Heme oxygenase mutant and bilirubin efficiently synthesized by multienzyme coupling system of heme oxygenase mutant

Abstract

The invention discloses a heme oxygenase mutant and bilirubin efficiently synthesized by a multienzyme coupling system of the heme oxygenase mutant, and belongs to the field of biological catalysis engineering. The invention provides a heme oxygenase mutant which is used for catalyzing heme to synthesize biliverdin. Coupling biliverdin reductase to catalyze biliverdin to synthesize bilirubin; cytochrome P450 reductase is introduced to provide electrons; removing competitive binding of the by-product to the substrate heme by carbon monoxide dehydrogenase; formate dehydrogenase is used for in-situ regeneration of coenzyme, and bilirubin is efficiently synthesized through a one-pot method. According to the heme oxygenase mutant, the two core enzymes are coupled with three external assistant enzymes, recombinant escherichia coli is constructed, heme is catalyzed, exogenous NADPH does not need to be added, and the yield of bilirubin within 4 h reaches 1611 mg / L and reaches the bilirubin synthesis level which is reported at present and has the highest yield and the shortest time. According to the technology, resource limitation is relieved, the synthesis method is easy to operate and high in yield, and efficient and green synthesis of bilirubin is achieved.

Description

Technical Field

[0001] The invention relates to a heme oxygenase mutant and a multi-enzyme coupling system thereof for efficiently synthesizing bilirubin, and belongs to the field of biocatalytic engineering. Background Art

[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 a variety of diseases. For example, the deposition of bilirubin in the central nervous system of the brain causes bilirubin encephalopathy in newborns, which damages hearing, movement and mental functions; and excessive free bilirubin concentration in plasma can easily cause jaundice. Therefore, bilirubin was considered a metabolic waste in the early days.

[0003] However, in recent years, with the in-depth study of bile pigments, its beneficial physiological effects have gradually been discovered and reported. Bilirubin has an antioxidant effect, which can physiologically prevent the oxidation of biological macromolecules such as fats and proteins and scavenge oxygen free radicals; appropriate concentrations of bilirubin in serum can prevent cardiovascular 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 used in the treatment of systemic complications of new coronavirus infection.

[0004] Based on the important role and potential market value of bilirubin, its production methods have been developed and reported accordingly. The traditional preparation method is to extract it from the bile of livestock such as pigs and cattle. Research progress in recent years has mainly focused on the development of extraction and purification processes. This method is limited by the resources of pig bile, the extraction rate is low, and the output is difficult to 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 bioorganic 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 oxidized by heme oxygenase (HO, EC 1.14.14.18) to form the intermediate product biliverdin, which is then reduced by biliverdin reductase (BVR, EC1.3.1.24) to form bilirubin. The reaction formula is as follows: Fig.12 shown.

[0006] So far, there are a few reports on the research of biological synthesis of bilirubin and its key intermediates. For example, Yan Sihan et al. constructed a heme oxygenase and glutamate dehydrogenase coenzyme regeneration system from Clostridium tetani and displayed it on the membrane surface. With E. coli whole cells as catalyst, the biliverdin production reached 76.3 mg / L; Jianfeng Mei et al. used biliverdin as substrate and E. coli as host to express cyanobacterial biliverdin reductase as whole cell catalyst, and the bilirubin production reached 325 mg / L. In the previous work, this technical team constructed a recombinant Escherichia coli containing heme oxygenase, biliverdin reductase, formate dehydrogenase and cytochrome P450 reductase, and the bilirubin production reached 415.5 mg / L (patent publication number CN117487733A).

[0007] Research reports show that the process of catalyzing the synthesis of bilirubin with heme as substrate is divided into two steps: the first step is the synthesis of biliverdin from heme catalyzed by heme oxygenase, and the second step is the synthesis of bilirubin from biliverdin catalyzed by biliverdin reductase. The reaction formula is as follows: Fig.12 As shown. The heme conversion rate catalyzed by heme oxygenase in the first step of the reaction is much lower than the biliverdin conversion rate catalyzed by biliverdin reductase in the second step. The catalytic functions of the two core enzymes are seriously unbalanced, resulting in the multi-enzyme coupling system constructed by the two to catalyze the substrate heme and synthesize bilirubin at a low efficiency. Therefore, it is urgent 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 carbon monoxide is produced as a byproduct, it is of great significance to couple the coenzyme regeneration system and the carbon monoxide byproduct elimination system in the reaction system of the two core enzymes to achieve a one-pot method for efficient catalysis of heme to synthesize bilirubin. Summary of the invention

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

[0009] Since the heme conversion rate catalyzed by heme oxygenase is much lower than the biliverdin conversion rate catalyzed by biliverdin reductase, ScHO was modified. Specifically, the substrate in the active center was obtained through crystal structure analysis. Alanine scanning and site-saturation mutagenesis were performed on amino acid residues in the range to obtain three single-point mutants with improved activity, and combined mutations of the single-point mutations were performed to obtain combined mutants with further improved activity.

