Chimeric protein containing Ho1 and PcyA and application of chimeric protein in production of phycocyanobilin

Abstract

The present invention provides a nucleic acid molecule encoding a chimeric protein useful for efficient production of phycocyanobilin, a genetic vector and a host cell comprising a nucleotide sequence encoding the chimeric protein, a modified cell capable of expressing the chimeric protein, the chimeric protein, and a method for producing phycocyanobilin using the chimeric protein.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical and biomedical technology. Specifically, the present invention relates to a chimeric protein that can be used to efficiently produce phycocyanobilin, and the use of the chimeric protein in producing phycocyanobilin. Background Art

[0002] Phycobiliproteins are unique photosynthetic light-harvesting antennae in cyanobacteria and red algae (Walter A. Slider et al, 1994). Allophycocyanin (APC), C-phycocyanin (CPC) and phycoerythrocyanin (PEC) are the three most common phycobiliproteins in cyanobacteria, all of which contain a covalently bonded chromophore, phycocyanobilin (PCB), and have different absorption and emission spectra in the visible light range. Phycobiliproteins are widely used as fluorescent probes (Patel A et al, 2005) and drugs (Ge B et al, 2005).

[0003] Studies have shown that PCB has antioxidant and anti-inflammatory effects (Benedetti S et al, 2010; BhatVB et al, 2001). Oral PCB can be converted into phycoerythrin in cells, which has the potential to inhibit NADPH oxidase. NADPH oxidase is related to the pathogenesis of many common diseases (McCarty MF et al, 2010). Since the stroke cascade involves inflammatory response and oxidative stress, the effect of PCB was evaluated using an animal experimental model of cerebral hypoperfusion and in vitro cell survival and cell differential expression experiments (Marín J et al, 2013; Pentón G et al, 2018), showing that PCB treatment can prevent H 2 O 2 and glutamate-induced cell damage, regulating 190 genes (93 up-regulated and 97 down-regulated) related to several immune and inflammatory processes.

[0004] PCB can be extracted from Spirulina (O'Carra P et al, 1966). However, although Spirulina is a rich source of PCB, the cultivation of algae takes a long time and requires a lot of space. At the same time, the presence of other light-harvesting pigments makes the extraction of PCB difficult. The above factors limit the large-scale production of PCB, making the production cost of high-purity PCB high.

[0005] Ferredoxin-dependent heme oxygenase 1 (Ho1) and phycocyanobilin:ferredoxin oxidoreductase (PcyA) are two key enzymes that convert heme into PCB in the enzymatic system [heme…Ho1…→…biliverdin…PcyA…→…PCB], that is, heme is catalyzed by Ho1 to produce biliverdin, and biliverdin is catalyzed by PcyA to produce PCB.

[0006] Ho1, also known as Hsp32, has been identified as a member of the heat shock protein family. Ho1 is not a heme protein and does not contain any heme prosthetic group (Kikuchi G et al, 2005). The soluble Ho1 protein secreted by cells can also act as a chaperone protein, participating in protein-protein interactions and other cellular functions besides its catalytic activity (Hopper CP et al, 2020). Regarding the catalytic activity of Ho1, in the rate-limiting multi-step reaction, Ho1 promotes the opening of the heme ring, releases iron, and converts heme into biliverdin (Masakazu S et al, 2000).

[0007] PcyA is a ferredoxin-dependent biliverdin reductase (FDBR), a free radical enzyme that does not require cofactors or metal ions. It can use four electrons from ferredoxin to continuously reduce the D-ring and A-ring vinyl groups of biliverdin IXα to generate PCB. PcyA is an α / β / α sandwich fold structure, in which biliverdin IXα in a ring conformation is located between the β-fold and the C-terminal α-helix. The basic patches on the surface of PcyA close to the biliverdin IXα molecule can provide binding sites for acidic ferredoxin, allowing direct electron transfer to biliverdin IXα. The position of biliverdin IXα is clearly fixed in PcyA by several hydrophilic interactions and the shape of the biliverdin IXα binding pocket in PcyA (Hagiwara Y et al, 2016). Summary of the invention

[0008] Based on the analysis of protein structure and function, the inventors of the present application designed and screened a large number of chimeric proteins containing different connecting peptides, Ho1 and PcyA using molecular modeling and protein engineering design methods, and obtained functional chimeric proteins that can be efficiently and soluble expressed in host cells (e.g., Escherichia coli cells) and have excellent Ho1 enzyme activity and PcyA enzyme activity. The chimeric proteins can achieve efficient heterologous production of phycocyanobilin in host cells (e.g., Escherichia coli cells).

[0009] Therefore, in one aspect, the present application provides a chimeric protein comprising:

[0010] (i) a first peptide segment, wherein the first peptide segment comprises or consists of heme oxygenase 1 (Ho1) or an active variant thereof;

[0011] (ii) a second peptide segment, the second peptide segment comprising or consisting of phycocyanobilin:ferredoxin oxidoreductase (PcyA) or an active variant thereof; and

[0012] (iii) a connecting peptide;

[0013] Wherein, the first peptide segment and the second peptide segment are connected via the connecting peptide.

[0014] In certain embodiments, the active variant of Hol is:

[0015] (a) having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) amino acid sequence identity; or,

[0016] (b) has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions.

[0017] In certain embodiments, the active variant of Ho1 retains at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the biological activity (e.g., the activity of catalyzing the conversion of heme to biliverdin) of the Ho1 from which it is derived.

[0018] In certain embodiments, the amino acid residues at positions corresponding to positions 6, 10, 17, 20, 21, 27, 28, 49, 125, 127, 130, 131, 133, 134, 137, 138, 164, 168, 196 and / or 199 of SEQ ID NO: 1 in the active variant of Ho1 are the same as the Ho1 from which it is derived.

[0019] In certain embodiments, the active variant of PcyA is:

[0020] (a) having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) amino acid sequence identity; or,

[0021] (b) has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions.

[0022] In certain embodiments, the active variant of PcyA retains at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the biological activity (e.g., the activity of catalyzing the conversion of biliverdin to phycocyanobilin) ​​of the PcyA from which it is derived.

[0023] In certain embodiments, the amino acid residues at positions corresponding to positions 76, 88, 105, 216 and / or 212 of SEQ ID NO: 2 in the active variant of PcyA are the same as the PcyA from which they are derived.

[0024] In certain embodiments, the connecting peptide is selected from peptides having the ability to form a helical structure.

[0025] In certain embodiments, the connecting peptide has a peptide sequence such as A(EAAAK) n The sequence shown in A, wherein n is 1, 2, 3 or 4; preferably, n is 2, 3 or 4.

[0026] In certain embodiments, the distance between the active site of the Ho1 or its active fragment and the active site of the PcyA or its active fragment is to within the range (for example, within to to or to within the range of ).

[0027] In certain embodiments, the distance between the active site of the Ho1 or its active fragment and the active site of the PcyA or its active fragment is to within the range (for example, within or within the range of ).

[0028] In certain embodiments, the distance between the active site of the Ho1 or its active fragment and the active site of the PcyA or its active fragment is to (For example, ) within the range of .

[0029] In certain embodiments, the connecting peptide is a flexible connecting peptide; and the connecting peptide comprises or consists of 4-36 (e.g., 4-10, 4-15, 4-20, 4-22, 4-25 or 4-30) glycines (G) and / or 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) serines (S).

[0030] In certain embodiments, the connecting peptide consists of 10-55 (e.g., 10-12, 10-15, 10-20, 10-25, 10-50, 10) amino acid residues.

[0031] In certain embodiments, the connecting peptide has a feature selected from the group consisting of:

[0032] (1) The connecting peptide has a structure such as (GGGGS) m The sequence shown, wherein m is 1, 2, 3, 4, 5, 6, 7, 8 or 9; preferably, m is 1, 2, 3, 4, 5 or 6;

[0033] (2) The connecting peptide has (GS) y The sequence shown, wherein y is 5, 6, 7, 8 or 9; preferably, y is 5 or 6.

[0034] In certain embodiments, the distance between the active site of the Ho1 or its active fragment and the active site of the PcyA or its active fragment is to within the range (for example, within to to to to to to or to within the range of ).

[0035] In certain embodiments, the distance between the active site of the Ho1 or its active fragment and the active site of the PcyA or its active fragment is to within the range (for example, within or within the range of ).

[0036] In certain embodiments, the distance between the active site of the Ho1 or its active fragment and the active site of the PcyA or its active fragment is to within the range (for example, within to to or to within the range of ).

