Chimeric protein containing ho1 and pcya and use thereof in producing phycocyanobilin

Abstract

A nucleic acid molecule encoding a chimeric protein which can be used for efficiently producing phycocyanobilin, a genetic vector and 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 by means of using the chimeric protein.

Description

Chimeric protein comprising Ho1 and PcyA and its application in producing phycocyanobilin

[0001] This application is based on the application with CN application number 202311300437.2 and application date October 9, 2023, and claims its priority. The disclosed content of the CN application is hereby introduced as a whole into this application. Technical Field

[0002] 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

[0003] Phycobiliproteins are unique photosynthetic light-harvesting antennae found 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 found in cyanobacteria. All contain a covalently bound chromophore, phycocyanobilin (PCB), and exhibit distinct absorption and emission spectra in the visible light range. Phycobiliproteins are widely used as fluorescent probes (Patel A et al., 2005) and pharmaceuticals (Ge B et al., 2005).

[0004] Studies have shown that PCBs have antioxidant and anti-inflammatory properties (Benedetti S et al., 2010; Bhat VB et al., 2001). Orally administered PCBs can be converted into phycoerythrin within cells, potentially inhibiting NADPH oxidase, an enzyme implicated in the pathogenesis of many common diseases (McCarty MF et al., 2010). Because the stroke cascade involves inflammation and oxidative stress, the effects of PCBs have been evaluated using animal models of cerebral hypoperfusion and in vitro cell survival and differential cell expression assays (Marín J et al., 2013; Pentón G et al., 2018). PCB treatment has shown that it can protect against H₂O₂- and glutamate-induced cell damage and regulate 190 genes (93 upregulated and 97 downregulated) associated with several immune and inflammatory processes.

[0005] PCBs can be extracted from Spirulina (O'Carra P et al., 1966). However, although Spirulina is a rich source of PCBs, the cultivation of the algae is long and requires a lot of space. Furthermore, the presence of other light-harvesting pigments makes PCB extraction difficult. These factors limit the large-scale production of PCBs, resulting in high production costs for high-purity PCBs.

[0006] Ferredoxin-dependent heme oxygenase 1 (Ho1) and phycocyanobilin:ferredoxin oxidoreductase (PcyA) are two key enzymes that convert heme into PCBs 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 PCBs.

[0007] 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 groups (Kikuchi G et al., 2005). Soluble Ho1 protein secreted by cells also acts as a chaperone, participating in protein-protein interactions and other cellular functions beyond its catalytic activity (Hopper CP et al., 2020). Regarding Ho1's catalytic activity, Ho1 promotes the opening of the heme ring in a rate-limiting multistep reaction, releasing iron and converting heme to biliverdin (Masakazu S et al., 2000).

[0008] PcyA, a ferredoxin-dependent biliverdin reductase (FDBR), is a free radical enzyme that does not require cofactors or metal ions. It uses four electrons from ferredoxin to sequentially reduce the D-ring and A-ring vinyl groups of biliverdin IXα to produce PCBs. PcyA has an α / β / α sandwich fold structure, in which the cyclic biliverdin IXα is located between the β-sheet and the C-terminal α-helix. A basic patch on the PcyA surface near the biliverdin IXα molecule provides a binding site for acidic ferredoxin, allowing direct electron transfer to biliverdin IXα. The location of biliverdin IXα in PcyA is clearly defined by several hydrophilic interactions and the shape of the biliverdin IXα binding pocket (Hagiwara Y et al., 2016).

[0009] Summary of the Invention

[0010] Based on the analysis of protein structure and function, the inventors of the present application used molecular modeling and protein engineering methods to design and screen a large number of chimeric proteins containing different connecting peptides, Ho1 and PcyA, 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).

[0011] Therefore, in one aspect, the present application provides a chimeric protein comprising:

[0012] (i) a first peptide segment, wherein the first peptide segment comprises or consists of heme oxygenase 1 (Ho1) or an active variant thereof;

[0013] (ii) a second peptide segment comprising or consisting of phycocyanobilin:ferredoxin oxidoreductase (PcyA) or an active variant thereof; and

[0014] (iii) a connecting peptide;

[0015] Wherein, the first peptide segment and the second peptide segment are connected via the connecting peptide.

[0016] In certain embodiments, the active variant of Hol is:

[0017] (a) has 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,

[0018] (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.

[0019] 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.

[0020] 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 those of the Ho1 from which they are derived.

[0021] In certain embodiments, the active variant of PcyA, compared to the PcyA from which it is derived:

[0022] (a) has 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,

[0023] (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.