[0010] The present invention provides a heme oxygenase mutant. The heme oxygenase mutant is obtained by mutating the glutamine at position 25 of the heme oxygenase as shown in the amino acid sequence of SEQ ID NO.1 to valine, and is named Q25V; or by mutating the histidine at position 156 of the heme oxygenase as shown in the amino acid sequence of SEQ ID NO.1 to asparagine, and is named H156N; or by mutating the asparagine at position 247 of the heme oxygenase as shown in the amino acid sequence of SEQ ID NO.1 to histidine, and is named N247H; or by mutating the histidine at position 156 of the heme oxygenase as shown in the amino acid sequence of SEQ ID NO.1 to asparagine, and simultaneously mutating the asparagine at position 247 to histidine, and is named H156N / N247H; or by mutating the histidine at position 156 of the heme oxygenase as shown in the amino acid sequence of SEQ ID NO.1 to asparagine, and simultaneously mutating the asparagine at position 247 to histidine, and is named H156N / N247H. The glutamine at position 25 of the heme oxygenase shown in NO.1 is mutated to valine, and the histidine at position 156 is mutated to asparagine, which is named Q25V / H156N; or the glutamine at position 25 of the heme oxygenase with an amino acid sequence as shown in SEQ ID NO.1 is mutated to valine, and the asparagine at position 247 is mutated to histidine, which is named Q25V / N247H.

[0011] The amino acid sequence of heme oxygenase is shown in SEQ ID NO.1:

[0012] MEDSSNTIIPSPTDVGALANRINFQTRDAHNKINTFMGIKMAIAMRHGFIYRQGILAYYYVFDAIEQEIDRLLNDPVTEEELQTSTILKQFWLEDFRRSTQIYKDLKLLYSNTFKSTESLNEFLATFQKPPLLQQFINNIHENI HKEPCTILSYCHVLYLALFAGGKLIRSNLYRRLGLFPNFEKLSQKELVKKGTNFFTFSDLGPTEETRLKWEYKKNYELATRTELTEAQKLQIISVAEGIFDWNFNIVAEIGELNRRELMGKFSFKCITYLYEEWMFNKDSATRR

[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] atggaagatagcagcaacaccatcatcccgtccccgaccgatgttggcgccctggcgaaccgtatcaacttccagacccgcgatgcgcacaacaaaatcaacaccttc atgggtatcaaaatggcgatcgcaatgcgtcacggcttcatctaccgccagggcatcctggcgtactactacgttttcgacgcgatcgaacaggaaatcgatcgtctg ctgaacgatccggtgaccgaagaagaattgcagacctccaccatcctgaaacagttctggctggaggacttccgccgtagcacccagatctacaaagatctgaaactg ctgtacagcaacaccttcaaatccaccgaaagcctgaacgagttcctggcgaccttccagaaaccgccgctgctgcaacagttcatcaacaacatccatgaaaacatcc acaaagaaccgtgcaccatcctgtcctactgcaatgttctgtacctggccctgttcgcgggcggcaaactgatccgttccaacctgtaccgtcgcctgggtctgttcc cgaacttcgaaaaactgtctcagaaagaactggttaaaaaaggcaccaacttcttcaccttctccgacctgggcccgaccgaagaaacccgcctgaaatgggaatataa aaagaactacgaactggcaacccgtaccgaactgaccgaagcgcagaaactgcaaatcatctccgttgcggaaggtatcttcgactggcattttaacatcgtggcgga aatcggcgaactgaaccgtcgtgaactgatgggcaaattctctttcaaatgcatcacctacctgtacgaagaatggatgttcaacaaagatagcgcgacccgccgttaa

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

[0024] The invention also provides a recombinant vector carrying the gene.

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

[0026] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as host cells.

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

[0028] (2) culturing a recombinant expression transformant containing the heme oxygenase mutant, isolating transformant cells containing the recombinant heme oxygenase mutant enzyme, and disrupting the transformant cells containing the recombinant heme oxygenase mutant enzyme to obtain a cell disrupted liquid;

[0029] (3) culturing a recombinant expression transformant containing the heme oxygenase mutant, isolating transformant cells containing the recombinant heme oxygenase mutant enzyme, disrupting the transformant cells containing the recombinant heme oxygenase mutant enzyme, obtaining a cell disrupted liquid, and freeze-drying the cell disrupted liquid of the recombinant heme oxygenase mutant enzyme to obtain a lyophilized enzyme powder.

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

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

[0032] In one embodiment of the present invention, the use 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 derived from Azospirillum palustre + Dependent formate dehydrogenase, cytochrome P450 reductase from Saccharomyces cerevisiae; the enzyme reaction system is: hemin is used as a substrate to prepare biliverdin, and NADP from Azospirillum palustre is added at the same time + dependent formate dehydrogenase, cytochrome P450 reductase from Saccharomyces cerevisiae;

[0033] In one embodiment of the present invention, the use 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 from Mus musculus, NADP from Azospirillumpalustre + The enzyme reaction system was as follows: bilirubin was prepared by using hemin as substrate, and biliverdin reductase from Mus musculus and NADP-450 reductase from Azospirillum palustre were added. + A formate dehydrogenase-dependent enzyme, a cytochrome P450 reductase from Saccharomyces cerevisiae, and a carbon monoxide dehydrogenase from Afipia carboxidovorans were also expressed.