[0037] In certain embodiments, the distance between the active site of the Ho1 or its active fragment and the active site of the PcyA or its active fragment is to within the range (for example, within or within the range of ).

[0038] In certain embodiments, the connecting peptide is selected from or consists of a peptide linker comprising one or more glycine (G).

[0039] In certain embodiments, the connecting peptide has a z The sequence shown, wherein z is 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14 or 15; preferably, z is 6, 7 or 10.

[0040] In certain embodiments, the chimeric protein has one or more of the following features:

[0041] (1) The Ho1 has: (a) an amino acid sequence as shown in SEQ ID NO: 1; (b) an amino acid sequence having at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 1; or, (c) a sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conservative substitutions), additions, or deletions compared to the amino acid sequence shown in SEQ ID NO: 1;

[0042] (2) The PcyA has: (a) an amino acid sequence as shown in SEQ ID NO: 2; (b) an amino acid sequence having at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 2; or (c) a sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conservative substitutions), additions, or deletions compared to the amino acid sequence shown in SEQ ID NO: 2;

[0043] (3) The connecting peptide has an amino acid sequence as shown in any one of SEQ ID NOs: 3-5, 7-8, 11-17, 22-23; In certain embodiments, the connecting peptide has an amino acid sequence as shown in any one of SEQ ID NOs: 3-5, 11, 13-14, 22;

[0044] (4) The first peptide segment is connected to the N-terminus or C-terminus of the second peptide segment through the peptide linker; in certain embodiments, the first peptide segment is connected to the N-terminus of the second peptide segment through the peptide linker;

[0045] (5) The active site distance between the Ho1 or its active variant and the PcyA or its active variant is within to (For example, ) within the range of .

[0046] As used in the present application, "active site distance" is used to refer to the distance from the center of the enzyme active cavity formed by Hol or its active variant to the center of the enzyme active cavity formed by PcyA or its active variant.

[0047] The enzyme activity cavity formed by Ho1 or its active variant has the meaning generally understood in the art, which generally refers to the enzyme activity region formed by the key amino acids involved in the enzyme activity reaction when Ho1 or its active variant is in a correctly folded spatial conformation with enzyme activity. For example, when describing with Ho1 shown in SEQ ID NO: 1 provided in the present application as an example, the enzyme activity cavity formed by Ho1 or its active variant generally means that when the Ho1 or its active variant is in a correctly folded spatial conformation with enzyme activity, the enzyme activity region formed by amino acid residues including but not limited to Ala6, Arg10, His17, Ala20, Glu21, Lys27, Cys28, Val49, Tyr125, Arg127, Gly130, Asp131, Ser133, Gly134, Ile137, Leu138, Glu164, Lys168, Phe196, Asn199.

[0048] The enzymatic activity cavity formed by PcyA or its active variant has the meaning generally understood in the art, which generally refers to the enzymatic activity region formed by the key amino acids involved in the enzymatic activity reaction when PcyA or its active variant is in a correctly folded spatial conformation with enzymatic activity. For example, when PcyA shown in SEQ ID NO: 2 provided in the present application is described as an example, the enzymatic activity cavity formed by PcyA or its active variant generally means that when the PcyA or its active variant is in a correctly folded spatial conformation with enzymatic activity, the enzymatic activity region formed by amino acid residues including but not limited to Glu76, His88, Asp105, Gln216 and Tyr212.

[0049] The sequence shown in SEQ ID NO:1 contains an amino acid (such as methionine (Met)) encoded by a start codon (such as ATG) at its N-terminus. The Ho1 of the present invention encompasses not only an amino acid sequence containing an amino acid (such as Met) encoded by a start codon at its N-terminus, but also an amino acid sequence that does not contain an amino acid (such as Met) encoded by a start codon at its N-terminus. Therefore, a sequence that does not contain an amino acid (such as Met) encoded by a start codon at the N-terminus of the above amino acid sequence is also within the protection scope of the present invention.

[0050] The sequence shown in SEQ ID NO:2 does not contain an amino acid (such as methionine (Met)) encoded by a start codon (such as ATG) at its N-terminus (due to cloning strategy and design). The PcyA of the present invention encompasses not only an amino acid sequence that does not contain an amino acid (such as Met) encoded by a start codon at its N-terminus, but also an amino acid sequence that contains an amino acid (such as Met) encoded by a start codon at its N-terminus. Therefore, a sequence that contains an amino acid (such as Met) encoded by a start codon at the N-terminus of the above amino acid sequence is also within the protection scope of the present invention.

[0051] In certain embodiments, the present invention provides the following embodiments:

[0052] Scheme 1. A chimeric protein comprising:

[0053] (i) a first peptide segment, wherein the first peptide segment comprises or consists of heme oxygenase 1 (Ho1) or an active variant thereof;

[0054] (ii) a second peptide segment, the second peptide segment comprising or consisting of phycocyanobilin:ferredoxin oxidoreductase (PcyA) or an active variant thereof; and

[0055] (iii) a connecting peptide;

[0056] Wherein, the first peptide segment and the second peptide segment are connected via the connecting peptide.

[0057] Scheme 2. The chimeric protein of Scheme 1, wherein the connecting peptide has a peptide sequence such as A(EAAAK) n The sequence shown in A, wherein n is 1, 2, 3 or 4; preferably, n is 2, 3 or 4.

[0058] Scheme 3. The chimeric protein of Scheme 2, wherein the active site of the Ho1 or its active fragment is 500 nm away from the active site of the PcyA or its active fragment. to within the range (for example, within to to or to within the scope of

[0059] Preferably, the active site of the Ho1 or its active fragment is at a distance of to within the range (for example, within or within the scope of

[0060] Preferably, the active site of the Ho1 or its active fragment is at a distance of to (For example, ) within the range of .

[0061] Scheme 4. The chimeric protein of Scheme 1, wherein the connecting peptide is a flexible connecting peptide;

[0062] Furthermore, the connecting peptide comprises or consists of 4-36 (e.g., 4-10, 4-15, 4-20, 4-22, 4-25 or 4-30) glycine (G) and / or 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) serine (S).

[0063] Scheme 5. The chimeric protein of Scheme 1, wherein the connecting peptide has a feature selected from the following:

[0064] (1) The connecting peptide has a structure such as (GGGGS) m The sequence shown, wherein m is 1, 2, 3, 4, 5, 6, 7, 8 or 9; preferably, m is 1, 2, 3, 4, 5 or 6;

[0065] (2) The connecting peptide has (GS) y The sequence shown, wherein y is 5, 6, 7, 8 or 9; preferably, y is 5 or 6.

[0066] Scheme 6. The chimeric protein of Scheme 4 or 5, wherein the active site of the Ho1 or its active fragment is 500 nm away from the active site of the PcyA or its active fragment. to within the range (for example, within to to to to to to or to within the scope of

[0067] Preferably, the active site of the Ho1 or its active fragment is at a distance of to within the range (for example, within or within the scope of

[0068] Preferably, the active site of the Ho1 or its active fragment is at a distance of to within the range (for example, within to to or to within the scope of

[0069] Preferably, the active site of the Ho1 or its active fragment is at a distance of to within the range (for example, within or within the range of ).

[0070] Scheme 7. The chimeric protein of Scheme 3, wherein the connecting peptide comprises or consists of one or more glycine (G);

[0071] Preferably, the connecting peptide has (G) z The sequence shown, wherein z is 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14 or 15; preferably, z is 6, 7 or 10.

[0072] Scheme 8. The chimeric protein of any one of Schemes 1-7, which has one or more selected from the following features:

[0073] (1) The Ho1 has: (a) an amino acid sequence as shown in SEQ ID NO: 1; (b) an amino acid sequence having at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 1; or, (c) a sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conservative substitutions), additions, or deletions compared to the amino acid sequence shown in SEQ ID NO: 1;

[0074] (2) The PcyA has: (a) an amino acid sequence as shown in SEQ ID NO: 2; (b) an amino acid sequence having at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 2; or (c) a sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conservative substitutions), additions, or deletions compared to the amino acid sequence shown in SEQ ID NO: 2;

[0075] (3) The connecting peptide has an amino acid sequence as shown in any one of SEQ ID NOs: 3-5, 7-8, 11-17, 22-23; preferably, the connecting peptide has an amino acid sequence as shown in any one of SEQ ID NOs: 3-5, 11, 13-14, 22;

[0076] (4) The first peptide segment is connected to the N-terminus or C-terminus of the second peptide segment through the peptide linker; preferably, the first peptide segment is connected to the N-terminus of the second peptide segment through the peptide linker;

[0077] (5) The active site distance between the Ho1 or its active variant and the PcyA or its active variant is within to (For example, ) within the range of .

