagttcctggcgaccttccagaaaccgccgctgctgcaacagttcatcaacaacatccatgaaaacatccacaaagaaccgtgcaccatcctgtcctactgccacgttctgtacctggccctgttcgcgggcggcaaactgatccgttccaacctgtaccgtcgcctgggtctgttcccgaacttcgaaaaactgtctcagaaagaactggttaaaaaaggcaccaacttcttcaccttctccgacctgggcccgaccgaagaaacccgcctgaaatgggaatataaaaagaactacgaactggcaacccgtaccgaactgaccgaagcgcagaaactgcaaatcatctccgttgcggaaggtatcttcgactggaactttaacatcgtggcggaaatcggcgaactgaaccgtcgtgaactgatgggcaaattctctttcaaatgcatcacctacctgtacgaagaatggatgttcaacaaagatagcgcgacccgccgttaa

[0017] SEQ ID NO.8 (Nucleotide sequence of H156N):

[0018] atggaagatagcagcaacaccatcatcccgtccccgaccgatgttggcgccctggcgaaccgtatcaacttccagacccgcgatgcgcacaacaaaatcaacaccttcatgggtatcaaaatggcgatcgcaatgcgtcacggcttcatctaccgccagggcatcctggcgtactactacgttttcgacgcgatcgaacaggaaatcgatcgtctgctgaacgatccggtgaccgaagaagaattgcagacctccaccatcctgaaacagttctggctggaggacttccgccgtagcacccagatctacaaagatctgaaactgctgtacagcaacaccttcaaatccaccgaaagcctgaacgagttcctggcgaccttccagaaaccgccgctgctgcaacagttcatcaacaacatccatgaaaacatccacaaagaaccgtgcaccatcctgtcctactgcaatgttctgtacctggccctgttcgcgggcggcaaactgatccgttccaacctgtaccgtcgcctgggtctgttcccgaacttcgaaaaactgtctcagaaagaactggttaaaaaaggcaccaacttcttcaccttctccgacctgggcccgaccgaagaaacccgcctgaaatgggaatataaaaagaactacgaactggcaacccgtaccgaactgaccgaagcgcagaaactgcaaatcatctccgttgcggaaggtatcttcgactggaactttaacatcgtggcggaaatcggcgaactgaaccgtcgtgaactgatgggcaaattctctttcaaatgcatcacctacctgtacgaagaatggatgttcaacaaagatagcgcgacccgccgttaa

[0019] SEQ ID NO.9 (Nucleotide sequence of N247H):

[0020] atggaagatagcagcaacaccatcatcccgtccccgaccgatgttggcgccctggcgaaccgtatcaacttccagacccgcgatgcgcacaacaaaatcaacaccttcatgggtatcaaaatggcgatcgcaatgcgtcacggcttcatctaccgccagggcatcctggcgtactactacgttttcgacgcgatcgaacaggaaatcgatcgtctgctgaacgatccggtgaccgaagaagaattgcagacctccaccatcctgaaacagttctggctggaggacttccgccgtagcacccagatctacaaagatctgaaactgctgtacagcaacaccttcaaatccaccgaaagcctgaacgagttcctggcgaccttccagaaaccgccgctgctgcaacagttcatcaacaacatccatgaaaacatccacaaagaaccgtgcaccatcctgtcctactgccacgttctgtacctggccctgttcgcgggcggcaaactgatccgttccaacctgtaccgtcgcctgggtctgttcccgaacttcgaaaaactgtctcagaaagaactggttaaaaaaggcaccaacttcttcaccttctccgacctgggcccgaccgaagaaacccgcctgaaatgggaatataaaaagaactacgaactggcaacccgtaccgaactgaccgaagcgcagaaactgcaaatcatctccgttgcggaaggtatcttcgactggcattttaacatcgtggcggaaatcggcgaactgaaccgtcgtgaactgatgggcaaattctctttcaaatgcatcacctacctgtacgaagaatggatgttcaacaaagatagcgcgacccgccgttaa

[0021] SEQ ID NO.10 (Nucleotide sequence of H156N / N247H):

[0022] atggaagatagcagcaacaccatcatcccgtccccgaccgatgttggcgccctggcgaaccgtatcaacttccagacccgcgatgcgcacaacaaaatcaacaccttcatgggtatcaaaatggcgatcgcaatgcgtcacggcttcatctaccgccagggcatcctggcgtactactacgttttcgacgcgatcgaacaggaaatcgatcgtctgctgaacgatccggtgaccgaagaagaattgcagacctccaccatcctgaaacagttctggctggaggacttccgccgtagcacccagatctacaaagatctgaaactgctgtacagcaacaccttcaaatccaccgaaagcctgaacgagttcctggcgaccttccagaaaccgccgctgctgcaacagttcatcaacaacatccatgaaaacatccacaaagaaccgtgcaccatcctgtcctactgcaatgttctgtacctggccctgttcgcgggcggcaaactgatccgttccaacctgtaccgtcgcctgggtctgttcccgaacttcgaaaaactgtctcagaaagaactggttaaaaaaggcaccaacttcttcaccttctccgacctgggcccgaccgaagaaacccgcctgaaatgggaatataaaaagaactacgaactggcaacccgtaccgaactgaccgaagcgcagaaactgcaaatcatctccgttgcggaaggtatcttcgactggcattttaacatcgtggcggaaatcggcgaactgaaccgtcgtgaactgatgggcaaattctctttcaaatgcatcacctacctgtacgaagaatggatgttcaacaaagatagcgcgacccgccgttaa

[0023] The present invention also provides a gene encoding the above-mentioned heme oxygenase mutant.

[0024] The present invention also provides a recombinant vector carrying the above-mentioned gene.

[0025] The present invention also provides a recombinant cell expressing the above mutant or carrying the above gene or the above recombinant vector.

[0026] In one embodiment of the present invention, the recombinant cell uses a bacterium or a fungus as the host cell.

[0027] The present invention also provides a recombinant enzyme catalyst containing the above heme oxygenase mutant, which is any one of the following forms: (1) culturing a recombinant expression transformant containing the heme oxygenase mutant, and separating the transformed somatic cells containing the recombinant heme oxygenase mutant enzyme;

[0028] (2) culturing a recombinant expression transformant containing the heme oxygenase mutant, separating the transformed somatic cells containing the recombinant heme oxygenase mutant enzyme, disrupting the transformed somatic cells containing the recombinant heme oxygenase mutant enzyme, and obtaining a cell lysate;

[0029] (3) culturing a recombinant expression transformant containing the heme oxygenase mutant, separating the transformed somatic cells containing the recombinant heme oxygenase mutant enzyme, disrupting the transformed somatic cells containing the recombinant heme oxygenase mutant enzyme, obtaining a cell lysate, and freeze-drying the cell lysate of the recombinant heme oxygenase mutant enzyme to obtain a freeze-dried enzyme powder.