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

[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 is a recombinant Escherichia coli. Preferably, the recombinant Escherichia coli is an E. coli BL21 (DE3) expression host. Preferably, the recombinant Escherichia coli is an expression vector of pET28a (+), pET21a (+), pET32a (+), pGEX, pETDuet-1, pRSFDuet-1 or pCDFDuet-1.

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

[0046] In one embodiment of the present invention, the enzyme activity of the heme oxygenase is measured by the peak area of ​​the product biliverdin detected by high pressure liquid chromatography using heme as substrate. The screening of the heme oxygenase mutants is carried out by comparing the enzyme activity.

[0047] The present invention also provides 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 gene; preferably, the recombinant vector uses pET28a(+), pET21a(+), pET32a(+), pGEX, pETDuet-1, pRSFDuet-1 or pCDFDuet-1 as an 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 an expression host.

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

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

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

[0054] The invention provides a method for improving the bilirubin synthesis ability of Escherichia coli. The method comprises the following steps: using Escherichia coli BL21 (DE3) as a host cell, expressing heme oxygenase and cytochrome P450 reductase derived from Saccharomyces cerevisiae, biliverdin reductase derived from Mus musculus, NADP-61 derived from Azospirillumpalustre, and the like. + A formate dehydrogenase-dependent enzyme was expressed, and a carbon monoxide dehydrogenase from Afipia carboxidovorans was also expressed.

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

[0056] The present invention also provides a method for whole-cell catalytic synthesis of bilirubin, which comprises taking heme as a substrate and, under ventilation conditions, using the above-mentioned recombinant cell lysate or the above-mentioned recombinant Escherichia coli whole cell to catalytically transform and synthesize bilirubin.

[0057] In one embodiment of the present invention, the whole-cell catalytic system includes but is not limited to 3 mM hemoglobin (equivalent to 1.953 g / L), aeration volume of 8 vvm, hemoglobin is pre-dissolved in a NaOH solution (100 mM) accounting for 50% of the total reaction volume, and 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 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 one embodiment of the present invention, the whole-cell catalyst is obtained by culturing the recombinant bacteria and inducing them to express heme oxygenase, biliverdin reductase, cytochrome P450 reductase, formate dehydrogenase and carbon monoxide dehydrogenase, and further collecting the recombinant bacterial cells.

[0059] In one embodiment of the present invention, the whole cell catalyst is obtained by inoculating 1% of the recombinant Escherichia coli co-expressing heme oxygenase, biliverdin reductase, cytochrome P450 reductase, formate dehydrogenase and carbon monoxide dehydrogenase into 100 mL LB medium and culturing at 37°C. When the OD 600 When the p-value reached 0.4-0.6, 0.5 mM IPTG was added to induce protein expression. After induction at 20°C for 16 h, the cells were collected by low-temperature centrifugation at 8,000 × g for 10 min and washed twice with 20 mM Tris-HCl (pH 7.5) buffer.

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

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

[0062] The present invention provides the use of the above recombinant Escherichia coli or the above method in preparing bilirubin or a product containing bilirubin.

[0063] Beneficial Effects

[0064] (1) The present invention successfully obtains a high-activity mutant of heme oxygenase and improves the catalytic efficiency of heme oxygenase. Heme oxygenase mutants, cytochrome P450 reductase, and biliverdin reductase are co-expressed in Escherichia coli, and high-activity formate dehydrogenase and carbon monoxide dehydrogenase are coupled at the same time, successfully realizing the reconstruction of a complete heme degradation cascade reaction in a single cell. At the same time, no exogenous addition of NADPH is required, and high catalytic activity can be achieved by using endogenous coenzymes in the cell, and there is no accumulation of intermediate product biliverdin during the catalytic process; in addition, the byproduct carbon monoxide produced during the catalytic process is eliminated in time, avoiding the accumulation of carbon monoxide-heme complexes that cannot be used by the enzyme. With heme as the substrate, after multi-enzyme one-pot cascade catalysis, the final bilirubin production can reach 1611 mg / L, and the conversion rate is greater than 91%. The multi-enzyme coupling system constructed by the present invention solves the problem of low conversion rate in the catalytic conversion of heme to synthesize biliverdin and bilirubin research.

[0065] (2) The establishment of this whole-cell conversion system eliminates the resource limitations of the pig bile extraction method and solves the problems of complicated 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 a substrate. According to all current data, the bilirubin production in the present invention is the highest level in the biological synthesis starting from the heme substrate. BRIEF 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 strains containing recombinant plasmids pETDuet-1-ScHO-ScCPR-ApFDH and pCDFDuet-1-MmBVR-AcCODH, lane 1 is the supernatant of cell disruption.