[0078] In another aspect, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric protein as described above.

[0079] In certain embodiments, the nucleotide sequence encoding the chimeric protein is codon-optimized or non-optimized according to the codon preference of the host cell (eg, E. coli cell).

[0080] In another aspect, the present application provides a vector comprising the isolated nucleic acid molecule as described above.

[0081] In certain embodiments, the vector is an expression vector or a cloning vector.

[0082] In another aspect, the present application provides a host cell comprising the isolated nucleic acid molecule or vector as described above.

[0083] In certain embodiments, the host cell is selected from prokaryotic cells (eg, E. coli cells), and eukaryotic cells (eg, yeast cells, insect cells, plant cells, mammalian cells).

[0084] In certain embodiments, the host cell is an E. coli cell.

[0085] On the other hand, the present application also provides a method for preparing the chimeric protein as described above, which comprises culturing the host cell as described above under conditions allowing protein expression, and recovering the chimeric protein from the cell culture.

[0086] In another aspect, the present application provides a modified cell capable of expressing the chimeric protein as described above.

[0087] In certain embodiments, the engineered cell comprises a nucleotide sequence encoding a chimeric protein as described above.

[0088] In certain embodiments, the engineered cells are capable of synthesizing phycocyanobilin molecules.

[0089] In certain embodiments, the modified cell is capable of synthesizing and / or transporting heme molecules. For example, the modified cell is capable of synthesizing heme molecules itself, and / or, the modified cell is capable of obtaining heme molecules from the environment (e.g., outside the cell) via a transport pathway.

[0090] In certain embodiments, the engineered cells do not contain endogenous Hol and / or PcyA genes.

[0091] In certain embodiments, the engineered cells are E. coli cells.

[0092] On the other hand, the present application provides a method for preparing the modified cells as described above, comprising: (1) providing cells to be modified; (2) introducing the isolated nucleic acid molecule or vector as described above into the cells to be modified in step (1), wherein the nucleic acid molecule or vector comprises a nucleotide sequence encoding the chimeric protein as described above, so as to obtain a modified cell capable of expressing the chimeric protein.

[0093] In certain embodiments, the vector is an expression vector.

[0094] In certain embodiments, the cell to be modified is capable of synthesizing and / or transporting heme molecules. For example, the cell to be modified is capable of synthesizing heme molecules by itself, and / or, the cell to be modified is capable of obtaining heme molecules from the environment (e.g., outside the cell) via a transport pathway.

[0095] In certain embodiments, the cell to be engineered does not contain endogenous Ho1 and / or PcyA genes.

[0096] In certain embodiments, the cell to be modified is an E. coli cell.

[0097] In another aspect, the present application provides a method for producing phycocyanobilin, comprising:

[0098] (1) Providing a modified cell as described above;

[0099] (2) culturing the modified cells as described above under conditions that allow protein expression; and,

[0100] (3) Recovery of phycocyanobilin from cell culture.

[0101] In certain embodiments, in step (2), the modified cells are cultured at an initial optical density of 0.05-0.5 (e.g., 0.05-0.1, 0.05-0.3, 0.08-0.1, 0.08-0.3, 0.08-0.5) for 10-50 h (e.g., 10-30 h) at 20-37 °C (e.g., 20-25 °C, 20-30 °C, 25-37 °C).

[0102] In certain embodiments, prior to step (2), the expression of the chimeric protein in the engineered cell is inhibited.

[0103] In certain embodiments, in step (3), the method recovers phycocyanobilin from the cell culture by comprising the steps of:

[0104] (a) recovering cells from the cell culture after culturing in step (2);

[0105] (b) contacting the recovered cells with an alcohol solution;

[0106] (c) extracting phycocyanobilin from the alcohol solution treated in step (b).

[0107] In some embodiments, the alcohol solution is methanol.In some embodiments, in step (b), the recovered cells are contacted with the alcohol solution for 0.5-4 h (e.g., 0.5-3 h, 0.5-2 h, 1-4 h, 1-3 h, 1-2 h) at a water bath temperature of 40-60° C. (e.g., 50° C.).

[0108] In another aspect, the present application provides the use of the chimeric protein, isolated nucleic acid molecule, vector, host cell, modified cell or method for preparing the modified cell as described above for producing phycocyanobilin.

[0109] Definition of terms

[0110] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the virology, biochemistry, and immunology laboratory operation steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0111] When the terms "for example," "such as," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0112] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0113] As used herein, heme oxygenase 1 (Ho1), also known as Hsp32, is a member of the heat shock protein family and has the meaning commonly understood by those skilled in the art. Generally speaking, in addition to catalytic activity, Ho1 protein can also act as a chaperone protein, participating in protein-protein interactions and other cellular functions other than its catalytic activity. It is easy for those skilled in the art to understand that the activity of the chimeric protein of the present application mainly depends on the catalytic activity of Ho1, that is, catalyzing the conversion of heme into biliverdin. Therefore, Ho1 having the activity of catalyzing the conversion of heme into biliverdin is within the scope of protection of the present application. In certain embodiments, the Ho1 can be selected from Ho1 derived from Synechocystis sp. (PCC6803), Arthrospir (Spirulina) subsalsa (FACHB-351), Arthrospira (Spirulina) platensis BP, cyanobacterial strain Limnothrix sp. NS01, cyanobacterium Synechocystis sp. PCC 6803, Cyanobacterial strains Nostoc commune TUBT05 and Oscillatoria okeni TISTR8549.

[0114] In the present invention, when referring to the amino acid sequence of heme oxygenase 1 (Ho1), the description is made with reference to the sequence shown in SEQ ID NO: 1. However, those skilled in the art understand that Ho1 may have a variety of different sources and natural or artificial mutations, and therefore, the Ho1 of the present application not only includes the Ho1 represented by SEQ ID NO: 1, but also Ho1 having a different source from SEQ ID NO: 1 or containing natural or artificial mutations is also within the protection scope of the present application.

[0115] As used herein, phycocyanobilin:ferredoxin oxidoreductase (PcyA) has the meaning commonly understood by those skilled in the art, which is a ferredoxin-dependent bilirin reductase (FDBR) that can use four electrons of ferredoxin to continuously reduce the D ring and A ring vinyl of biliverdin IXα to generate phycocyanobilin. It is easy for those skilled in the art to understand that the activity of the chimeric protein of the present application mainly depends on the catalytic activity of PcyA, that is, catalyzing the conversion of biliverdin into phycocyanobilin. Therefore, PcyA having the activity of catalyzing the conversion of biliverdin into phycocyanobilin is within the scope of protection of the present application. In certain embodiments, the PcyA can be selected from PcyA derived from Synechocystis sp., (PCC6803), Arthrospir (Spirulina) subsalsa (FACHB-351), Arthrospira (Spirulina) platensis BP, cyanobacterial strain Limnothrix sp. NS01, cyanobacterium Synechocystis sp. PCC6803, Cyanobacterial strains Nostoc commune TUBT05 and Oscillatoria okeniTISTR8549.

[0116] In the present invention, when referring to the amino acid sequence of phycocyanobilin:ferredoxin oxidoreductase (PcyA), the description is made with reference to the sequence shown in SEQ ID NO: 2. However, those skilled in the art understand that PcyA can have a variety of different sources and natural or artificial mutations, and therefore, the PcyA of the present application not only includes the PcyA represented by SEQ ID NO: 2, but also PcyA having a different source from SEQ ID NO: 2 or containing natural or artificial mutations is also within the scope of protection of the present application.

[0117] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0118] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0119] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0120] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0121] Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the desired expression level, etc. A vector can be introduced into a host cell to thereby produce transcripts, proteins, or peptides, including proteins, isolated nucleic acid molecules, etc. as described herein.

[0122] In the present invention, the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0123] In the present invention, unless otherwise specified herein or clearly contradicted by the context, the terms "chimeric protein" and "fusion protein" have the same meaning and can be used interchangeably.

[0124] Advantageous Effects of the Invention

[0125] The chimeric protein provided in the present application can be efficiently and soluble expressed in host cells (e.g., Escherichia coli cells), and the expressed chimeric protein has excellent Ho1 enzyme activity and PcyA enzyme activity at the same time, which can achieve efficient heterologous production of phycocyanobilin in host cells (e.g., Escherichia coli cells), and is expected to improve the current status of phycocyanobilin extraction in the art, such as difficulty, high cost, and long cycle.