[0030] The present invention also provides a method for improving the conversion rate of heme oxygenase to a substrate and enhancing the enzyme activity. The method is to mutate glutamine at position 25 of the heme oxygenase with the amino acid sequence shown in SEQ ID NO.1 to valine; or mutate histidine at position 156 of the heme oxygenase with the amino acid sequence shown in SEQ ID NO.1 to asparagine; or mutate asparagine at position 247 of the heme oxygenase with the amino acid sequence shown in SEQ ID NO.1 to histidine; or mutate histidine at position 156 of the heme oxygenase with the amino acid sequence shown in SEQ ID NO.1 to asparagine and simultaneously mutate asparagine at position 247 to histidine; or mutate glutamine at position 25 of the heme oxygenase with the amino acid sequence shown in SEQ ID NO.1 to valine and simultaneously mutate histidine at position 156 to asparagine; or mutate glutamine at position 25 of the heme oxygenase with the amino acid sequence shown in SEQ ID NO.1 to valine and simultaneously mutate asparagine at position 247 to histidine.

[0031] The present invention also provides the use of the above mutant or the above recombinant cell or the above recombinant enzyme catalyst in the preparation of biliverdin or bilirubin.

[0032] In one embodiment of the present invention, for the application in the preparation of biliverdin, the reaction system includes a whole-cell reaction and an enzyme reaction system, wherein: the whole-cell reaction is: using hemin as a substrate, the whole cell catalyzes the substrate to prepare biliverdin, and the recombinant cell also expresses NADP + -dependent formate dehydrogenase derived from Azospirillum palustre and cytochrome P450 reductase derived from Saccharomyces cerevisiae; the enzyme reaction system is: using hemin as a substrate to prepare biliverdin, and simultaneously adding NADP + -dependent formate dehydrogenase derived from Azospirillum palustre and cytochrome P450 reductase derived from Saccharomyces cerevisiae;

[0033] In one embodiment of the present invention, for the application in the preparation of bilirubin, the reaction system includes a whole-cell reaction and an enzyme reaction system, wherein: the whole-cell reaction is: using hemin as a substrate, the whole cell catalyzes the substrate to prepare bilirubin, and the recombinant cell also expresses biliverdin reductase derived from Mus musculus, NADP + -dependent formate dehydrogenase derived from Azospirillum palustre, cytochrome P450 reductase derived from Saccharomyces cerevisiae, and also expresses carbon monoxide dehydrogenase derived from Afipia carboxidovorans; the enzyme reaction system is: using hemin as a substrate to prepare bilirubin, and simultaneously adding biliverdin reductase derived from Mus musculus, NADP + -dependent formate dehydrogenase derived from Azospirillum palustre, cytochrome P450 reductase derived from Saccharomyces cerevisiae, and also expresses carbon monoxide dehydrogenase derived from Afipia carboxidovorans.

[0034] The present invention provides a genetically engineered bacterium, which expresses heme oxygenase or the above mutant derived from Saccharomyces cerevisiae, and its wild-type nucleotide sequence is as shown in SEQ ID NO.2; cytochrome P450 reductase derived from Saccharomyces cerevisiae, and its nucleotide sequence is as shown in SEQ ID NO.3; biliverdin reductase derived from Mus musculus, and its nucleotide sequence is as shown in SEQ ID NO.4; NADP +The nucleotide sequence of the NAD-dependent formate dehydrogenase is as shown in SEQ ID NO.5; meanwhile, a carbon monoxide dehydrogenase derived from Afipia carboxidovorans was also expressed, and its nucleotide sequence is as shown in SEQ ID NO.6.

[0035] SEQ ID NO.3:

[0036]

[0037] SEQ ID NO.4:

[0038] atgagcaccgaaccgaaacgtaaattcggcgttgttgttgttggtgttggtcgcgcgggcagcgttcgtatccgtgatctgaaagatccgcactctagcgcgttcctgaacctgatcggttacgtttctcgtcgtgaactgggcagcctggataacgttcgtcagatctctctggaagatgcgctgcgtagccaggaagttgatgttgcatacatctgcactgaatcctcttcccacgaggactacatccgtcagttcctccaggcgggtaaacacgttctggttgaatacccgatggcgctgagcttcgcggcggcgcaggaactgtgggaactggcggcgcagaaaggtcgtgttctgcacgaagaacacatcgaactgctgatggaagaatttgagttcctgaaacgtgaagttgcaggcaaagaactgctgaaaggcagcctgcgtttcaccgcgagcccgctggaagaagaaaaattcggcttcccggcgttcagcggcatctctcgtctgacctggctggtgtctctgttcggtgaactgtctctgatcagcgcgactatggaaaaccgtaaagaagatcagtatatgaaaatgaccgttcagctggaaacccagaacaaatctccgctgagctggattgaagaaaaaggtccgggcctgaaacgtaaccgtcacatcagcatccacttcaaatccggcagcctggaagaagttccgaacgttggtgttaacaaaaacatcttcctgaaagatcaggatattttcatccagaaactgctgggccaggttagcgcggaagatctggcggcggaaaagaaacgtatcctgcactgcctggaactggcgagcgatattcagcgtctgtgccaccgtaaacagtaa

[0039] SEQ ID NO.5:

[0040]

[0041] SEQ ID NO.6:

[0042] atggcaaaggcgcacatcgaactgactatcaacggccatccggtagaagcgctggttgaaccacgtaccctgctgattcacttcatccgcgaacagcagaacctgaccggtgcccacatcggctgcgacacgtctcactgtggtgcgtgcactgttgacctggacggcatgagcgtcaaatcctgcactatgttcgcagtacaggcgaacggtgcatccatcaccactatcgaaggtatggcggcgccagatggcactctgtctgcactgcaggagggctttcgcatgatgcacggtctgcagtgcggttactgtactccgggcatgatcatgcgcagccaccgtctgctgcaggaaaacccgagcccgactgaagcggagatccgttttggcattggcggcaacctgtgccgctgcaccggttaccagaacatcgtcaaggcgatccagtatgccgctgcaaaaatcaacggtgtgccgttcgaggaagcagcggaa

[0043] 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.

[0044] In one embodiment of the present invention, the genetically engineered bacteria are recombinant Escherichia coli. Preferably, the recombinant Escherichia coli uses E. coli BL21(DE3) as the expression host. Preferably, the recombinant Escherichia coli uses pET28a(+), pET21a(+), pET32a(+), pGEX, pETDuet-1, pRSFDuet-1, or pCDFDuet-1 as the expression vector.

[0045] In one embodiment of the present invention, the high-performance liquid detection method refers to detecting the content of biliverdin, the product catalyzed by heme oxygenase, or detecting the content of bilirubin, the product of the two-step reaction.

[0046] In one embodiment of the present invention, the enzyme activity of the heme oxygenase is measured by reacting with heme as a substrate and detecting the peak area of its product biliverdin by high-pressure liquid chromatography. The screening of the heme oxygenase mutant is carried out by comparing the enzyme activities.

[0047] The present invention also provides the base sequences encoding the above-mentioned heme oxygenase, biliverdin reductase, formate dehydrogenase and chaperone protein.

[0048] The present invention also provides a recombinant vector carrying the above genes; preferably, the recombinant vector uses pET28a(+), pET21a(+), pET32a(+), pGEX, pETDuet-1, pRSFDuet-1 or pCDFDuet-1 as the expression vector.

[0049] The present invention also provides one or more recombinant cells expressing the above genes or carrying the above recombinant vectors.

[0050] In one embodiment of the present invention, the recombinant cell uses Escherichia coli as the expression host.