[0068] Figure 3 :The active center of heme oxygenase ScHO and its distance to substrate Amino acid residues within the range.

[0069] Figure 4 : Standard curve of biliverdin determined by ELISA.

[0070] Figure 5 :The distance between the active center of heme oxygenase ScHO and the substrate Results of high-throughput screening of saturation mutagenesis of amino acid residues within a range.

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

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

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

[0074] Fig. 9 :pH optimization results of bilirubin synthesis catalyzed by recombinant Escherichia coli.

[0075] Fig.10 : Reaction process diagram of bilirubin synthesis catalyzed by recombinant Escherichia coli at different substrate concentrations.

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

[0077] Fig.12 : Bilirubin reaction formula. DETAILED DESCRIPTION

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

[0079] 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 fully dissolve and shake well, then add 57.1mL of glacial acetic acid, shake well, and add deionized water to 1L. Prepare 50×TAE buffer, then dilute 50 times to make 1×TAE buffer, use 1×TAE buffer as solvent, prepare agarose solution with a mass concentration of 0.9% (heat to dissolve), add nucleic acid dye (1.5μL / 20mL) after agarose is completely dissolved, shake gently to mix well, avoid bubbles, pour into nucleic acid gel mold to cool, and obtain agarose gel.

[0080] The 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 160V, 15min, and place the agarose gel under UV light to observe the bands after the electrophoresis. Digestion template: Take 50μL of PCR product that is positive by electrophoresis, add 1μL of endonuclease DpnⅠ and 5μL of the corresponding buffer to remove the methylated template DNA. The reaction conditions are 37℃, 3h. After the reaction is completed, keep it at 65℃ for 10min to inactivate DpnⅠ.

[0082] The product purification involved in the following examples: The digestion product was purified according to the product instructions of FastPure Gel DNA ExtractionMini Kit.

[0083] Homologous recombination involved in the following embodiments: The product instructions of ClonExpress IIOne Step Cloning Kit were used for homologous recombination, and the truncated and mutated DNA fragments were connected to the linearized vector to obtain the recombinant plasmid.

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

[0085] Add all the homologous recombination reaction liquid to E.coli BL21 (DE3) competent cells, mix well, and place in an ice bath for 30 minutes; place in a 42°C water bath for heat shock for 90 seconds, quickly transfer to an ice bath for cooling for 5 minutes, add 600 μL LB medium, and culture at 37°C, 200 rpm for 1 hour; aspirate 50 μL and spread on an LB plate containing the corresponding antibiotics, and culture inverted at 37°C for 12 hours; pick a single colony for sequencing verification, and preserve the strain with the correct recombinant plasmid introduced.

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

[0087] SDS-PAGE gel electrophoresis:

[0088] Prepare BBI non-blocking SDS-PAGE denaturing acrylamide color gel rapid preparation kit and prepare protein gel according to the instructions. Sample preparation: Take wet bacteria containing the target protein, prepare 20g / L cell suspension with Tris-HCl buffer (pH 7.5, 100mM), and place it in an ice bath for ultrasonic disruption (450W, work 1s, pause 4s, total 10min). Centrifuge at 12,000×g, 4℃ for 1min to separate the supernatant and precipitate. Take 20μL supernatant, mix with 8μL Loading buffer, heat in a boiling water bath for 10min, centrifuge at 12,000×g for 5min, and set aside. Electrophoresis: Protein gel loading volume, sample 10μL, Marker 3μL; electrophoresis conditions: 160V, 60min; electrophoresis buffer (g / L): glycine 14.4, SDS1, Tris 3. Staining: Soak the protein gel in staining solution (Coomassie Brilliant Blue R250 1g, methanol 450mL, acetic acid 100mL, water 450mL) for 15min, and wash twice with water. Decolorization: Soak in decolorizing solution (ethanol 10%, acetic acid 10%, water 80%) and shake slightly, replace with fresh decolorizing solution until the bands are clearly visible.

[0089] Bilirubin test

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

[0091] Detection of heme oxygenase activity

[0092] In a 1mL reaction system, add 100mg of purified heme oxygenase or its mutant, 100mg of purified cytochrome P450 reductase, 30mM NADPH, 1mM hemin chloride, use Tris-HCl (100mM, pH 7.5) as solvent, place in a 35℃ constant temperature oscillator, react at 500rpm for 20min. Then take a sample of 50μL, mix it evenly with 950μL methanol, centrifuge it at 12,000×g for 1min, take 200μL of supernatant, place it in a 96-well plate to measure the absorbance at 690nm, and calculate the product biliverdin concentration according to the standard curve, and further calculate the activity unit U and specific activity U / mg.