[0126] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0127] Figure 1 :[A, B] show the three-dimensional structural model of heme oxygenase 1 (Ho1) and heme (upper figure) and two different side schematic diagrams of the enzyme active site (lower figure); [C, D] show phycocyanin:

[0128] The three-dimensional structural model of ferredoxin oxidoreductase (PcyA) and biliverdin (top) and two different side views of the enzyme active site (bottom);

[0129] Figure 2 : The three-dimensional structural model of the chimeric protein (Ho1-connector peptide-PcyA), wherein the connector peptide includes: (A) AEK2, (B) AEK3, (C) AEK4, (D) AEK5, (E) G6, (F) G7, (G) G8, (H) G9, (I) G10, (J) (GGGGS)1, (K) (GGGGS)2, (L) (GGGGS)3, (M) (GGGGS)4, (N) (GGGGS)5, (O) (GGGGS)6, (P) L1, (Q) L2, (R) GS3, (S) GS4, (T) GS5 and (U) GS6;

[0130] Figure 3 : PCR schematic diagram of inserting the linker into the ho1-pcyA gene construct;

[0131] Figure 4 : Schematic diagram of cloning of ho1-linker-pcyA gene in pET30a vector for further expressing recombinant chimeric protein containing linker peptide;

[0132] Figure 5: Schematic diagram of SDS-PAGE analysis of samples of the growth process before and after induction of the recombinant chimeric protein; (1) low molecular weight protein marker, (2) BL21 (DE3) Escherichia coli transformed with pET30a before induction, (3) BL21 (DE3) Escherichia coli transformed with pET-30a after induction, (4) Escherichia coli BL21 (DE3) strain transformed with pET30a-ho1-pcyA before induction; after induction of recombinant chimeric protein expression, (5) pET30a_ho1_ AEK2_pcyA, (6)pET30a_ho1_AEK3_pcyA, (7)pET30a_ho1_AEK4_pcyA, (8)pET30a_ho1_AEK5_pcyA, (9)pET3 0a_ho1_G6_pcyA, (10)pET30a_ho1_G7_pcyA, (11)pET30a_ho1_G8_pcyA, (12)pET30a_ho1_G9_pcyA, (13)pE T30a_ho1_G10_pcyA, (14)pET30a_ho1_(GGGGS)1_pcyA, (15)pET30a_ho1_(GGGGS)2_pcyA, (16)pET30a_ho 1_(GGGGS)3_pcyA, (17)pET30a_ho1_(GGGGS)4_pcyA, (18)pET30a_ho1_(GGGGS)5_pcyA, (19)pET30a_ho1_( BL21(DE3) Escherichia coli transformed with pET30a_ho1_(GS)6_pcyA, (20) pET30a_ho1_L1_pcyA, (21) pET30a_ho1_L2_pcyA, (22) pET30a_ho1_(GS)3_pcyA, (23) pET30a_ho1_(GS)4_pcyA, (24) pET30a_ho1_(GS)5_pcyA, and (25) pET30a_ho1_(GS)6_pcyA;

[0133] Figure 6 : Schematic representation of the representative antioxidant activity of PCBs extracted from Ho1-linker peptide-PcyA positive BL21 (DE3) Escherichia coli cells;

[0134] Figure 7: Schematic diagram of the relationship between the expression level of the recombinant chimeric protein (Ho1-(GGGGGS)2 connecting peptide-PcyA) in Escherichia coli and the PCB yield; [A] Schematic diagram of SDS-PAGE of samples after 12 hours and 20 hours of growth: (1) low molecular weight protein marker, (2) Escherichia coli cells before induction, (3) recombinant Escherichia coli cells expressing (Ho1-(GGGGGS)2 connecting peptide-PcyA) after 20 hours of growth, (4) Escherichia coli cells before induction, (5) recombinant Escherichia coli cells expressing (Ho1-(GGGGGS)2 connecting peptide-PcyA) after 12 hours of growth; and the PCB yield of each variant; [B] Growth curve of recombinant Escherichia coli cells expressing (Ho1-(GGGGGS)2 connecting peptide-PcyA);

[0135] Figure 8 : A schematic diagram showing the relationship between the solubility of the chimeric protein containing the connecting peptide and the production of PCBs by E. coli cells expressing the chimeric protein, wherein each protein is represented by the connecting peptide it contains. DETAILED DESCRIPTION

[0136] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0137] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Compiled Molecular Biology Laboratory Manual, 3rd Edition, John Wiley & Sons, Inc., 1995. It is understood by those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the invention claimed.

[0138] Example 1 Design of chimeric proteins linking heme oxygenase 1 (Ho1) and phycocyanobilin:ferredoxin oxidoreductase (PcyA) by different linker peptides

[0139] The relationship between structure and function is a core concept in science and engineering (Hartwell L et al, 1999). Therefore, the theory that molecular structure determines biological function promotes the experimental detection of protein structure and function. A chimeric protein for phycocyanobilin (PCB) synthesis was designed through molecular modeling and protein engineering, and the enzyme structure determinants of two enzymes involved in PCB synthesis were studied at the same time. The chimeric protein consists of three modules.

[0140] The three module structures of the chimeric protein are as follows:

[0141] (a) The first module is heme oxygenase 1 (Ho1), and the amino acid sequence of Ho1 in this embodiment is SEQ ID NO: 1;

[0142] (b) The second module is a connecting peptide formed by helices of different lengths and structures, wherein the connecting peptide is selected from the following table:

[0143]

[0144] (c) The third module is phycocyanobilin:ferredoxin oxidoreductase (PcyA). The amino acid sequence of PcyA in this example is SEQ ID NO: 2.

[0145] In the above manner, the structure of the chimeric protein is composed of three modules, and module (b) connects the two domains (a) and (c).

[0146] The rationality of the chimeric protein module structure

[0147] Module (a) is the Ho1 domain, and module (c) is the PcyA domain. Both domains must be able to fold independently, and the fusion in a single protein must ensure that the folding of the two enzymes is not affected, so as to exert their functionality. In addition, the two enzymes must maintain a distance to effectively transport the compounds obtained by the enzymatic reaction to generate PCBs. Therefore, it is not easy to form a functional chimeric protein by connecting the two enzymes.

[0148] Ho1 is a 27 kDa enzyme, whose protein sequence includes the first 240 amino acid residues encoded by the ho1 gene isolated from Synechocystis sp. (strain PCC 6803 / Kazusa) (Ge B et al, 2009) [NCBI Reference Sequence Accession Number: WP_010871494.1]. PcyA is a 28 kDa enzyme, whose protein sequence includes the first 248 amino acid residues encoded by the pcyA gene isolated from Synechocystis sp. (strain PCC 6803 / Kazusa) [NCBI Reference Sequence Accession Number: WP_010873881.1].

[0149] The construction of chimeric proteins involves connecting two or more protein domains through a connecting peptide (Chen X et al, 2013). The choice of the connecting peptide sequence is very important for the construction of functional chimeric proteins. Although the flexibility and hydrophilicity of the connecting peptide are very important, it has been verified that other peptides with the ability to form a monomeric α-helical structure will not interfere with the functionality of the bifunctional chimeric protein (Arachi R et al, 2001).

[0150] The key of the present invention is to use the connecting peptide to connect Ho1 and PcyA ( Figure 1 :The three-dimensional structural model of Ho1 and PcyA and the schematic diagram of the enzyme active site) were used to model and identify the regularity, so as to further screen the active recombinant chimeric protein for PCB production. First, the structural model of Ho1 and PcyA was obtained using the fully automatic protein structure homology modeling software (SWISS-MODEL), and the SWISS-MODEL software can be accessed through the Expasy website server (https: / / swissmodel.expasy.org / ). The three-dimensional structure of the connecting peptide contained in the active recombinant chimeric protein can be obtained by three different methods: (1) homology modeling (Krieger E et al, 2003) (https: / / swissmodel.expasy.org / ), (2) stringing / folding recognition (Rost B et al, 1997) (https: / / zhanglab.ccmb.med.umich.edu / LOMETS / ) and (3) ab initio predictive modeling using the Robetta server (Bonneau R et al, 2001) (http: / / robetta.bakerlab.org / ). The obtained structures were aligned and the average of the determined structures was calculated for further structural analysis.