[0024] 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 of the PcyA from which it is derived (e.g., the activity of catalyzing the conversion of biliverdin to phycocyanobilin).

[0025] 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 it is derived.

[0026] In certain embodiments, the connecting peptide is selected from peptides having the ability to form a helical structure.

[0027] In certain embodiments, the connecting peptide has a 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.

[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 within the range (e.g., to to or to within the scope of ).

[0029] 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 (e.g., or within the scope of ).

[0030] 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 scope of .

[0031] 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).

[0032] 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.

[0033] In certain embodiments, the connecting peptide has a feature selected from the group consisting of:

[0034] (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;

[0035] (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.

[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 (e.g., to to to to to to or to within the scope 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 (e.g., or within the scope of ).

[0038] 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 (e.g., to to or to within the scope of ).

[0039] 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 (e.g., or within the scope of ).

[0040] In certain embodiments, the connecting peptide is selected from or consists of a peptide linker comprising one or more glycine (G).

[0041] 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.

[0042] In certain embodiments, the chimeric protein has one or more of the following characteristics:

[0043] (1) The Ho1 has: (a) the 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 as 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 as shown in SEQ ID NO: 1;

[0044] (2) The PcyA has: (a) the amino acid sequence 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;

[0045] (3) The connecting peptide has an amino acid sequence as shown in any one of SEQ ID NOs: 3-5, 7-8, 11-17, and 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, and 22;

[0046] (4) The first peptide segment is connected to the N-terminus or C-terminus of the second peptide segment via the peptide linker; in certain embodiments, the first peptide segment is connected to the N-terminus of the second peptide segment via the peptide linker;

[0047] (5) The distance between the active sites of the Ho1 or its active variant and the PcyA or its active variant is to (For example, ) within the scope of .

[0048] As used herein, the "active site distance" refers 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.

[0049] The enzymatic activity cavity formed by Ho1 or its active variant has the meaning commonly 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 Ho1 or its active variant is in a spatial conformation that is correctly folded and has enzymatic activity. For example, when describing Ho1 shown in SEQ ID NO: 1 provided in the present application as an example, the enzymatic activity cavity formed by Ho1 or its active variant generally refers to the enzymatic 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, and Asn199 when the Ho1 or its active variant is in a spatial conformation that is correctly folded and has enzymatic activity.

[0050] The enzymatically active cavity formed by PcyA or its active variants has a meaning generally understood in the art, and generally refers to the enzymatically active region formed by key amino acids involved in the enzymatic activity reaction when PcyA or its active variants are in a properly folded and enzymatically active spatial conformation. For example, when describing PcyA as shown in SEQ ID NO: 2 provided herein, the enzymatically active cavity formed by PcyA or its active variants generally refers to the enzymatically active region formed by, including but not limited to, amino acid residues Glu76, His88, Asp105, Gln216, and Tyr212, when the PcyA or its active variants are in a properly folded and enzymatically active spatial conformation.

[0051] The sequence shown in SEQ ID NO: 1 includes 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 amino acid sequences that include an amino acid (such as Met) encoded by a start codon at its N-terminus, but also amino acid sequences that do not include an amino acid (such as Met) encoded by a start codon at its N-terminus. Therefore, sequences that do not include an amino acid (such as Met) encoded by a start codon at the N-terminus of the above amino acid sequence are also within the scope of protection of the present invention.

[0052] The sequence shown in SEQ ID NO: 2 does not contain an amino acid (e.g., methionine (Met)) encoded by a start codon (e.g., ATG) at its N-terminus (due to cloning strategy and design). The PcyA of the present invention encompasses not only amino acid sequences that do not contain an amino acid (e.g., Met) encoded by a start codon at its N-terminus, but also amino acid sequences that contain an amino acid (e.g., Met) encoded by a start codon at its N-terminus. Therefore, sequences containing an amino acid (e.g., Met) encoded by a start codon at the N-terminus of the above amino acid sequences are also within the scope of protection of the present invention.

[0053] In certain embodiments, the present invention provides the following embodiments:

[0054] Scheme 1. A chimeric protein comprising:

[0055] (i) a first peptide segment, wherein the first peptide segment comprises or consists of heme oxygenase 1 (Ho1) or an active variant thereof;

[0056] (ii) a second peptide segment comprising or consisting of phycocyanobilin:ferredoxin oxidoreductase (PcyA) or an active variant thereof; and

[0057] (iii) a connecting peptide;

[0058] Wherein, the first peptide segment and the second peptide segment are connected via the connecting peptide.