[0051] In one embodiment of the present invention, the recombinant bacterium is constructed by PCR amplifying heme oxygenase (ScHO) or its mutant (ScHOmut), biliverdin reductase (MmBVR), cytochrome P450 reductase (ScCPR) or formate dehydrogenase (ApFDH), cloning them into the plasmid pETDuet-1, and constructing the recombinant plasmid pETDuet-1-ScHO-ScCPR-ApFDH, or pETDuet-1-ScHOmut-ScCPR-ApFDH, to construct a recombinant Escherichia coli catalyst.

[0052] In another embodiment of the present invention, the recombinant bacterium is constructed by PCR amplifying formate dehydrogenase (ApFDH) and carbon monoxide dehydrogenase (AcCODH), cloning them into the plasmid pCDFDuet-1, and constructing the recombinant plasmid pCDFDuet-1-ApFDH-AcCODH, to construct a recombinant Escherichia coli whole-cell catalyst.

[0053] In another embodiment of the present invention, the recombinant bacterium is constructed by co-transforming the recombinant plasmid pETDuet-1-ScHO-ScCPR-ApFDH or pETDuet-1-ScHOmut-ScCPR-ApFDH with the recombinant plasmid pCDFDuet-1-ApFDH-AcCODH into E. coli BL21(DE3), to construct a recombinant Escherichia coli whole-cell catalyst.

[0054] The present invention provides a method for improving the bilirubin synthesis ability of Escherichia coli. The method is to use Escherichia coli E. coli BL21(DE3) as the host cell to express heme oxygenase and cytochrome P450 reductase derived from Saccharomyces cerevisiae, biliverdin reductase derived from Mus musculus, NADP + -dependent formate dehydrogenase derived from Azospirillum palustre, and at the same time, express carbon monoxide dehydrogenase derived from Afipia carboxidovorans.

[0055] In an embodiment of the present invention, the heme oxygenase is the heme oxygenase obtained by deleting the amino acids at positions 289-318 at the C-terminus of the heme oxygenase with NCBI accession number GHM92019.1 or the mutant of the above heme oxygenase; the NCBI accession number of the biliverdin reductase is XP_030102511.1, and the cytochrome P450 reductase is the cytochrome P450 reductase obtained by deleting the amino acids at positions 1-24 at the N-terminus of the cytochrome P450 reductase with NCBI accession number NP_011908.1; the NADP + -dependent formate dehydrogenase has NCBI accession number WP_098736599.1; the carbon monoxide dehydrogenase has NCBI accession number WP_013913729.1.

[0056] The present invention also provides a method for synthesizing bilirubin by whole-cell catalysis. The method is to use heme as a substrate and, under aeration conditions, use the above recombinant cell lysate or the above recombinant Escherichia coli whole cells for catalytic conversion to synthesize bilirubin.

[0057] In an embodiment of the present invention, the whole-cell catalysis system includes but is not limited to 3 mM heme (equivalent to 1.953 g / L), aeration rate of 8 vvm, heme is pre-dissolved in a NaOH solution (100 mM) accounting for 50% of the total reaction volume, after complete dissolution, the pH is adjusted to 6.5 with HCl, 50 mM ammonium formate is added, and the reaction volume is made up with a 100 mM citric acid-sodium citrate solution (pH 6.5). The reaction time is 4 h and the reaction temperature is 35°C.

[0058] In an embodiment of the present invention, the whole-cell catalyst is obtained by culturing the recombinant bacterium and inducing it to express heme oxygenase, biliverdin reductase, cytochrome P450 reductase, formate dehydrogenase, and carbon monoxide dehydrogenase, and further collecting the recombinant bacterium cells.

[0059] In one embodiment of the present invention, the whole-cell catalyst is obtained by inoculating recombinant Escherichia coli co-expressing heme oxygenase, biliverdin reductase, cytochrome P450 reductase, formate dehydrogenase and carbon monoxide dehydrogenase into 100 mL of LB medium at an inoculation amount of 1%, culturing at 37 °C, and when the OD 600 reaches 0.4 - 0.6, adding 0.5 mM IPTG to induce protein expression, inducing at 20 °C for 16 h, then centrifuging at 8,000×g at low temperature for 10 min, collecting the cells, and washing the cells twice with 20 mM Tris-HCl (pH 7.5) buffer to obtain the whole-cell catalyst.

[0060] In one embodiment of the present invention, the reaction conditions for the whole-cell catalysis are: stirring speed 200 r / min, temperature 35 °C, pH 6.5, aeration rate 8 vvm, and reaction time 1 - 4 h.

[0061] In one embodiment of the present invention, the addition amount of heme is: 1 - 4 mM (corresponding to 651 - 2604 mg / L), and the addition amount of the catalyst is: 20 g / L (dry cell weight).

[0062] The present invention provides the application of the above recombinant Escherichia coli or the above method in the preparation of bilirubin or products containing bilirubin.

[0063] Beneficial effects

[0064] (1) In the present invention, a high-activity mutant of heme oxygenase was successfully obtained, and the catalytic efficiency of heme oxygenase was improved. Heme oxygenase mutant, cytochrome P450 reductase, and biliverdin reductase were co-expressed in Escherichia coli, and at the same time, high-activity formate dehydrogenase and carbon monoxide dehydrogenase were coupled. The complete heme degradation cascade reaction was successfully reconstructed in a single cell. At the same time, without the exogenous addition of NADPH, high catalytic activity can be achieved using endogenous coenzymes in the cell, and there is no accumulation of the intermediate product biliverdin during the catalytic process; in addition, the by-product carbon monoxide generated during the catalytic process is eliminated in time, avoiding the accumulation of carbon monoxide-heme complex that cannot be utilized by the enzyme. Using heme as the substrate, through multi-enzyme one-pot cascade catalysis, the final yield of bilirubin can reach 1611 mg / L, and the conversion rate > 91%. The multi-enzyme coupling system constructed in the present invention solves the problem of low conversion rate in the research of catalytic conversion of heme to synthesize biliverdin and bilirubin.

[0065] (2) The establishment of this whole-cell transformation system eliminates the resource limitations of the pig bile extraction method, solves the problems of cumbersome procedures, low yield, and large wastewater discharge in the production of bilirubin by traditional biological tissue extraction methods, has good atom economy, and realizes the efficient and green synthesis of bilirubin using heme as a substrate. According to all the available data, the bilirubin yield in the present invention is the highest level in the biological synthesis starting from heme substrate. Description of the Drawings

[0066] Figure 1 : Protein expression of the strain containing the recombinant plasmid pETDuet-1-ScHO-ScCPR-ApFDH, lane 1 is the supernatant of cell disruption.

[0067] Figure 2 : Protein expression of the strain containing the recombinant plasmids pETDuet-1-ScHO-ScCPR-ApFDH and pCDFDuet-1-MmBVR-AcCODH, lane 1 is the supernatant of cell disruption.

[0068] Figure 3 : Active center of heme oxygenase ScHO and amino acid residues within the range from the substrate range.

[0069] Figure 4 : Standard curve of biliverdin measured by microplate reader.

[0070] Figure 5 : High-throughput screening results of saturation mutagenesis of amino acid residues within the range from the substrate near the active center of heme oxygenase ScHO range.