[0093] The activity unit U is defined as: the amount of enzyme required to catalyze the synthesis of 1 μmol of biliverdin per minute at 35°C and pH 7.5 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 present invention uses the heme oxygenase obtained by deleting the C-terminal 289-318 amino acids of the heme oxygenase with NCBI accession number GHM92019.1 and the ScCPR obtained by deleting the N-terminal 1-24 amino acid sequence of the ScCPR with 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 vector

[0101] (1) Gene acquisition

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

[0103] (2) Construction of recombinant vector

[0104] The synthesized ScHO gene, ScCPR gene, and ApFDH gene were sequentially inserted into the pETDuet-1 vector, wherein the ScHO nucleotide sequence was inserted between the restriction enzyme sites BamH I and Sac I of pETDuet-1 in the order of 5'→3', the ScCPR nucleotide sequence was added with a ribosome binding sequence (5'-tttgtttaactttaagaaggagatatacc-3') at the 5' end, and then inserted between the restriction enzyme sites Sal I and Not I of pETDuet-1 in the order of 5'→3', and the ApFDH nucleotide sequence was inserted between the restriction enzyme sites Nde I and Xho I of pETDuet-1 in the order of 5'→3', 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, wherein the nucleotide sequence of MmBVR was inserted between the restriction enzyme cutting sites BamH I and Sac I of pCDFDuet-1 in the order of 5'→3', and the nucleotide sequence of AcCODH was inserted between the restriction enzyme cutting sites Nde I and Xho I of pCDFDuet-1 in the order of 5'→3', 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, and the recombinant plasmids obtained in the above step (1) were respectively introduced into the competent cells of E. coli BL21 (DE3) and spread on LB solid culture medium plates containing ampicillin or streptomycin, or containing the above two antibiotics, and the positive transformants were selected 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 inoculated into LB liquid culture medium test tubes containing (both at a concentration of 50 mg / L) ampicillin or streptomycin, or both antibiotics, and cultured at 37°C and 200 rpm for 8 to 10 hours to obtain seed solution. The seed solution was inoculated into a 1% (v / v) inoculum containing (both at a concentration of 50 mg / L) ampicillin or streptomycin, or LB liquid culture medium shake flask (100 mL of liquid / 500 mL of volume) containing (both at a concentration of 50 mg / L) ampicillin or streptomycin, or both antibiotics, and cultured at 37°C and 200 rpm until OD 600 When the pH value reaches 0.4-0.6, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.5 mM, adjust the temperature to 20°C, and continue culturing for 16 hours to induce protein expression; and obtain fermentation broth respectively.

[0110] The obtained fermentation broth was centrifuged at low temperature, the centrifugation conditions were 8,000×g, 10 min, 4°C, the supernatant was discarded after centrifugation, and the precipitate was washed twice with physiological saline to obtain wet bacteria containing the target protein; the obtained wet bacteria were ultrasonically disrupted under ultrasonic conditions of 400W, 0°C, working for 2s, resting for 4s, and a total time of 10min to obtain a cell lysate, which is the catalyst to be added in the embodiment of the present invention.

[0111] The obtained cell lysate was subjected to SDS-PAGE gel electrophoresis to verify the soluble expression of the protein. Figure 1 and Figure 2 As 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, the amino acid residues around the ScHO active center (SEQ ID NO.1) were analyzed. Figure 3 ), get the distance to the substrate The sites within the range were mutated using degenerate PCR primers, and the mutation sites and corresponding primer sequences are shown in Table 2. N represents a random one of the four nucleotides of ATCG, and K represents a random one of the two nucleotides of GT. The primer sequences were submitted to Qingke Biotechnology (Suzhou) for synthesis.

[0115] Table 2: Mutation sites and primers

[0116]

[0117] Using pETDuet-1-ScHO-ScCPR-ApFDH constructed in Example 1 as a template and the nucleotide sequences in Table 2 as primers, PCR amplification was performed to construct a mutation 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; and finally, the reaction was extended at 72°C for 5 min and then cooled to 16°C to terminate the reaction.

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

[0120] The PCR product was treated with Dpn I to eliminate the template plasmid. The system and reaction conditions were: 50 μL of PCR product, 1 μL of Takaraquickcut Dpn I, 5 μL of 10×quickcut buffer, mixed and placed in a 37°C constant temperature water bath for 2 hours.

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

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

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

[0124] Establishment of high-throughput detection method:

[0125] Dissolve the biliverdin standard in methanol and prepare a gradient of 0-200 mg / L biliverdin standard solution. Take 200 μL of each concentration and place it in a 96-well plate. Use an ELISA 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 the following: Figure 4 The standard curve is shown.

[0126] High-throughput screening process:

[0127] The monoclonal clone obtained in Example 2 was selected and inoculated into a 48-deep-well plate containing 1 mL of LB medium in each well, and 50 mg / L of ampicillin 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-deep-well plate containing fresh LB medium at an inoculation amount of 1% (v / v), and 50 mg / L of ampicillin 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 shaking table at 20°C and 200 rpm for further culturing for 16 h. After the cultivation was completed, the plate was placed in a centrifuge with a matching rotor, centrifuged at 8000 × g, 4°C for 10 min, the supernatant was discarded, and the plate containing the bacterial precipitate was placed in a -20°C refrigerator for 24 h to increase the permeability of the cell membrane.