[0059] Scheme 2. The chimeric protein of Scheme 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.

[0060] Scheme 3. The chimeric protein of Scheme 2, wherein the distance between the active sites of the Ho1 or its active fragment and the PcyA or its active fragment is to within the range (e.g., to to or to within the scope of

[0061] Preferably, the distance between the active sites of the Ho1 or its active fragment and the PcyA or its active fragment is to within the range (e.g., or within the scope of

[0062] Preferably, the distance between the active sites of the Ho1 or its active fragment and the PcyA or its active fragment is to (For example, ) within the scope of .

[0063] Scheme 4. The chimeric protein of Scheme 1, wherein the connecting peptide is a flexible connecting peptide;

[0064] 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).

[0065] Scheme 5. The chimeric protein of Scheme 1, wherein the connecting peptide has a feature selected from the following:

[0066] (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;

[0067] (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.

[0068] Scheme 6. The chimeric protein of Scheme 4 or 5, wherein the distance between the active sites of the Ho1 or its active fragment and the PcyA or its active fragment is to within the range (e.g., to to to to to to or to within the scope of

[0069] Preferably, the distance between the active sites of the Ho1 or its active fragment and the PcyA or its active fragment is to within the range (e.g., or within the scope of

[0070] Preferably, the distance between the active sites of the Ho1 or its active fragment and the PcyA or its active fragment is to within the range (e.g., to to or to within the scope of

[0071] Preferably, the distance between the active sites of the Ho1 or its active fragment and the PcyA or its active fragment is to within the range (e.g., or within the scope of ).

[0072] Scheme 7. The chimeric protein of Scheme 3, wherein the connecting peptide comprises or consists of one or more glycine (G);

[0073] 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.

[0074] Scheme 8. The chimeric protein of any one of Schemes 1-7, which has one or more features selected from the following:

[0075] (1) The Ho1 has: (a) the 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 as 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 as shown in SEQ ID NO: 1;

[0076] (2) The PcyA has: (a) the amino acid sequence 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;

[0077] (3) The connecting peptide has an amino acid sequence as shown in any one of SEQ ID NOs: 3-5, 7-8, 11-17, and 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, and 22;

[0078] (4) The first peptide segment is connected to the N-terminus or C-terminus of the second peptide segment via the peptide linker; preferably, the first peptide segment is connected to the N-terminus of the second peptide segment via the peptide linker;

[0079] (5) The distance between the active sites of the Ho1 or its active variant and the PcyA or its active variant is to (For example, ) within the scope of .

[0080] In another aspect, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric protein as described above.

[0081] 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).

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

[0083] In certain embodiments, the vector is an expression vector or a cloning vector.

[0084] In another aspect, the present application provides a host cell comprising the isolated nucleic acid molecule or vector as described above.

[0085] 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).

[0086] In certain embodiments, the host cell is an E. coli cell.

[0087] 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.

[0088] In another aspect, the present application provides engineered cells capable of expressing the chimeric protein described above.

[0089] In certain embodiments, the engineered cell comprises a nucleotide sequence encoding a chimeric protein as described above.

[0090] In certain embodiments, the engineered cells are capable of synthesizing phycocyanobilin molecules.

[0091] In certain embodiments, the modified cell is capable of synthesizing and / or transporting a heme molecule. For example, the modified cell is capable of synthesizing a heme molecule itself, and / or the modified cell is capable of obtaining a heme molecule from the environment (e.g., extracellularly) via a transport pathway.

[0092] In certain embodiments, the engineered cells do not contain endogenous Hol and / or PcyA genes.

[0093] In certain embodiments, the engineered cells are E. coli cells.

[0094] 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, to obtain modified cells capable of expressing the chimeric protein.

[0095] In certain embodiments, the vector is an expression vector.

[0096] 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 itself, and / or the cell to be modified is capable of obtaining heme molecules from the environment (e.g., extracellularly) via a transport pathway.

[0097] In certain embodiments, the cell to be engineered does not contain endogenous Hol and / or PcyA genes.

[0098] In certain embodiments, the cell to be modified is an Escherichia coli cell.

[0099] In another aspect, the present application provides a method for producing phycocyanobilin, comprising:

[0100] (1) Providing a modified cell as described above;

[0101] (2) culturing the modified cells as described above under conditions that allow protein expression; and,

[0102] (3) Recovery of phycocyanin from cell culture.

[0103] 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).

[0104] In certain embodiments, prior to step (2), expression of the chimeric protein in the engineered cell is suppressed.