[0071] Figure 6 : Fitting results of Michaelis-Menten equation for ScHO wild type and mutants.

[0072] Figure 7 : Ni 2+ Heme oxygenase ScHO and cytochrome P450 reductase ScCPR purified by Ni affinity chromatography.

[0073] Figure 8 : Temperature optimization results of recombinant Escherichia coli catalyzing the synthesis of bilirubin.

[0074] Figure 9 : pH optimization results of recombinant Escherichia coli catalyzing the synthesis of bilirubin.

[0075] Figure 10 : Reaction progress diagram of recombinant Escherichia coli catalyzing the synthesis of bilirubin at different substrate concentrations.

[0076] Figure 11: HPLC chromatogram and standard curve of bilirubin; wherein, a, chromatogram of bilirubin; b, standard curve of bilirubin.

[0077] Figure 12 : Reaction formula of bilirubin. Specific implementation mode

[0078] Preparation of nucleic acid gel involved in the following examples:

[0079] 50×TAE buffer: Accurately weigh 242 g of Tris and 37.2 g of Na2EDTA·2H2O into a 1 L volumetric flask, add 800 mL of ultrapure water, dissolve and shake well, then add 57.1 mL of glacial acetic acid, shake well, and make up the volume to 1 L with deionized water. After preparing 50×TAE buffer, dilute it 50 times to prepare 1×TAE buffer. Using 1×TAE buffer as the solvent, prepare an agarose solution with a mass concentration of 0.9% (dissolve by heating). After the agarose is completely dissolved, add nucleic acid dye (1.5 μL / 20 mL), gently shake and mix evenly to avoid generating bubbles, and pour it into a nucleic acid gel mold to cool to obtain an agarose gel.

[0080] Gel electrophoresis involved in the following examples:

[0081] Place the gel in an electrophoresis tank filled with 1×TAE buffer, set the electrophoresis conditions to 160 V and 15 min. After electrophoresis, observe the bands of the agarose gel under ultraviolet light. Digest the template: Take 50 μL of the PCR product detected as positive by electrophoresis, add 1 μL of restriction enzyme DpnⅠ and 5 μL of the corresponding buffer to remove methylated template DNA. The reaction conditions are 37 °C for 3 h. After the reaction, incubate at 65 °C for 10 min to inactivate DpnⅠ.

[0082] Product purification involved in the following examples: According to the product instruction manual of FastPure Gel DNA Extraction Mini Kit to purify the digestion product.

[0083] Homologous recombination involved in the following examples: According to the product instruction manual of ClonExpress II One Step Cloning Kit for homologous recombination, ligate the truncated mutant DNA fragment into the linearized vector to obtain a recombinant plasmid.

[0084] Transformation of the recombinant plasmid involved in the following examples:

[0085] Add all the reaction solution of homologous recombination into E. coli BL21(DE3) competent cells, mix well, and place on ice bath for 30 min; place in a 42 °C water bath for heat shock for 90 s, quickly transfer to an ice bath for cooling for 5 min, add 600 μL of LB medium, and culture at 37 °C and 200 rpm for 1 h; pipette 50 μL and spread it on an LB plate containing the corresponding antibiotic, and culture inverted at 37 °C for 12 h; pick single colonies for sequencing verification, and preserve the strains into which the correct recombinant plasmid has been introduced.

[0086] The detection methods involved in the following examples are as follows:

[0087] SDS-PAGE gel electrophoresis:

[0088] 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 the wet bacterial cells containing the target protein, prepare a 20 g / L cell suspension with Tris-HCl buffer (pH 7.5, 100 mM), and place it on an ice bath for ultrasonic disruption (450 W, working for 1 s, pausing for 4 s, for a total of 10 min). Centrifuge at 12,000×g and 4 °C for 1 min to separate the supernatant and precipitate. Take 20 μL of the supernatant, mix it with 8 μL of Loading buffer, heat it in a boiling water bath for 10 min, and centrifuge at 12,000×g for 5 min for standby. Electrophoresis: The sample loading volume of the protein gel is 10 μL for the sample and 3 μL for the Marker; electrophoresis conditions: 160 V, 60 min; electrophoresis buffer (g / L): glycine 14.4, SDS 1, Tris 3. Staining: Immerse the protein gel in the staining solution (1 g of Coomassie Brilliant Blue R250, 450 mL of methanol, 100 mL of acetic acid, 450 mL of water) for 15 min, and wash it twice with water. Decolorization: Immerse it in the decolorizing solution (10% ethanol, 10% acetic acid, 80% water) and gently shake it, and replace the fresh decolorizing solution until the bands are clearly visible.

[0089] Bilirubin detection

[0090] Mobile phase: A (25 mM ammonium acetate, adjusted to pH 3.5 with acetic acid); B (acetonitrile: methanol: isopropanol = 8:1:1); A:B = 5:95; column temperature: 35 °C, chromatographic column: C18, injection volume: 5 μL, running time 10 min, detection wavelength 400 nm. The chromatogram of bilirubin and the corresponding standard curve are as shown in the instruction manual Figure 11 as follows.

[0091] Detection of heme oxygenase enzyme activity

[0092] 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 hemin were added. Using Tris-HCl (100 mM, pH 7.5) as the solvent, it was placed in a constant temperature oscillator at 35 °C and reacted at 500 rpm for 20 min. Subsequently, 50 μL of the sample was taken, mixed evenly with 950 μL of methanol, centrifuged at 12,000×g for 1 min, 200 μL of the supernatant was aspirated, placed in a 96-well plate to measure the absorbance at 690 nm, and the concentration of the product biliverdin was calculated according to the standard curve, and further the activity unit number U and specific activity U / mg were calculated.

[0093] The definition of the activity unit U is: under the conditions of 35 °C and pH 7.5, the amount of enzyme required to catalyze the synthesis of 1 μmol of biliverdin per minute is 1 U. The specific activity is: the number of activity units per milligram of heme oxygenase or its mutant, and its unit is U / mg.

[0094] The protein sequence information involved in the following examples is shown in Table 1.

[0095] Table 1: Protein Information

[0096] 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 Carbon monoxide dehydrogenase (AcCODH) Afipia carboxidovorans WP_013913729.1

[0097] Among them, the heme oxygenase used in the present invention is the heme oxygenase obtained by deleting the amino acids at positions 289-318 at the C-terminus of the heme oxygenase with the NCBI accession number GHM92019.1, and the ScCPR obtained by deleting the amino acid sequence at positions 1-24 at the N-terminus of ScCPR with the NCBI accession number NP_011908.1.

[0098] Example 1: Gene Synthesis and Expression

[0099] The specific steps are as follows:

[0100] 1. Construction of Recombinant Vectors

[0101] (1) Acquisition of Genes

[0102] Heme oxygenase (ScHO) with a nucleotide sequence as shown in SEQ ID NO.2, biliverdin reductase (MmBVR) with a nucleotide sequence as shown in SEQ ID NO.4, cytochrome P450 reductase (ScCPR) with a nucleotide sequence as shown in SEQ ID NO.3, formate dehydrogenase (ApFDH) with a nucleotide sequence as shown in SEQ ID NO.5, and carbon monoxide dehydrogenase (AcCODH) with a nucleotide sequence as shown in SEQ ID NO.6 were synthesized respectively. They were submitted to Sangon Biotech (Shanghai) Co., Ltd. for codon optimization, gene synthesis, and construction of recombinant vectors.