[0128] Then, add the following reaction system (total volume 0.5mL) to each well of the well plate: 1mM hemin chloride, 30mM ammonium formate, and Tris-HCl (100mM, pH 7.5) as solvent, and shake to evenly suspend the bacterial precipitate. Place in a constant temperature incubator at 35°C and 200rpm for 1 hour. After the reaction, add 0.5mL of methanol to each well to terminate the reaction. Place the well plate in a centrifuge with a matching rotor, centrifuge at 8000×g, 4°C for 10min, take 200μL of supernatant, add to a 96-well plate, and use an enzyme reader to detect the absorbance of the product biliverdin catalyzed by heme oxygenase at 690nm, and substitute the absorbance value into Figure 4 The standard curve is shown in Figure 2, and the concentration is calculated. The saturation mutation results of the 20 mutation sites selected in Example 3 are shown in Figure 2. Figure 5 As shown. Figure 5 It can be seen that compared with the wild type, there are mutants with improved catalytic function at sites 28, 142 and 210, so the monoclones with the highest catalytic ability in the three sites were selected and sent to Qingke Bio (Suzhou) for sequencing to obtain the corresponding mutant sequences, and the corresponding monoclones were deposited as strains. Among them, the amino acid changes before and after the mutation at the mutation site are shown in Table 3.

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

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

[0131] Example 4: Combination mutation of ScHO

[0132] The three forward mutations Q25V, H156N, and N247H obtained in Example 3 were combined 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 the tube was cultured in a constant temperature shaker at 37°C and 200 rpm for 8 h, and then the plasmid was extracted using Vazyme FastPurePlasmid Mini Kit-BOX 2. The extraction steps were referred to the instructions provided in the kit.

[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 mutation primers shown in Table 4, the above three plasmids were used as templates to carry out PCR reaction to introduce the second / third mutation site.

[0136] Table 4 Site-directed mutagenesis primers

[0137]

[0138] The PCR, template digestion and product purification processes refer to Example 2. Obtaining double mutant recombinant plasmid

[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 plasmid

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

[0141] Table 5: Concentration of biliverdin synthesis catalyzed 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 the ability of the positively induced Q25V and H156N, N247H or H156N / N247H combined mutations to catalyze the synthesis of biliverdin was weaker than that of the single point mutations H156N and N247H; while the ability of the single point mutations H156N and N247H to catalyze the synthesis of biliverdin was further improved after combined mutation, which was 2.1 times higher than that of the wild type.

[0144] Example 5: Analysis of Enzymatic Properties 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 plasmid containing mutants screened in Example 3, the mutant sequences were amplified using PCR primers designed according to Table 6 and connected to pET28a(+) by 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, and pET28a(+)-ScCPR were constructed, and the recombinant strains were constructed and the proteins were expressed according to Example 1, and the proteins were purified according to the following method.

[0151] (1) Preparation of protein purification reagents

[0152] Binging Buffer: Tris-HCl buffer (100mM, pH 7.5). Washing Buffer: Take 1L Tris-HCl buffer (100mM, pH 7.5), add 3.4g imidazole (final concentration 50mM), and adjust the pH of the buffer to 7.5 with hydrochloric acid or sodium hydroxide. Elution Buffer: Take 1L Tris-HCl buffer (100mM, pH 7.5), add 34g imidazole (final concentration 500mM), and adjust the pH of the buffer 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 crushing was purified using a Ni affinity column (40×12.6mm, Bio-Rad, USA). The specific steps are as follows: (I) The Ni column was rinsed with Binging Buffer at a flow rate of 0.4 mL / min until the UV baseline was balanced, and the amount used was approximately 15 times the volume of the nickel column. (II) After the baseline was flushed with Binging Buffer, the sample was loaded at a flow rate of 0.2 mL / min to allow the target protein to fully bind to the Ni column. (III) The impurities were rinsed with Washing Buffer at a flow rate of 0.3 mL / min until the UV baseline was balanced, and the amount used was approximately 10 times the volume of the nickel column. (IV) The target protein was rinsed and eluted with Elution Buffer at a flow rate of 0.25 mL / min. When the absorbance reached 0.1, collection began, and when the absorbance dropped to 0.1, collection stopped. Continue to rinse with Elution Buffer and adjust the flow rate to 0.3mL / min until the UV baseline is balanced. (V) Replace Elution Buffer with Binging Buffer and continue to rinse. Use Binging Buffer to balance the Ni column and adjust the flow rate to 0.35mL / min until the UV baseline is balanced. The amount used is approximately 10 times the volume of the nickel column. (VI) Use 8 times the column volume of 20% ethanol to preserve the Ni column and store the Ni column in a 4°C refrigerator. (VII) Remove salt ions: Place the collected target protein eluate in Tris-HCl buffer (100mM, pH 7.5) and dialyze overnight to obtain a purified enzyme solution. Use SDS-PAGE to analyze the purification results. The results are as shown below. Figure 7 shown.