[0105] In certain embodiments, in step (3), the method recovers phycocyanobilin from the cell culture by the steps comprising:

[0106] (a) recovering cells from the cell culture after culturing in step (2);

[0107] (b) contacting the recovered cells with an alcohol solution;

[0108] (c) extracting phycocyanobilin from the alcohol solution treated in step (b).

[0109] In certain embodiments, the alcohol solution is methanol.In certain embodiments, in step (b), the recovered cells are contacted with the alcohol solution at a water bath temperature of 40-60° C. (e.g., 50° C.) for 0.5-4 h (e.g., 0.5-3 h, 0.5-2 h, 1-4 h, 1-3 h, 1-2 h).

[0110] 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.

[0111] Definition of terms

[0112] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virology, biochemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0113] When the terms "for example," "such as," "including," "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."

[0114] 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) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0115] 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 its catalytic activity, the Ho1 protein also functions as a chaperone, participating in protein-protein interactions and other cellular functions beyond its catalytic activity. Those skilled in the art will readily appreciate that the activity of the chimeric protein of the present application primarily depends on the catalytic activity of Ho1, namely, the conversion of heme to biliverdin. Therefore, any Ho1 protein that exhibits the activity of catalyzing the conversion of heme to 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.

[0116] 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 set forth in SEQ ID NO: 1. However, those skilled in the art will appreciate that Ho1 can have a variety of different origins and naturally occurring or artificially generated mutations. Therefore, the Ho1 of the present application encompasses not only the Ho1 represented by SEQ ID NO: 1, but also Ho1 having a different origin from SEQ ID NO: 1 or containing naturally occurring or artificially generated mutations.

[0117] As used herein, phycocyanobilin:ferredoxin oxidoreductase (PcyA) has the meaning generally understood by those skilled in the art. It is a ferredoxin-dependent bile reductase (FDBR) that can use four electrons from ferredoxin to continuously reduce the D-ring and A-ring vinyl groups of biliverdin IXα to produce phycocyanobilin. It is readily understood by those skilled in the art that the activity of the chimeric protein of the present application primarily depends on the catalytic activity of PcyA, namely, catalyzing the conversion of biliverdin to phycocyanobilin. Therefore, any PcyA capable of catalyzing the conversion of biliverdin to 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. PCC 6803, Cyanobacterial strains Nostoc commune TUBT05 and Oscillatoria okeni TISTR8549.

[0118] 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 set forth in SEQ ID NO: 2. However, those skilled in the art will appreciate that PcyA can have a variety of different sources and naturally occurring or artificially generated mutations. Therefore, the PcyA of the present application encompasses not only the PcyA set forth in SEQ ID NO: 2, but also PcyAs having different sources from SEQ ID NO: 2 or containing naturally occurring or artificially generated mutations.

[0119] As used herein, the term "identity" refers to the match 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 6 positions match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using 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.

[0120] 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 of residues physically or functionally similar to 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 the 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).

[0121] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material elements it carries to be 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 (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage; and animal viruses. 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 virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (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, a vector may also contain an initiation of replication site.

[0122] 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.

[0123] 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 a transcript, protein, or peptide, including proteins, isolated nucleic acid molecules, etc., as described herein.

[0124] In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, 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.

[0125] 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 are used interchangeably.

[0126] Advantageous Effects of the Invention

[0127] The chimeric protein provided in the present application can be efficiently and solublely expressed in host cells (e.g., Escherichia coli cells), and the expressed chimeric protein has excellent Ho1 enzyme activity and PcyA enzyme activity. It 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 field, such as difficulty, high cost, and long cycle.

[0128] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the 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

[0129] Figure 1: [A, B] Shows the three-dimensional structural model of heme oxygenase 1 (Ho1) and heme (top) and two schematic diagrams of the enzyme active site (bottom); [C, D] Shows the three-dimensional structural model of phycocyanobilin:ferredoxin oxidoreductase (PcyA) and biliverdin (top) and two schematic diagrams of the enzyme active site (bottom);

[0130] Figure 2: Three-dimensional structural model of the chimeric protein (Ho1-connector peptide-PcyA), wherein the connecting 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;

[0131] Figure 3: PCR schematic diagram of inserting the linker into the ho1-pcyA gene construct;

[0132] Figure 4: Schematic diagram of cloning of the ho1-linker-pcyA gene into the pET30a vector for further expressing a recombinant chimeric protein containing a linker peptide;