[0103] (2) Construction of recombinant vectors

[0104] The synthesized ScHO gene, ScCPR gene, and ApFDH gene were successively inserted into the pETDuet-1 vector. Among them, the nucleotide sequence of ScHO was inserted between the restriction enzyme sites BamH I and Sac I of pETDuet-1 in the 5’→3’ order. After adding the ribosome binding sequence (5’-tttgtttaactttaagaaggagatatacc-3’) to the 5’ end of the nucleotide sequence of ScCPR, it was inserted between the restriction enzyme sites Sal I and Not I of pETDuet-1 in the 5’→3’ order. The nucleotide sequence of ApFDH was inserted between the restriction enzyme sites Nde I and Xho I of pETDuet-1 in the 5’→3’ order to obtain the recombinant plasmid pETDuet-1-ScHO-ScCPR-ApFDH;

[0105] The synthesized MmBVR gene and AcCODH gene were inserted into the pCDFDuet-1 vector. Among them, the nucleotide sequence of MmBVR was inserted between the restriction enzyme sites BamH I and Sac I of pCDFDuet-1 in the 5’→3’ order, and the nucleotide sequence of AcCODH was inserted between the restriction enzyme sites Nde I and Xho I of pCDFDuet-1 in the 5’→3’ order to obtain the recombinant plasmid pCDFDuet-1-MmBVR-AcCODH.

[0106] 2. Construction of recombinant strains

[0107] E. coli BL21(DE3) was selected as the expression host. The recombinant plasmids obtained in the above step (1) were respectively introduced into the competent cells of E. coli BL21(DE3) and spread on the LB solid medium plates containing ampicillin or streptomycin, or containing both of the above two antibiotics. Positive transformants were picked for strain preservation to obtain the corresponding engineering strains: E. coli BL21(DE3) / pETDuet-1-ScHO-ScCPR-ApFDH, E. coli BL21(DE3) / pCDFDuet-1-MmBVR-AcCODH.

[0108] 3. Protein expression

[0109] The engineered strains obtained in step 2 were respectively inoculated into test tubes of LB liquid medium containing ampicillin or streptomycin (both at a concentration of 50 mg / L), or containing both of the above two antibiotics, and cultured at 37 °C and 200 rpm for 8 - 10 h to obtain seed solutions. The seed solutions were inoculated into shake flasks of LB liquid medium containing ampicillin or streptomycin (both at a concentration of 50 mg / L), or containing both of the above two antibiotics (100 mL liquid loading / 500 mL volume) at an inoculation amount of 1% (v / v), and cultured at 37 °C and 200 rpm until 600 the OD reached 0.4 - 0.6, then isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.5 mM was added, the temperature was adjusted to 20 °C, and the culture was continued for 16 h to induce protein expression; fermentation broths were obtained respectively.

[0110] The obtained fermentation broths were centrifuged at low temperature, the centrifugation conditions were 8,000×g, 10 min, 4 °C. 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 ultrasonic conditions of 400 W, 0 °C, working for 2 s, intermittent for 4 s, and a total time of 10 min to obtain cell lysates, which are the catalysts to be added in the examples of the present invention.

[0111] The obtained cell lysates were verified for soluble expression of proteins by SDS-PAGE gel electrophoresis. The protein expression conditions are as Figure 1 and Figure 2 shown. The results showed that all five proteins were successfully expressed.

[0112] Example 2: Construction of heme oxygenase mutants

[0113] The specific steps are as follows:

[0114] First, analyze the amino acid residues ( Figure 3 ) around the active center (SEQ ID NO.1) of ScHO, obtain the sites within the range from the substrate , and use degenerate PCR primers for mutagenesis. The mutagenesis sites and the corresponding primer sequences are shown in Table 2. Among them, N represents any one of the four nucleotides ATCG, and K represents any one of the two nucleotides GT. The primer sequences were submitted to Tsingke Biological (Suzhou) for synthesis.

[0115] Table 2: Mutagenesis sites and primers

[0116]

[0117] Using the pETDuet-1-ScHO-ScCPR-ApFDH constructed in Example 1 as a template and the nucleotide sequences in Table 2 as primers, PCR amplification was carried out to construct a mutant library.

[0118] The PCR reaction conditions were as follows: (1) pre-denaturation at 95°C for 3 min; (2) denaturation at 95°C for 15 s; (3) annealing at 55°C for 15 s; (4) extension at 72°C for 2 min; steps (2) to (4) were cycled 30 times; finally, extension was performed at 72°C for 5 min, and the reaction was terminated by cooling to 16°C.

[0119] PCR system: 1 μL of plasmid template pETDuet-1-ScHO-ScCPR-ApFDH; 1 μL each of forward and reverse primers; 25 μL of Takara primeSTAR Max high-fidelity enzyme; 22 μL of ddH2O.

[0120] 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, 5 μL of 10×quickcut buffer. After mixing, it was placed in a 37°C constant temperature water bath and allowed to stand for 2 h.

[0121] After the reaction was completed, the reaction solution was purified using the Vazyme FastPure Gel DNA Extraction Mini Kit and referring to its instruction manual to obtain a high-purity recombinant plasmid: pETDuet-1-ScHO mutant-ScCPR-ApFDH.

[0122] According to the recombinant strain construction method described in Example 1, the purified product was introduced into E. coli BL21(DE3) competent cells and spread on an LB plate containing ampicillin resistance. After culturing at 37°C for 12 h, monoclonal strains containing the mutant were obtained.

[0123] Example 3: High-throughput screening of forward mutants

[0124] Establishment of a high-throughput detection method:

[0125] Take biliverdin standard and dissolve it in methanol, and prepare biliverdin standard solutions with gradients of 0 - 200 mg / L. Take 200 μL of each concentration and place it in a 96-well plate, and use an enzyme-linked immunosorbent assay reader to read the absorbance of each well at 690 nm. Use the well containing 200 μL of methanol as a blank control to obtain a standard curve as Figure 4 shown.

[0126] High-throughput screening process:

[0127] The monoclonal antibody obtained in Example 2 was picked and inoculated into a 48-well deep-well plate containing 1 mL of LB medium per well, and ampicillin at a concentration of 50 mg / L was added. After culturing at 37 °C and 200 rpm for 8 h, a seed solution was obtained. The obtained seed solution was transferred to another 48-well deep-well plate containing fresh LB medium at an inoculation amount of 1% (v / v), and ampicillin at a concentration of 50 mg / L was added. After culturing at 37 °C and 200 rpm for 2 h, IPTG was added to each well to a final concentration of 0.5 mM, and the plate was transferred to a shaker at 20 °C and 200 rpm and cultured for another 16 h. After the culture was completed, the plate was placed in a centrifuge equipped with a rotor, centrifuged at 8000×g and 4 °C for 10 min, the supernatant was discarded, and the plate containing the cell pellet was placed in a -20 °C refrigerator and frozen for 24 h to increase the cell membrane permeability.