[0155] (3) After obtaining the purified ScHO wild-type enzyme, Q25V, H156N, N247H, Q25V / H156N, Q25V / N247H, H156N / N247H, Q25V / H156N / N247H, and ScCPR wild-type enzyme, the protein concentration was determined using an ultra-micro spectrophotometer and the reaction system was prepared as follows:

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

[0157] Reaction progress determination process: Place the 96-well plate in an ELISA reader, set the temperature to 35°C, shake for 2 seconds every 5 seconds, scan the absorbance value of each well at 690nm every 30 seconds, and press Figure 4 The standard curve shown is converted to biliverdin concentration. The first 10% of each reaction process is taken to calculate the reaction rate, and the reaction rates under different substrate concentrations are fitted according to the Michaelis-Menten equation. The results are shown in Figure 6 The kinetic parameters of the wild type and mutant ScHO calculated based on the fitting results are shown in Table 6, and the enzyme activity data were detected at the same time, and the results are shown in Table 7.

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

[0159]

[0160]

[0161] Kinetic parameter determination experiments showed that the ScHO combined mutant H156N / N247H had the highest catalytic efficiency and specific activity.

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

[0163] Preferred ScHO mutants H156N / N247H Integrate the remaining proteins into the same strain of E. coli. The specific steps are as follows:

[0164] (1) Preparation of plasmid

[0165] The preferred ScHO mutant ScHO obtained in Example 4 H156N / N247H The strain was extracted with VazymeFastPure Plasmid Mini Kit-BOX 2. The extraction steps were referred to the instructions provided in the kit. The recombinant plasmid pETDuet-1-ScHO H156N / N247H -ScCPR-ApFDH;

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

[0167] (2) Construction of recombinant strains

[0168] According to the method for constructing the recombinant strain described in Example 1, the above two plasmids were simultaneously transformed into the same E. coli BL21 (DE3) competent cells to obtain a recombinant strain containing ScHO H156N / N247H Five enzyme co-expression strain: E. coli BL21 (DE3) /

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

[0170] Example 7: Establishment of an efficient reaction system

[0171] The recombinant 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 of Example 1, a fermentation broth was prepared, and the obtained fermentation broth was centrifuged at low temperature, and the centrifugation conditions were 8,000×g, 10min, 4°C. After centrifugation, the supernatant was discarded, and the precipitate was washed twice with physiological saline to obtain whole cells; the dry weight of the whole cells was weighed to 20g / L, and ultrasonically crushed under ultrasonic conditions of 400W, 0°C, working for 2s, resting for 4s, and a total time of 10min to obtain cell lysate. The cell lysate was used as a catalyst to optimize the reaction conditions. The specific operation steps are as follows:

[0172] 1. Optimization of reaction conditions

[0173] (1) Optimization of optimal reaction temperature

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

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

[0176] The reaction results at different temperatures are as follows Figure 8 The test results show that the optimal reaction temperature is 35° C. At this time, 1 mM hemin is converted into 541 mg / L bilirubin within 1 hour, and the yield reaches 92.6%.

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

[0178] The reaction conditions are as follows: 100 mL reaction system, 20 g / L recombinant Escherichia coli cell lysate (the amount of the lysate added is: first weigh 20 g / L of the whole cell dry weight, and after crushing, it is the corresponding lysate to be added), the solvent is Tris-HCl solution (pH 7.5-8.0, 100 mM) or citric acid-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), EDTA-Na2 5 mM, hemin 2 mM, 35°C, 200 rpm.

[0179] Under the above reaction conditions, three buffer solutions were used to prepare reaction systems with pH values ​​of 6.0, 6.5, 7.0, 7.5, and 8.0, respectively. Samples were taken after 1 hour of reaction. 950 μL of a mixture of DMSO and dichloromethane (volume ratio 5:1) was added to 50 μL of the reaction solution sample, and the mixture was 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 are as follows Fig. 9 The test results show that the optimal reaction pH is 6.5, at which time, 2 mM hemin is converted into 812 mg / L bilirubin within 1 hour, and the yield reaches 69.5%.

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

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

[0183] 2. The best reaction conditions are:

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

[0185] The results showed that when the substrate concentration was 3 mM, the product bilirubin reached 1611 mg / L at 4 h, and the yield was as high as 91.9%. Continuing to increase the substrate concentration did not lead to further accumulation of the product bilirubin.