[0151]

[0152]

[0153] The structural characterization of the chimeric protein model was as follows Figure 2 Table 1 shows the extended geometric structure characteristics and length of the connecting peptide. The length is calculated from the distance of the flanking α carbon atom amino acid, ranging from The length obtained is not the actual length expected in the folded protein. Therefore, the effect of the connecting peptide on the constructed chimeric protein model is shown by the average distance between the active site cavities of the two enzymes Ho1 and PcyA, as follows: The distance between the active site cavities of the two enzymes Ho1 and PcyA in 5 corresponding structural models was calculated using the Cavity Plus web server (http: / / www.pkumdl.cn:8000 / cavityplus / index.php) to obtain the average value. The average values ​​obtained range from

[0154] Example 2 Cloning of genes for expressing bifunctional chimeric proteins in E. coli

[0155] The gene structure encoding the chimeric protein was cloned into the pET-30a expression vector and further expressed in E. coli under the control of the T7 promoter. The chimeric protein contained the connecting peptide designed in Example 1. The gene structure was then expressed in BL21 (DE3) E. coli. The BL21 (DE3) E. coli was incubated with Luria-Bertani (LB) medium, and the expression of the recombinant chimeric protein was induced using ZY autoinduction medium or 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG).

[0156] To obtain all variants, the tandem ho1-pcyA genes (encoding a chimeric protein) were chemically synthesized using the method studied by Agarwal and his team (Agarwal KL et al, 1970), and oligonucleotides were prepared in solid phase by phosphoramidite chemistry for DNA synthesis (Beaucage SL et al, 1981). The DNA fragment flanked by the BamH I-Xho I restriction sites was obtained and inserted into the pET-30a expression vector (Invitrogen Corp. USA) for expression under the control of the T7 promoter.

[0157] A two-round PCR experimental strategy was used to generate the ho1-linker-pcyA gene variant, and the primers used included the linker to be inserted between the ho1 and pcyA genes. Figure 3 This is a simplified schematic diagram of the strategy. Figure 3 As shown, two rounds of PCR experiments are required. In the first round of PCR experiments, three PCR experiments are performed: (A, B) primers or DNA fragments that insert the linker and the flanking sequences of the ho1 and pcyA genes are used, respectively, and (C) annealed primers or DNA fragments encoding the linker are used, and the primers contain extensions of ho1 and pcyA overhang / flanking ends to be filled. In the second round of PCR experiments, the purified products of the three PCR experiments (A, B, C) are used as templates for this PCR experiment, and the primers used are ho1 and pcyA flanking primers, which contain restriction enzyme sites for further cloning.

[0158] Table 2 lists the PCR amplification primers used for the gene constructs. Figure 3 Table 2 shows the protein sequences corresponding to the linkers and their DNA primers: (1) 5'BamH I-ho1, (2) 3'RC pcyA-XhoⅠ, (3) 5'linker-pcyA, (4) 5'RC ho1 C-terminal-linker, (5) 5'ho1 C-terminal-linker, (6) 5'RC linker-pcyA. All primer sequences are in the 5' to 3' direction, and RC indicates the reverse complementary sequence of a specific primer. The gene constructs inserted with the linker were sequenced to verify their integrity and sequence.

[0159] After digestion with specific restriction enzymes, the ho1-linker-pcyA DNA gene fragment was ligated to the pET-30a expression vector using T4 DNA ligase (Promega Corp. USA) according to the supplier's instructions.

[0160] Table 2 DNA primers used to insert the linker between ho1-pcyA genes in PCR experiments

[0161]

[0162]

[0163]

[0164]

[0165] All experimental products were transformed into XL-1-Blue E. coli strain. DNA of individual colonies was purified and analyzed by restriction enzyme digestion. Then, DNA of colonies matching the required pattern of each gene construct was sequenced using an automatic sequencer, and a representative clone was selected for each gene construct. The selected clones were transformed into BL21 (DE3) E. coli strain for protein expression. General cloning methods are as follows Figure 4 shown.

[0166] The transformed BL21 (DE3) Escherichia coli strain was cultured in LB medium, and 1 mM IPTG or ZY autoinduction medium was used to induce the expression of the recombinant chimeric protein. IPTG induction was used for small-capacity screening, while ZY autoinduction medium was used for large-capacity screening. First, the recombinant BL21 (DE3) Escherichia coli strain transformed with the gene construct containing the linker was cultured in an LB plate containing 50 μg / mL kanamycin and 1% glucose at 37°C for 16 hours to inhibit the expression of the recombinant chimeric protein. The culture was then inoculated at an initial optical density of 0.1 and cultured for 20 hours. The culture was then centrifuged, the cells were collected, and the cells were washed with phosphate buffer at pH 7.0 and centrifuged again before PCB extraction. The expression of the recombinant chimeric protein is as follows. Figure 5 The whole recombinant E. coli cells were analyzed by SDS-PAGE and found that the recombinant chimeric proteins in all experiments were expressed, and the expression level was between 5% and 30% of the total cell protein content, which was sufficient to catalyze the conversion of heme into biliverdin and finally into PCB in a multi-stage reaction.

[0167] For PCB analysis, the cell pellets of 1L of the recombinant E. coli strain expressing the recombinant chimeric protein containing the connecting peptide were resuspended in methanol in a 50°C water bath for about 1 to 2 hours until the cell pellet turned white. Subsequently, the cell pellet was removed by centrifugation at 8000×g for 10 minutes, and the supernatant was collected for chloroform-water extraction. After the solution was freeze-dried, the PCB precipitate was collected. For quantitative and in-depth analysis, the PCB precipitate was dissolved in methanol and concentrated hydrochloric acid was added (V CH3OH :V HCl =95:5) using an ultraviolet spectrophotometer to measure absorbance at 680 nm (Bennett AB L, 1973). The equation "c[mM] = Abs 680nm / 37.9×dilution factor” to calculate PCB concentration (Glazer AN et al, 1973; Ge B et al, 2013). Table 3 shows the results of PCB production by recombinant E. coli cells.

[0168] As an essential component of the recombinant chimeric protein of the present invention, the connecting peptide plays an important role in obtaining stable and biologically active PCB molecules. The present invention effectively verifies the relationship between protein structure and function. Not all connecting peptides used to construct recombinant chimeric proteins can generate functional molecules and then synthesize PCB. The selected connecting peptides have a certain influence on the expression level of the recombinant chimeric protein. At the same time, the overall solubility of the recombinant chimeric protein and the folding of the independent domains it contains may be affected by the connecting peptide.

[0169] For the high-PCB-producing recombinant E. coli strain variants, the average value of PCB production in the culture flask ranged from 2.5 ± 0.21 mg / L culture, wherein the chimeric protein used contained a linker peptide with an extended geometric length of The connecting peptide shortens the distance between the active sites of Ho1 and PcyA to After transformation with gene construct, it contains short connecting peptide (extended geometric length is ) and long linker peptides (extended geometric length is ) did not produce PCB, and the short linker peptide and the long linker peptide shortened the distance between the active sites of Ho1 and PcyA to The reason for not generating PCBs may be incorrect folding or the distance between the two enzyme active sites is not optimal, which is not conducive to the dual enzymatic reaction of generating PCBs. In general, recombinant E. coli strains that produce chimeric proteins containing AEK2, AEK3, AEK4, G10, (GGGGS)2, (GGGGS)3 and (GS)5 connecting peptides have higher PCB yields.

[0170] Table 3 PCB production of E. coli strains expressing Ho1-connector peptide-PcyA recombinant chimeric protein

[0171]

[0172] Example 3 Antioxidant activity of PCB produced by recombinant Escherichia coli strains

[0173] Antioxidant capacity is an important factor in many human diseases and health damage. A quick, simple and low-cost method for evaluating antioxidant capacity is to use 2,2-biphenyl-1-picrylhydrazyl (DPPH). DPPH is a stable free radical that can act as a hydrogen free radical scavenger ( Kedare SB et al, 2011).

[0174] In the test, the dark blue DPPH radical extracts a hydrogen atom in a single electron reaction to generate light yellow DPPH-H. The color change is related to the decrease in absorbance at 517nm. At the same time, the decrease in absorbance at 517nm is proportional to the antioxidant capacity of the sample. The DPPH radical scavenging activity was determined according to the method studied by Shimada et al. (Shimada K, et al. (1992) J Agric and Food Chem 40: 945-948). In short, 100 μL of the test sample was mixed with 100 μL of 0.1 mM DPPH, shaken vigorously for 30 minutes, and then the absorbance at 517nm was measured using an ELISA reader. The scavenging activity was calculated as follows: DPPH scavenging rate = [1-(AC) / B]*100%, where A is the absorbance of a mixture of 100 μL DPPH and 100 μL sample at 517 nm, B is the absorbance of a mixture of 100 μL DPPH and 100 μL methanol at 517 nm, and C is the absorbance of a mixture of 100 μL sample and 100 μL methanol at 517 nm.