[0128] Subsequently, the following reaction system (total volume 0.5 mL) was added to each small well of the plate: hemin 1 mM, ammonium formate 30 mM, with Tris-HCl (100 mM, pH 7.5) as the solvent, and shaken to evenly suspend the cell pellet. The reaction was carried out in a constant temperature incubator at 35 °C and 200 rpm for 1 hour. After the reaction was completed, 0.5 mL of methanol was added to each well to terminate the reaction. The plate was placed in a centrifuge equipped with a rotor, centrifuged at 8000×g and 4 °C for 10 min, 200 μL of the supernatant was aspirated and added to a 96-well plate, and the absorbance of the product biliverdin catalyzed by heme oxygenase at 690 nm was detected with an enzyme-linked immunosorbent assay (ELISA) reader. The absorbance value was substituted into Figure 4 the standard curve shown, and the concentration was calculated. The saturation mutagenesis results of the 20 mutation sites selected in Example 3 are as shown in Figure 5 shown. It can be seen from Figure 5 that compared with the wild type, mutants with improved catalytic function exist at positions 28, 142, and 210. Therefore, the monoclonal antibodies with the highest catalytic ability among the three positions were respectively selected and sent to Tsingke Biotechnology (Suzhou) for sequencing to obtain the corresponding mutant sequences, and the corresponding monoclonal antibodies were preserved as strains. Among them, the amino acid changes at the mutation sites before and after mutation are shown in Table 3.

[0129] Table 3: Sequencing results of mutants and concentration of catalytic synthesis of biliverdin

[0130] Mutation Site Product biliverdin concentration (mg / L) Q25V 25.95 H156N 37.30 N247H 39.15 WT 22.17

[0131] Example 4: Combinatorial mutation of ScHO

[0132] The three positive mutations Q25V, H156N, and N247H obtained in Example 3 were subjected to combinatorial mutation to further obtain high-activity mutants. The specific steps are as follows:

[0133] The recombinant strain containing three forward mutants obtained in Example 3 was inoculated into a test tube containing 5 mL of LB medium, ampicillin was added, and it was cultured in a constant temperature shaker at 37 °C and 200 rpm for 8 h. Subsequently, plasmid extraction was carried out using Vazyme FastPure Plasmid Mini Kit - BOX 2, and the extraction steps were referred to the instructions provided in the kit. Three recombinant plasmids were obtained.

[0134] pETDuet-1-ScHO Q25V -ScCPR-ApFDH, pETDuet-1-ScHO H156N -ScCPR-ApFDH and

[0135] pETDuet-1-ScHO N247H -ScCPR-ApFDH. Subsequently, according to the mutant primers shown in Table 4, using the above three plasmids as templates, PCR reactions were carried out to introduce the second / third mutation sites.

[0136] Table 4 Site-directed mutagenesis primers

[0137]

[0138] Among them, the PCR, template digestion, and product purification processes were referred to Example 2. Double-mutant recombinant plasmids were obtained.

[0139] pETDuet-1-ScHO Q25V / H156N -ScCPR-ApFDH, pETDuet-1-ScHO Q25V / N247H -ScCPR-ApFDH, pETDuet-1-ScHO H156N / N247H -ScCPR-ApFDH; and triple-mutant recombinant plasmids

[0140] pETDuet-1-ScHO Q25V / H156N / N247H -ScCPR-ApFDH. Subsequently, according to the recombinant strain construction method described in Example 1, the corresponding recombinant strains were obtained, and according to the screening method described in Example 3, the double-point combination mutants and triple-point combination mutants were screened, and the results are shown in Table 5.

[0141] Table 5: Concentrations of biliverdin catalyzed and synthesized by combined mutants

[0142] Mutation Site Product biliverdin concentration (mg / L) Q25V / H156N 36.07 Q25V / N247H 31.63 H156N / N247H 45.58 Q25V / H156N / N247H 22.81 WT 21.55

[0143] The results showed that after the combined mutations of Q25V and H156N, N247H or H156N / N247H with positive effects, the ability to catalytically synthesize biliverdin was weaker than that of the single-point mutations H156N and N247H; while for the single-point mutations H156N and N247H after combined mutations, the ability to catalytically synthesize biliverdin was further improved, which was 2.1 times higher than that of the wild type.

[0144] Example 5: Enzymatic property analysis of mutants and wild-type ScHO

[0145] The specific steps are as follows:

[0146] For the wild-type heme oxygenase ScHO (SEQ ID NO.1) and cytochrome P450 reductase ScCPR, as well as the recombinant plasmids containing mutants screened in Example 3, the mutant sequences were amplified using the PCR primers designed in Table 6 and ligated to pET28a(+) through homologous recombination technology.

[0147] Table 6: Homologous recombination primers

[0148]

[0149]

[0150] Recombinant plasmids pET28a(+)-ScHO, pET28a(+)-Q25V, pET28a(+)-H156N, pET28a(+)-N247H, pET28a(+)-Q25V / H156N, pET28a(+)-Q25V / N247H, pET28a(+)-H156N / N247H, pET28a(+)-Q25V / H156N / N247H, pET28a(+)-ScCPR were constructed. The construction of recombinant strains and protein expression were carried out according to Example 1, and protein purification was carried out according to the following method.

[0151] (1) Preparation of protein purification reagents

[0152] Binging Buffer: Tris-HCl buffer (100 mM, pH 7.5). Washing Buffer: Take 1 L of Tris-HCl buffer (100 mM, pH 7.5), add 3.4 g of imidazole (final concentration 50 mM), and adjust the buffer pH to 7.5 with hydrochloric acid or sodium hydroxide. Elution Buffer: Take 1 L of Tris-HCl buffer (100 mM, pH 7.5), add 34 g of imidazole (final concentration 500 mM), and adjust the buffer pH to 7.5 with hydrochloric acid or sodium hydroxide.

[0153] (2) Protein purification process

[0154] 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 are as follows: (Ⅰ) Rinse the Ni column with Binging Buffer at a flow rate of 0.4 mL / min until the UV baseline is balanced, with an approximate usage of 15 times the volume of the nickel column. (Ⅱ) After flushing the baseline flat with Binging Buffer, load the sample at a flow rate of 0.2 mL / min to allow the target protein to fully bind to the Ni column. (Ⅲ) Rinse the impurity proteins with WashingBuffer at a flow rate of 0.3 mL / min until the impurity proteins are rinsed clean and the UV baseline is balanced, with an approximate usage of 10 times the volume of the nickel column. (Ⅳ) Rinse and 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. Continue to rinse with Elution Buffer and adjust the flow rate to 0.3 mL / min until the UV baseline is balanced. (Ⅴ) Replace Elution Buffer with Binging Buffer and continue rinsing. Balance the Ni column with Binging Buffer at a flow rate of 0.35 mL / min until the UV baseline is balanced, with an approximate usage of 10 times the volume of the nickel column. (Ⅵ) Store the Ni column with 8 times the column volume of 20% ethanol and keep the Ni column in a 4°C refrigerator. (Ⅶ) Remove salt ions: Place the collected target protein eluate in Tris-HCl buffer (100 mM, pH 7.5) and dialyze overnight to obtain the purified enzyme solution. Analyze the purification result by SDS-PAGE, and the result is as Figure 7 shown.