[0186] Although the present invention has been disclosed as above in the form of a preferred embodiment, 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 scope of protection of the present invention should be based on the definition of 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 with the amino acid sequence as shown in SEQ ID NO.1 to valine; or obtained by mutating the histidine at position 156 of the heme oxygenase with the amino acid sequence as shown in SEQ ID NO.1 to asparagine; or by mutating the asparagine at position 247 of the heme oxygenase with the amino acid sequence as shown in SEQ ID NO.1 to histidine; Or the amino acid sequence of the heme oxygenase shown in SEQ ID NO.1 is obtained by mutating the histidine at position 156 to asparagine, and the asparagine at position 247 to histidine; Or the amino acid sequence of the heme oxygenase shown in SEQ ID NO.1 is obtained by mutating the glutamine at position 25 to valine and the histidine at position 156 to asparagine; Or the amino acid sequence of the heme oxygenase shown in SEQ ID NO.1 is obtained by mutating the glutamine at position 25 to valine and 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, preferably, the recombinant cell uses bacteria or fungi as host cells.

4. A recombinant enzyme catalyst comprising the heme oxygenase mutant according to claim 1, characterized in that: Is any of the following: (1) culturing a recombinant expression transformant containing the heme oxygenase mutant, and isolating a transformant cell containing the recombinant heme oxygenase mutant enzyme; (2) culturing a recombinant expression transformant containing the heme oxygenase mutant, isolating transformant cells containing the recombinant heme oxygenase mutant enzyme, and disrupting the transformant cells containing the recombinant heme oxygenase mutant enzyme to obtain a cell disrupted liquid; (3) culturing a recombinant expression transformant containing the heme oxygenase mutant, isolating transformant cells containing the recombinant heme oxygenase mutant enzyme, disrupting the transformant cells containing the recombinant heme oxygenase mutant enzyme, obtaining a cell disrupted liquid, and freeze-drying the cell disrupted liquid of the recombinant heme oxygenase mutant enzyme to obtain a lyophilized enzyme powder.

5. A method for improving the conversion rate of heme oxygenase to a substrate and / or improving its enzyme activity, characterized in that: The method comprises mutating the glutamine at position 25 of the heme oxygenase whose amino acid sequence is shown in SEQ ID NO.1 to valine; or the histidine at position 156 of the heme oxygenase whose amino acid sequence is shown in SEQ ID NO.1 is mutated to asparagine; or mutating the asparagine at position 247 of the heme oxygenase whose amino acid sequence is shown in SEQ ID NO.1 to histidine; Or the histidine at position 156 of the heme oxygenase with the amino acid sequence as shown in SEQ ID NO.1 is mutated to asparagine, and the asparagine at position 247 is mutated to histidine; Or the glutamine at position 25 of the heme oxygenase with the amino acid sequence as shown in SEQ ID NO.1 is mutated to valine, and the histidine at position 156 is mutated to asparagine; Or the glutamine at position 25 of the heme oxygenase with an amino acid sequence as shown in SEQ ID NO.1 is mutated to valine, and the asparagine at position 247 is mutated to histidine.

6. Use of the mutant according to claim 1, the recombinant cell according to claim 3, or the recombinant enzyme catalyst according to claim 4 in the preparation of biliverdin or bilirubin, characterized in that: 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 is transformed to prepare biliverdin, and the recombinant cell also expresses NADP from Azospirillum palustre + dependent formate dehydrogenase, cytochrome P450 reductase from Saccharomyces cerevisiae; The enzyme reaction system is: using hemin as substrate to catalyze the preparation of biliverdin, and adding NADP from Azospirillum palustre + dependent formate dehydrogenase, cytochrome P450 reductase from Saccharomyces cerevisiae; 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 is converted to prepare bilirubin, and the recombinant cell also expresses biliverdin reductase from Mus musculus and NADP from Azospirillum palustre. + dependent formate dehydrogenase, cytochrome P450 reductase from Saccharomyces cerevisiae, and carbon monoxide dehydrogenase from Afipia carboxidovorans were also expressed; The enzyme reaction system is as follows: using hemin as substrate to catalyze the preparation of bilirubin, while adding biliverdin reductase from Mus musculus and NADP from Azospirillum palustre + A formate dehydrogenase-dependent enzyme, a cytochrome P450 reductase from Saccharomyces cerevisiae, and a carbon monoxide dehydrogenase from Afipia carboxidovorans were also expressed.

7. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria express the heme oxygenase mutant according to claim 1, biliverdin reductase from Mus musculus, NADP from Azospirillum palustre + Dependent formate dehydrogenase and cytochrome P450 reductase from Saccharomyces cerevisiae, and carbon monoxide dehydrogenase from Afipia carboxidovorans were also expressed; Preferably, 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 dehydrogenase-dependent enzyme is shown in SEQ ID NO.5; the nucleotide sequence of the carbon monoxide dehydrogenase is shown in SEQ ID NO.6; Preferably, the genetically engineered bacteria include but are not limited to Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and yeast.

8. A method for synthesizing bilirubin, characterized in that: The method comprises the following steps: using heme as a substrate and using the genetically engineered bacteria or its lysate or fermentation liquid as claimed in claim 7 as a catalyst to catalytically convert and synthesize bilirubin.

9. The method according to claim 8, characterized in that The added amount of hemoglobin 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.

10. Use of the genetically engineered bacteria according to claim 7 or the method according to claim 8 or 9 in the preparation of bilirubin or a product containing bilirubin.

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