[0175] The PCB obtained in the E. coli cells described in Example 2 was purified. E. coli cells expressing recombinant chimeric proteins containing connecting peptides AEK2, AEK3, AEK4, G10, (GGGGS)2, (GGGGS)3 and (GS)5 had a higher PCB yield. After the PCB obtained from the above E. coli cells was purified, it was dissolved in methanol at a concentration of 1 mg / mL, and after gradient dilution at a ratio of 1:2, the free radical scavenging rate was detected by the above experiment. The activity curves obtained in all experiments were similar. Figure 6A schematic diagram of the DPPH radical scavenging rate curve of PCB produced by E. coli cells containing the connecting peptide (GGGGS) 2 is shown. In the PCB concentration range of 1.9 to 1000 μg / mL, the free radical scavenging rate is 12.5% ​​to 92.3%, indicating that PCB has excellent antioxidant activity and meets the requirements of industrial and biopharmaceutical applications.

[0176] Example 4 Correlation between the expression level of the recombinant chimeric protein and the PCB yield

[0177] The gene construct was transformed into Escherichia coli to obtain a recombinant chimeric protein containing a (GGGGS)2 connecting peptide. The gene construct was used to analyze the relationship between the expression level of the recombinant chimeric protein and the PCB yield, and the results showed that the gene construct was one of the gene constructs with a high PCB yield.

[0178] When the recombinant E. coli strain was cultured in ZY autoinduction medium, the expression level of the recombinant chimeric protein was Figure 7 As shown. Cell growth was stopped at two different time points: one was after 12 hours of incubation (variant 1), when the optical density of the culture was 1.8; the other was after 20 hours of incubation (variant 2), when the optical density of the culture was 5.92. The expression levels of the recombinant chimeric protein at these two growth stages were 13% and 36%, respectively. PCB was purified from the bacteria of 1L culture, and 0.015mM (0.009mg / mL) and 0.053mM (0.031mg / mL) PCB could be obtained under each condition, respectively. It was found that the expression level of the recombinant chimeric protein had a significant linear correlation with the PCB yield of Escherichia coli cells. In this experiment, the same gene expression variant (Ho1-(GGGGS)2 connecting peptide-PcyA) was used to obtain functional chimeric proteins. Therefore, when making comparisons, the effect of protein folding on PCB yield can be minimized.

[0179] Example 5 Correlation between the solubility of the recombinant chimeric protein and PCB production

[0180] The main purpose of recombinant chimeric protein expression is usually to obtain high yields of soluble products in bacterial cells ( HP et al, 2005). Under normal cellular conditions, some cytoplasmic proteins are able to fold spontaneously. However, due to the accumulation of high concentrations of folding intermediates or inefficient processing by molecular chaperones during the folding process, aggregation patterns of overexpressed recombinant chimeric proteins in bacterial cells have been detected (Hartl FU et al, 2002). Usually, only folded and soluble proteins are functional, while proteins do not obtain the expected three-dimensional structure (referred to as protein misfolding), which usually results in the formation of protein aggregates that are inactive and insoluble (Skretas G et al, 2020).

[0181] The recombinant E. coli strain was cultured in ZY autoinduction medium for 20 hours to induce the expression of the recombinant chimeric protein. Then, 1L of the culture was centrifuged to collect the cells. Half of the cells were used to purify PCB (refer to Example 2 for the purification method), and half of the cells were used for the solubility study of the chimeric protein. The E. coli cells were then broken by ultrasound, and the soluble and insoluble components were separated by centrifugation. The recombinant chimeric protein content of the soluble component in the broken cells was compared with the recombinant chimeric protein content of the insoluble component in the broken cells by SDS-PAGE analysis. All recombinant chimeric proteins had excellent solubility, with a solubility of over 75%, such as Figure 8 However, it was observed that the PCB yields of different E. coli recombinant strains were different. Together with the results obtained in Example 4, it was shown that the folding structure of the obtained recombinant chimeric protein is a key factor in the generation of PCBs, which is determined by the structural characteristics of the connecting peptide connecting the Ho1 and PcyA domains.

[0182] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

[0183] Sequence information

[0184] SEQ ID NO: 1:

[0185] MSVNLASQLREGTKKSHSMAENVGFVKCFLKGVVEKNSYRKLVGNLYFVYSAMEEEMAKFKDHPILSHIYFPELNRKQSLEQDLQFYYGSNWRQEVKISAAGQAYVDRVRQVAATAPELL VAHSYTRYLGDLSGGQILKKIAQNAMNLHDGGTAFYEFADIDDEKAFKNTYRQAMNDLPIDQATAERIVDEANDAFAMNMKMFNELEGNLIKAIGIMVFNSLTRRRSQGSTEVGLATSEG

[0186] SEQ ID NO:2:

[0187] SAVTDLSLTNSSLMPTLNPMIQQLALAIAASWQSLPLKPYQLPEDLGYVEGRLEGEKLVIENRCYQTPQFRKMHLELAKVGKGLDILHCVMFPEPLYGLPLFGCDIVAGPGGVSAAIADLSPTQSDRQLPAAYQKSLAELGQPEFEQQRELPPWGEIFSEYCLFIRPSNVTEEERFVQRVVDFLQIHCHQSIVAEPLSEAQTLEHRQGQIHYCQQQQKNDKTRRVLEKAFGEAWAERYMSQVLFDVIQ

[0188] SEQ ID NO:3:AEAAAKEAAAKA

[0189] SEQ ID NO:4:AEAAAKEAAAKEAAAKA

[0190] SEQ ID NO:5:AEAAAKEAAAKEAAAKEAAAKA

[0191] SEQ ID NO:6:AEAAAKEAAAKEAAAKEAAAKEAAAKA

[0192] SEQ ID NO:7:GGGGGG

[0193] SEQ ID NO:8:GGGGGGG

[0194] SEQ ID NO:9:GGGGGGGG

[0195] SEQ ID NO:10:GGGGGGGGG

[0196] SEQ ID NO:11:GGGGGGGGGG

[0197] SEQ ID NO:12:GGGGS

[0198] SEQ ID NO:13:GGGGSGGGGS

[0199] SEQ ID NO:14:GGGGSGGGGSGGGGS

[0200] SEQ ID NO:15:GGGGSGGGGSGGGGSGGGGS

[0201] SEQ ID NO:16:GGGGSGGGGSGGGGSGGGGSGGGGS

[0202] SEQ ID NO:17:GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS

[0203] SEQ ID NO:18:GGGGSLVPRGSGGGGS

[0204] SEQ ID NO:19:GGSGGHMGSGG

[0205] SEQ ID NO:20:GSGSGS

[0206] SEQ ID NO:21:GSGSGSGS

[0207] SEQ ID NO:22:GSGSGSGSGS

[0208] SEQ ID NO:23:GSGSGSGSGSGS

[0209] SEQ ID NO:24:

[0210] GGCCTCGCCACCTCCGAAGGCGCAGAAGCAGCAGCAAAAGAAGCAGCAGCAAAAGCA

[0211] SEQ ID NO:25:

[0212] TGCTTTTGCTGCTGCTTCTTTTGCTGCTGCTTCTGCGCCTTCGGAGGTGGCGAGGCCSEQ ID NO:26:

[0213] GCAGAAGCAGCAGCAAAAGAAGCAGCAGCAAAAGCATCTGCAGTTACAGATCTGAGC

[0214] SEQ ID NO:27:

[0215] GCTCAGATCTGTAACTGCAGATGCTTTTGCTGCTGCTTCTTTTGCTGCTGCTTCTGCSEQ ID NO:28:

[0216] GGCCTCGCCACCTCCGAAGGCGCAGAAGCAGCAGCAAAAGAAGCAGCAGCAAAAGAAGCAGCAGCAAAAGCA

[0217] SEQ ID NO:29:

[0218] TGCTTTTGCTGCTGCTTCTTTTGCTGCTGCTTCTTTTGCTGCTGCTTCTGCGCCTTCGGAGGTGGCGAGGCC

[0219] SEQ ID NO:30:

[0220] GCAGAAGCAGCAGCAAAAGAAGCAGCAGCAAAAGAAGCAGCAGCAAAAGCATCTGCAGTTACAGATCTGAGC

[0221] SEQ ID NO:31:

[0222] GCTCAGATCTGTAACTGCAGATGCTTTTGCTGCTGCTTCTTTTGCTGCTGCTTCTTTTGCTGCTGCTTCTGC

[0223] SEQ ID NO:32:

[0224] GGCCTCGCCACCTCCGAAGGCGCAGAAGCAGCAGCAAAAGAAGCAGCAGCAAAAGAAGCAGCAGCAAAAGAAGCAGCAGCAAAAGCA

[0225] SEQ ID NO:33:

[0226] TGCTTTTGCTGCTGCTTCTTTTGCTGCTGCTTCTTTTGCTGCTGCTTCTTTTGCTGCTGCTTCTGCGCCTTCGGAGGTGGCGAGGCC

[0227] SEQ ID NO:34:

[0228] GCAGAAGCAGCAGCAAAAGAAGCAGCAGCAAAAGAAGCAGCAGCAAAAGAAGCAGCAGCAAAAGCATCTGCAGTTACAGATCTGAGC

[0229] SEQ ID NO:35:

[0230] GCTCAGATCTGTAACTGCAGATGCTTTTGCTGCTGCTTCTTTTGCTGCTGCTTCTTTTGCTGCTGCTTCTTTTGCTGCTGCTTCTGC

[0231] SEQ ID NO:36:

[0232] GGCCTCGCCACCTCCGAAGGCGCAGAAGCAGCAGCAAAAGAAGCAGCAGCAAAAAGCAGCAGCAAAAAGCAGCAGCAAAAGAAGCAGCAGCAAAAGCA

[0233] SEQ ID NO:37:

[0234] TGCTTTTGCTGCTGCTTCTTTTGCTGCTGCTTCTTTTGCTGCTGCTTCTTTTGCTGCTTCTTTTGCTGCTTCTGCGCCTTCGGAGGTGGCGAGGCC

[0235] SEQ ID NO:38:

[0236] GCAGAAGCAGCAGCAAAAGAAGCAGCAGCAAAAAGCAGCAGCAAAAGAAGCAGCAGCAAAAGAAGCAGCAAAGCATCTGCAGTTACAGATCTGAGC

[0237] SEQ ID NO:39:

[0238] GCTCAGATCTGTAACTGCAGATGCTTTTGCTGCTGCTTCTTTTGCTGCTGCTTCTTGCTGCTGCTTCTTTTGCTGCTGCTTCTTTTGCTGCTGCTTCTGC

[0239] SEQ ID NO:40:GGCCTCGCCACCTCCGAAGGCGGAGGAGGAGGAGGAGGA

[0240] SEQ ID NO:41:TCCTCCTCCTCCTCCTCCGCCTTCCGGAGGTGGCGAGGCC

[0241] SEQ ID NO:42:GGAGGAGGAGGAGGAGGATCTGCAGTTACAGATCTGAGC

[0242] SEQ ID NO:43:GCTCAGATCTGTAACTGCAGATCCTCCTCCTCCTCCTCC

[0243] SEQ ID NO:44:GGCCTCGCCACCTCCGAAGGCGGAGGAGGAGGAGGAGGAGGA

[0244] SEQ ID NO:45:TCCTCCTCCTCCTCCTCCTCCGCCTTCGGAGGTGGCGAGGCC

[0245] SEQ ID NO:46:GGAGGAGGAGGAGGAGGAGGATCTGCAGTTACAGATCTGAGC

[0246] SEQ ID NO:47:GCTCAGATCTGTAACTGCAGATCCTCCTCCTCCTCCTCCTCC

[0247] SEQ ID NO:48:GGCCTCGCCACCTCCGAAGGCGGAGGAGGAGGAGGAGGAGGAGGA

[0248] SEQ ID NO:49:TCCTCCTCCTCCTCCTCCTCCTCCGCCTTCGGAGGTGGCGAGGCC

[0249] SEQ ID NO:50:GGAGGAGGAGGAGGAGGAGGAGGATCTGCAGTTACAGATCTGAGC

[0250] SEQ ID NO:51:GCTCAGATCTGTAACTGCAGATCCTCCTCCTCCTCCTCCTCCTCC

[0251] SEQ ID NO:52:

[0252] GGCCTCGCCACCTCCGAAGGCGGAGGAGGAGGAGGAGGAGGAGGAGGA

[0253] SEQ ID NO:53:TCCTCCTCCTCCTCCTCCTCCTCCTCCGCCTTCGGAGGTGGCGAGGCC

[0254] SEQ ID NO:54:

[0255] GGAGGAGGAGGAGGAGGAGGAGGAGGATCTGCAGTTACAGATCTGAGC

[0256] SEQ ID NO:55:GCTCAGATCTGTAACTGCAGATCCTCCTCCTCCTCCTCCTCCTCCTCC

[0257] SEQ ID NO:56:

[0258] GGCCTCGCCACCTCCGAAGGCGGAGGAGGAGGAGGAGGAGGAGGAGGAGGA

[0259] SEQ ID NO:57:

[0260] TCCTCCTCCTCCTCCTCCTCCTCCTCCTCCGCCTTCGGAGGTGGCGAGGCC

[0261] SEQ ID NO:58:

[0262] GGAGGAGGAGGAGGAGGAGGAGGAGGAGGATCTGCAGTTACAGATCTGAGC

[0263] SEQ ID NO:59:

[0264] GCTCAGATCTGTAACTGCAGATCCTCCTCCTCCTCCTCCTCCTCCTCCTCC

[0265] SEQ ID NO:60:GGCCTCGCCACCTCCGAAGGCGGAGGAGGAGGAAGC

[0266] SEQ ID NO:61:GCTTCCTCCTCCTCCGCCTTCGGAGGTGGCGAGGCC

[0267] SEQ ID NO:62:GGAGGAGGAGGAAGCTCTGCAGTTACAGATCTGAGC

[0268] SEQ ID NO:63:GCTCAGATCTGTAACTGCAGAGCTTCCTCCTCCTCC

[0269] SEQ ID NO:64:

[0270] GGCCTCGCCACCTCCGAAGGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGC

[0271] SEQ ID NO:65:

[0272] GCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCCTTCGGAGGTGGCGAGGCC

[0273] SEQ ID NO:66:

[0274] GGAGGAGGAGGAAGCGGAGGAGGAGGAAGCTCTGCAGTTACAGATCTGAGC

[0275] SEQ ID NO:67:

[0276] GCTCAGATCTGTAACTGCAGAGCTTCCTCCTCCTCCGCTTCCTCCTCCTCC

[0277] SEQ ID NO:68:

[0278] GGCCTCGCCACCTCCGAAGGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGC

[0279] SEQ ID NO:69:

[0280] GCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCCTTCGGAGGTGGCGAGGCC

[0281] SEQ ID NO:70:

[0282] GGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCTCTGCAGTTACAGATCTGAGC

[0283] SEQ ID NO:71:

[0284] GCTCAGATCTGTAACTGCAGAGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCC

[0285] SEQ ID NO:72:

[0286] GGCCTCGCCACCTCCGAAGGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGC

[0287] SEQ ID NO:73:

[0288] GCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCCTTCGGAGGTGGCGAGGCC

[0289] SEQ ID NO:74:

[0290] GGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCTCTGCAGTTACAGATCTGAGC

[0291] SEQ ID NO:75:

[0292] GCTCAGATCTGTAACTGCAGAGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCC

[0293] SEQ ID NO:76:

[0294] GGCCTCGCCACCTCCGAAGGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGC

[0295] SEQ ID NO:77:

[0296] GCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCCTTCGGAGGTGGCGAGGCC

[0297] SEQ ID NO:78:

[0298] GGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCTCTGCAGTTACAGATCTGAGC

[0299] SEQ ID NO:79:

[0300] GCTCAGATCTGTAACTGCAGAGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCC

[0301] SEQ ID NO:80:

[0302] GGCCTCGCCACCTCCGAAGGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGC

[0303] SEQ ID NO:81:

[0304] GCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCCTTCGGAGGTGGCGAGGCC

[0305] SEQ ID NO:82:

[0306] GGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCTCTGCAGTTACAGATCTGAGC

[0307] SEQ ID NO:83:

[0308] GCTCAGATCTGTAACTGCAGAGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCCGCTTCCTCCTCCTCC

[0309] SEQ ID NO:84:

[0310] GGCCTCGCCACCTCCGAAGGCGGAGGAGGAGGAAGCCTAGTACCAAGAGGAAGCGGAGGAGGAGGAAGC

[0311] SEQ ID NO:85:

[0312] GCTTCCTCCTCCTCCGCTTCCTCTTGGTACTAGGCTTCCTCCTCCTCCGCCTTCGGAGGTGGCGAGGCC

[0313] SEQ ID NO:86:

[0314] GGAGGAGGAGGAAGCCTAGTACCAAGGAAGCGGAGGAGGAGGAAGCTCTGCAGTTACAGATCTGAGC

[0315] SEQ ID NO:87:

[0316] GCTCAGATCTGTAACTGCAGAGCTTCCTCCTCCTCCGCTTCCTCTTGGTACTAGGCTTCCTCCTCCTCC

[0317] SEQ ID NO:88:

[0318] GGCCTCGCCACCTCCGAAGGCGGAGGAAGCGGAGGACACATGGGAAGCGGAGGA

[0319] SEQ ID NO:89:

[0320] TCCTCCGCTTCCCATGTGTCCTCCGCTTCCTCCGCCTTCCGGAGGTGGCGAGGCC

[0321] SEQ ID NO:90:

[0322] GGAGGAAGCGGAGGACACATGGGAAGCGGAGGATCTGCAGTTACAGATCTGAGC

[0323] SEQ ID NO:91:

[0324] GCTCAGATCTGTAACTGCAGATCCTCCGCTTCCCATGTGTCCTCCGCTTCCTCC

[0325] SEQ ID NO:92:GGCCTCGCCACCTCCGAAGGCGGAAGCGGAAGCGGAAGC

[0326] SEQ ID NO:93:GCTTCCGCTTCCGCTTCCGCCTTCCGGAGGTGGCGAGGCC

[0327] SEQ ID NO:94:GGAAGCGGAAGCGGAAGCTCTGCAGTTACAGATCTGAGC

[0328] SEQ ID NO:95:GCTCAGATCTGTAACTGCAGAGCTTCCGCTTCCGCTTCC

[0329] SEQ ID NO:96:GGCCTCGCCACCTCCGAAGGCGGAAGCGGAAGCGGAAGCGGAAGC

[0330] SEQ ID NO:97:GCTTCCGCTTCCGCTTCCGCTTCCGCCTTCGGAGGTGGCGAGGCC

[0331] SEQ ID NO:98:GGAAGCGGAAGCGGAAGCGGAAGCTCTGCAGTTACAGATCTGAGC

[0332] SEQ ID NO:99:GCTCAGATCTGTAACTGCAGAGCTTCCGCTTCCGCTTCCGCTTCC

[0333] SEQ ID NO:100:

[0334] GGCCTCGCCACCTCCGAAGGCGGAAGCGGAAGCGGAAGCGGAAGCGGAAGC

[0335] SEQ ID NO:101:

[0336] GCTTCCGCTTCCGCTTCCGCTTCCGCTTCCGCCTTCGGAGGTGGCGAGGCC

[0337] SEQ ID NO:102:

[0338] GGAAGCGGAAGCGGAAGCGGAAGCGGAAGCTCTGCAGTTACAGATCTGAGC

[0339] SEQ ID NO:103:

[0340] GCTCAGATCTGTAACTGCAGAGCTTCCGCTTCCGCTTCCGCTTCCGCTTCC

[0341] SEQ ID NO:104:

[0342] GGCCTCGCCACCTCCGAAGGCGGAAGCGGAAGCGGAAGCGGAAGCGGAAGCGGAAGC

[0343] SEQ ID NO:105:

[0344] GCTTCCGCTTCCGCTTCCGCTTCCGCTTCCGCTTCCGCCTTCGGAGGTGGCGAGGCC

[0345] SEQ ID NO:106:

[0346] GGAAGCGGAAGCGGAAGCGGAAGCGGAAGCGGAAGCTCTGCAGTTACAGATCTGAGC

[0347] SEQ ID NO:107:

[0348] GCTCAGATCTGTAACTGCAGAGCTTCCGCTTCCGCTTCCGCTTCCGCTTCCGCTTCC

[0349] SEQ ID NO:108:GGGGGATCCAGCGCTATAGTTGTTGAC

[0350] SEQ ID NO:109:CCCCCTCGAGTAGGGCTAATAAAAAGCCAC。

Claims

1. A chimeric protein comprising: (i) a first peptide segment, wherein the first peptide segment comprises or consists of heme oxygenase 1 (Ho1) or an active variant thereof; (ii) a second peptide segment, the second peptide segment comprising or consisting of phycocyanobilin:ferredoxin oxidoreductase (PcyA) or an active variant thereof; and (iii) a connecting peptide; in, The first peptide segment and the second peptide segment are connected via the connecting peptide.

2. The chimeric protein of claim 1, wherein The connecting peptide has a structure such as A(EAAAK) n The sequence shown in A, wherein n is 1, 2, 3 or 4; preferably, n is 2, 3 or 4.

3. The chimeric protein of claim 1, wherein The connecting peptide is a flexible connecting peptide; Furthermore, the connecting peptide comprises or consists of 4-36 (e.g., 4-10, 4-15, 4-20, 4-22, 4-25 or 4-30) glycine (G) and / or 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) serine (S); Preferably, the connecting peptide consists of 10-55 (e.g., 10-12, 10-15, 10-20, 10-25, 10-50, 10) amino acid residues.

4. The chimeric protein of claim 1, wherein The connecting peptide has a feature selected from the following: (1) The connecting peptide has a structure such as (GGGGS) m The sequence shown, wherein m is 1, 2, 3, 4, 5, 6, 7, 8 or 9; preferably, m is 1, 2, 3, 4, 5 or 6; (2) The connecting peptide has (GS) y The sequence shown, wherein y is 5, 6, 7, 8 or 9; preferably, y is 5 or 6.

5. The chimeric protein of claim 1, wherein The connecting peptide comprises or consists of a peptide linker of one or more glycine (G); Preferably, the connecting peptide has (G) z The sequence shown, wherein z is 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14 or 15; preferably, z is 6, 7 or 10.

6. The chimeric protein of any one of claims 1 to 5, which has one or more of the following characteristics: (1) The Ho1 has: (a) an amino acid sequence as shown in SEQ ID NO: 1; (b) an amino acid sequence having at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 1; or, (c) a sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conservative substitutions), additions, or deletions compared to the amino acid sequence shown in SEQ ID NO: 1; (2) The PcyA has: (a) an amino acid sequence as shown in SEQ ID NO: 2; (b) an amino acid sequence having at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 2; or (c) a sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conservative substitutions), additions, or deletions compared to the amino acid sequence shown in SEQ ID NO: 2; (3) The connecting peptide has an amino acid sequence as shown in any one of SEQ ID NOs: 3-5, 7-8, 11-17, 22-23; preferably, the connecting peptide has an amino acid sequence as shown in any one of SEQ ID NOs: 3-5, 11, 13-14, 22; (4) The first peptide segment is connected to the N-terminus or C-terminus of the second peptide segment through the peptide linker; preferably, the first peptide segment is connected to the N-terminus of the second peptide segment through the peptide linker; (5) The active site distance between the Ho1 or its active variant and the PcyA or its active variant is within to (For example, ) within the range of .

7. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric protein of any one of claims 1-6.

8. A vector comprising the isolated nucleic acid molecule of claim 7; Preferably, the vector is an expression vector or a cloning vector.

9. A host cell comprising the isolated nucleic acid molecule of claim 7 or the vector of claim 8.

10. A modified cell capable of expressing the chimeric protein of any one of claims 1 to 6; Preferably, the modified cell comprises a nucleotide sequence encoding the chimeric protein of any one of claims 1 to 6; Preferably, the engineered cells are capable of synthesizing phycocyanobilin molecules; Preferably, the modified cells are Escherichia coli cells.

11. A method for preparing the modified cell of claim 10, comprising: (1) Providing cells to be transformed; (2) introducing the isolated nucleic acid molecule of claim 7 or the vector of claim 8 into the cell to be transformed in step (1), wherein the nucleic acid molecule or the vector comprises a nucleotide sequence encoding the chimeric protein of any one of claims 1 to 6, to obtain a transformed cell capable of expressing the chimeric protein; Preferably, the vector is an expression vector; Preferably, the cells to be transformed are Escherichia coli cells.

12. A method for producing phycocyanobilin, comprising: (1) Providing the modified cell of claim 10; (2) culturing the modified cell of claim 10 under conditions that allow protein expression; and, (3) Recovery of phycocyanobilin from cell culture.

13. Use of the chimeric protein of any one of claims 1 to 6, the isolated nucleic acid molecule of claim 7, the vector of claim 8, the host cell of claim 9, the modified cell of claim 10 or the method of claim 11 for producing phycocyanobilin.

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