[0155] (3) After obtaining the purified wild-type ScHO enzyme, Q25V, H156N, N247H, Q25V / H156N, Q25V / N247H, H156N / N247H, Q25V / H156N / N247H, and wild-type ScCPR enzyme, measure the protein concentration using an ultra-micro spectrophotometer and prepare according to the following reaction system:

[0156] 20 μg of wild-type or mutant ScHO enzyme, 20 μg of ScCPR, the final concentration of NADPH is 15 mM, and the final concentration of hemin in the reaction system is (0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0) mM respectively. Use Tris-HCl buffer (100 mM, pH 7.5) to control the total volume to 200 μL and place it in a 96-well plate to measure the reaction process.

[0157] Reaction process determination procedure: Place the 96-well plate in a microplate reader, set the temperature to 35 °C, shake for 2 s every 5 s, scan the absorbance value of each well at 690 nm every 30 s, and convert it to biliverdin concentration according to the standard curve shown in Figure 4 . Take the first 10% of each reaction process for the calculation of the reaction rate, and fit the reaction rates at different substrate concentrations according to the Michaelis-Menten equation. The results are shown in Figure 6 . The kinetic parameters of wild-type and mutant ScHO calculated according to the fitting results are shown in Table 6, and the enzyme activity data are also detected. The results are shown in Table 7.

[0158] Table 7: Kinetic parameters of wild-type and mutant ScHO

[0159]

[0160]

[0161] The kinetic parameter determination experiment shows that the combined mutant H156N / N247H of ScHO has the highest catalytic efficiency and specific activity.

[0162] Example 6: Construction of multi-enzyme co-expression engineering strain

[0163] The preferred ScHO mutant ScHO H156N / N247H and the remaining proteins are integrated into the same Escherichia coli strain. The specific operation steps are as follows:

[0164] (1) Preparation of plasmids

[0165] For the strain containing the preferred ScHO mutant ScHO H156N / N247H obtained in Example 4, extract the plasmid using Vazyme FastPure Plasmid Mini Kit-BOX 2. The extraction steps refer to the instructions provided in the kit. Obtain the recombinant plasmid pETDuet-1-ScHO H156N / N247H -ScCPR-ApFDH;

[0166] According to the method of Example 1, construct the recombinant plasmid pCDFDuet-1-MmBVR-AcCODH;

[0167] (2) Construction of recombinant strains

[0168] According to the recombinant strain construction method described in Example 1, transform the above two plasmids into the same E. coli BL21(DE3) competent cells at the same time to obtain the five-enzyme co-expression strain containing ScHO H156N / N247H : E. coli BL21(DE3) /

[0169] pETDuet-1-ScHO H156N / N247H -ScCPR-ApFDH / pCDFDuet-1-MmBVR-AcCODH, named as E. coli BL21(DE3) Mut .

[0170] Example 7: Establishing an efficient reaction system

[0171] Using the recombinant E. coli E. coli BL21(DE3) obtained in Example 6 Mut as the preferred catalyst, the reaction system was established. According to the method of Step 3 in Example 1, the fermentation broth was prepared. The obtained fermentation broth was centrifuged at low temperature, and the centrifugation conditions were 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; 20 g / L of the dry weight of whole cells was weighed, and ultrasonic disruption was carried out under ultrasonic conditions of 400 W, 0°C, working for 2 s, intermittent for 4 s, and a total time of 10 min to obtain cell lysate. The cell lysate was used as the catalyst to optimize the reaction conditions. The specific operation steps are as follows:

[0172] 1. Optimization of reaction conditions

[0173] (1) Optimization of the optimal reaction temperature

[0174] The reaction conditions were as follows: a 100 mL reaction system, 20 g / L of recombinant E. coli cell lysate (the addition amount of the lysate was: first weigh 20 g / L of the dry weight of whole cells, and after disruption, it was the corresponding lysate to be added), the solvent was Tris-HCl solution (pH 7.5, 100 mM), 5 mM of EDTA-Na2, 1 mM of hemin, and the reaction rotation speed was 200 rpm.

[0175] Under the above reaction conditions, they were respectively placed in a constant temperature water bath stirrer at 25°C, 30°C, 35°C, 40°C, and 45°C. After reacting for 1 hour, samples were taken. For a 50 μL reaction solution sample, 950 μL of a DMSO and dichloromethane mixed solution (volume ratio 5:1) was added, centrifuged at 12,000×g for 1 min, and the supernatant was taken. The bilirubin concentration was detected by HPLC.

[0176] The reaction results at different temperatures were as Figure 8 shown. The detection results showed that the preferred reaction temperature was 35°C. At this time, 1 mM of hemin was converted into 541 mg / L of bilirubin within 1 hour, and the yield reached 92.6%.

[0177] (2) Optimization of the optimal reaction pH

[0178] The reaction conditions are as follows: a 100 mL reaction system, 20 g / L of recombinant Escherichia coli cell lysate (the addition amount of the lysate is: first weigh 20 g / L of the dry weight of whole cells, and after disruption, it is the corresponding lysate to be added), the solvent is Tris-HCl solution (pH 7.5 - 8.0, 100 mM) or citrate-sodium citrate buffer (pH 6.0 - 6.5, 100 mM) or disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 6.5 - 7.5, 100 mM), 5 mM of EDTA-Na2, 2 mM of hemin, 35 °C, 200 rpm.

[0179] Under the above reaction conditions, reaction systems with pH values of 6.0, 6.5, 7.0, 7.5, and 8.0 were respectively prepared using three buffers. After reacting for 1 hour, samples were taken. For a 50 μL reaction solution sample, 950 μL of a DMSO and dichloromethane mixture (volume ratio 5:1) was added, centrifuged at 12,000×g for 1 min, the supernatant was taken, and the bilirubin concentration was detected by HPLC.

[0180] The reaction results at different pH values are as Figure 9 shown. The detection results show that the preferred reaction pH is 6.5. At this time, 2 mM of hemin is converted into 812 mg / L of bilirubin within 1 hour, and the yield reaches 69.5%.

[0181] (3) Catalyzing different concentrations of hemin under the optimal reaction conditions

[0182] The reaction conditions are as follows: a 100 mL reaction system, 20 g / L of recombinant Escherichia coli cell lysate (the addition amount of the lysate is: first weigh 20 g / L of the dry weight of whole cells, and after disruption, it is the corresponding lysate to be added), the solvent is citrate-sodium citrate buffer (pH 6.5, 100 mM), 5 mM of EDTA-Na2, 35 °C, 200 rpm. Under the above reaction conditions, 1 mM, 2 mM, 3 mM, and 4 mM of hemin were respectively added. Samples were continuously taken during the reaction process. For each sample, a 50 μL reaction solution sample was taken, 950 μL of a DMSO and dichloromethane mixture (volume ratio 5:1) was added, centrifuged at 12,000×g for 1 min, the supernatant was taken, and the bilirubin concentration was detected by HPLC. The reaction progress curves at different substrate concentrations are as Figure 10 shown.

[0183] 2. The optimal reaction conditions are:

[0184] 100 mL reaction system, recombinant Escherichia coli cell lysate at 20 g / L (the addition amount of the lysate is: first weigh the dry weight of whole cells at 20 g / L, and after disruption, it is the corresponding lysate to be added), the solvent is citric acid-sodium citrate buffer (pH 6.5, 100 mM), EDTA-Na2 at 5 mM, the concentration of hemin is 3 mM, at 35 °C, 200 rpm.

[0185] The results show that: when the substrate concentration is 3 mM, the product bilirubin reaches 1611 mg / L at 4 h, and the yield is as high as 91.9% at this time. Further increasing the substrate concentration cannot further accumulate the concentration of the product bilirubin.

[0186] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A heme oxygenase mutant, characterized in that, The heme oxygenase mutant is obtained by mutating the glutamine at position 25 of the heme oxygenase having the amino acid sequence shown in SEQ ID NO.1 to valine; or by mutating the histidine at position 156 of the heme oxygenase having the amino acid sequence shown in SEQ ID NO.1 to asparagine; or by mutating the asparagine at position 247 of the heme oxygenase having the amino acid sequence shown in SEQ ID NO.1 to histidine; or by mutating the histidine at position 156 of the heme oxygenase having the amino acid sequence shown in SEQ ID NO.1 to asparagine and simultaneously mutating the asparagine at position 247 to histidine; or by mutating the glutamine at position 25 of the heme oxygenase having the amino acid sequence shown in SEQ ID NO.1 to valine and simultaneously mutating the histidine at position 156 to asparagine; or by mutating the glutamine at position 25 of the heme oxygenase having the amino acid sequence shown in SEQ ID NO.1 to valine and simultaneously mutating the asparagine at position 247 to histidine.

2. A gene encoding the heme oxygenase mutant according to claim 1 or a recombinant vector carrying the gene.

3. A recombinant cell expressing the mutant according to claim 1 or carrying the gene or the recombinant vector according to claim 2.

4. The recombinant cell according to claim 3, wherein The recombinant cell uses bacteria or fungi as host cells.

5. A recombinant enzyme catalyst containing the heme oxygenase mutant described in claim 1, characterized in that, It is any one of the following forms: (1) Culturing a recombinant expression transformant containing the heme oxygenase mutant and isolating the transformed cells containing the recombinant heme oxygenase mutant enzyme; (2) Culturing a recombinant expression transformant containing the heme oxygenase mutant, isolating the transformed cells containing the recombinant heme oxygenase mutant enzyme, lysing the transformed cells containing the recombinant heme oxygenase mutant enzyme to obtain a cell lysate; (3) Culturing a recombinant expression transformant containing the heme oxygenase mutant, isolating the transformed cells containing the recombinant heme oxygenase mutant enzyme, lysing the transformed cells containing the recombinant heme oxygenase mutant enzyme to obtain a cell lysate, and freeze-drying the cell lysate of the recombinant heme oxygenase mutant enzyme to obtain a lyophilized enzyme powder.

6. A method for increasing the conversion rate of heme oxygenase to a substrate and / or increasing its enzyme activity, characterized in that, The method is to mutate the glutamine at position 25 of the heme oxygenase having the amino acid sequence shown in SEQ ID NO.1 to valine; or to mutate the histidine at position 156 of the heme oxygenase having the amino acid sequence shown in SEQ ID NO.1 to asparagine; or to mutate the asparagine at position 247 of the heme oxygenase having the amino acid sequence shown in SEQ ID NO.1 to histidine; or to mutate the histidine at position 156 of the heme oxygenase having the amino acid sequence shown in SEQ ID NO.1 to asparagine and simultaneously mutate the asparagine at position 247 to histidine; Alternatively, glutamine at position 25 of heme oxygenase with the amino acid sequence shown in SEQ ID NO.1 is mutated to valine, and histidine at position 156 is mutated to asparagine at the same time; Alternatively, glutamine at position 25 of heme oxygenase with the amino acid sequence shown in SEQ ID NO.1 is mutated to valine, and asparagine at position 247 is mutated to histidine at the same time.

7. Use of the mutant according to claim 1, or the recombinant cell according to claim 3 or 4, or the recombinant enzyme catalyst according to claim 5, for the preparation of biliverdin or bilirubin, characterized in that, The application in the preparation of biliverdin, wherein the reaction system includes whole-cell reaction and enzyme reaction system, and: The whole-cell reaction is as follows: Using hemin as a substrate, biliverdin is prepared by whole-cell conversion. The recombinant cell also expresses NADP + -dependent formate dehydrogenase derived from Azospirillum palustre and cytochrome P450 reductase derived from Saccharomyces cerevisiae; The enzyme reaction system is as follows: using hemin as a substrate, biliverdin is catalytically prepared, and NADP derived from Azospirillum palustre is added at the same time + -dependent formate dehydrogenase and cytochrome P450 reductase derived from Saccharomyces cerevisiae; The application in the preparation of bilirubin, wherein the reaction system includes whole-cell reaction and enzyme reaction system, and: The whole-cell reaction is as follows: Using hemin as a substrate, bilirubin is prepared by whole-cell conversion. The recombinant cell also expresses biliverdin reductase derived from Mus musculus, NADP + -dependent formate dehydrogenase derived from Azospirillum palustre, cytochrome P450 reductase derived from Saccharomyces cerevisiae, and also expresses carbon monoxide dehydrogenase derived from Afipia carboxidovorans; The enzyme reaction system is as follows: using hemin as a substrate, catalyzing the preparation of bilirubin, and simultaneously adding biliverdin reductase from Mus musculus, NADP + -dependent formate dehydrogenase from Azospirillum palustre, cytochrome P450 reductase from Saccharomyces cerevisiae, and simultaneously expressing carbon monoxide dehydrogenase from Afipia carboxidovorans.

8. A genetically engineered bacterium, characterized in that, The genetically engineered bacterium expresses the heme oxygenase mutant described in claim 1, biliverdin reductase derived from Mus musculus, NADP + -dependent formate dehydrogenase derived from Azospirillum palustre, and cytochrome P450 reductase derived from Saccharomyces cerevisiae, and also expresses carbon monoxide dehydrogenase derived from Afipia carboxidovorans.

9. The genetically engineered bacterium according to claim 8, characterized in that, The nucleotide sequence of the cytochrome P450 reductase is as shown in SEQ ID NO.3; the nucleotide sequence of the biliverdin reductase is as shown in SEQ ID NO.4; the + nucleotide sequence of the NADP -dependent formate dehydrogenase is as shown in SEQ ID NO.5; the nucleotide sequence of the carbon monoxide dehydrogenase is as shown in SEQ ID NO.

6.

10. The genetically engineered bacterium according to claim 8, characterized in that, The genetically engineered bacterium is an Escherichia coli genetically engineered bacterium, a Bacillus subtilis genetically engineered bacterium, a Corynebacterium glutamicum genetically engineered bacterium, or a yeast genetically engineered bacterium.

11. A method for synthesizing bilirubin, characterized in that, The method is to use heme as a substrate and use the genetically engineered bacterium or its lysate or its fermentation broth described in any one of claims 8 to 10 as a catalyst to catalyze the conversion and synthesis of bilirubin.

12. The method according to claim 11, wherein The addition amount of the heme is: 1-4 mM, the stirring speed is 100-500 r / min, the temperature is 25-40 °C, the pH is 6.0-8.0, and the reaction time is 1-4 h.

13. The application of the genetically engineered bacterium described in any one of claims 8 to 10 or the method described in claim 11 or 12 in the preparation of bilirubin or a product containing bilirubin.

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