[0133] Figure 5: Schematic diagram of SDS-PAGE analysis of samples from the growth process before and after induction of the recombinant chimeric protein; (1) low molecular weight protein marker, (2) BL21 (DE3) E. coli transformed with pET30a before induction, (3) BL21 (DE3) E. coli transformed with pET-30a after induction, (4) E. coli BL21 (DE3) strain transformed with pET30a-ho1-pcyA before induction; after induction of recombinant chimeric protein expression, (5) pET30a-ho1-pcyA 1_AEK2_pcyA, (6)pET30a_ho1_AEK3_pcyA, (7)pET30a_ho1_AEK4_pcyA, (8)pET30a_ho1_AEK5_pcyA, (9)pET 30a_ho1_G6_pcyA, (10)pET30a_ho1_G7_pcyA, (11)pET30a_ho1_G8_pcyA, (12)pET30a_ho1_G9_pcyA, (13)p ET30a_ho1_G10_pcyA, (14)pET30a_ho1_(GGGGS)1_pcyA, (15)pET30a_ho1_(GGGGS)2_pcyA, (16)pET30a_h o1_(GGGGS)3_pcyA, (17)pET30a_ho1_(GGGGS)4_pcyA, (18)pET30a_ho1_(GGGGS)5_pcyA, (19)pET30a_ho1_ BL21(DE3) Escherichia coli transformed with (GGGGS)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;

[0134] Figure 6: Schematic representation of the representative antioxidant activity of PCBs extracted from Ho1-linker peptide-PcyA-positive BL21(DE3) E. coli cells;

[0135] Figure 7: Schematic diagram of the relationship between the expression level of the recombinant chimeric protein (Ho1-(GGGGGS)2 connecting peptide-PcyA) in E. 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) E. coli cells before induction, (3) recombinant E. coli cells expressing (Ho1-(GGGGGS)2 connecting peptide-PcyA) after 20 hours of growth, (4) E. coli cells before induction, (5) recombinant E. 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 E. coli cells expressing (Ho1-(GGGGGS)2 connecting peptide-PcyA);

[0136] FIG8 is 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. In the schematic diagram, each protein is represented by the connecting peptide it contains. DETAILED DESCRIPTION

[0137] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0138] 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., Molecular Biology: A Compendium of Laboratory Manuals, 3rd edition, John Wiley & Sons, Inc., 1995. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0139] Example 1 Design of a chimeric protein linking heme oxygenase 1 (Ho1) and phycocyanobilin:ferredoxin oxidoreductase (PcyA) by different linker peptides

[0140] 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 has facilitated experimental investigations of protein structure and function. Through molecular modeling and protein engineering, a chimeric protein for phycocyanobilin (PCB) synthesis was designed, simultaneously investigating the structural determinants of two enzymes involved in PCB synthesis. The chimeric protein consists of three modules.

[0141] The three modular structures of the chimeric protein are as follows:

[0142] (a) The first module is heme oxygenase 1 (Ho1), and the amino acid sequence of Ho1 in this example is SEQ ID NO: 1;

[0143] (b) The second module is a connecting peptide formed by helices of different lengths and structures, and the connecting peptide is selected from the following table:

[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 structural 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 their fusion into a single protein must ensure that the folding of both enzymes is not affected to achieve their functionality. Furthermore, the two enzymes must maintain a distance to effectively transport the compounds obtained by the enzymatic reaction to form PCBs. Therefore, forming a functional chimeric protein by linking these two enzymes is not a simple task.

[0148] Ho1 is a 27 kDa enzyme whose protein sequence comprises 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 No.: WP_010871494.1]. PcyA is a 28 kDa enzyme whose protein sequence comprises the first 248 amino acid residues encoded by the pcyA gene isolated from Synechocystis sp. (strain PCC 6803 / Kazusa) [NCBI Reference Sequence Accession No.: WP_010873881.1].

[0149] The construction of chimeric proteins involves linking two or more protein domains via a linker peptide (Chen X et al., 2013). The choice of linker peptide sequence is crucial for the construction of functional chimeric proteins. While the flexibility and hydrophilicity of the linker peptide are crucial, other peptides that form monomeric α-helical structures have been shown to not interfere with the functionality of bifunctional chimeric proteins (Arachi R et al., 2001).

[0150] The key to the present invention is the use of the connecting peptide to connect Ho1 and PcyA (Figure 1: 3D structural models of Ho1 and PcyA, and schematic diagram of the enzyme active site), modeling, and identifying regularities, thereby further screening for active recombinant chimeric proteins for use in PCB production. First, the structural models of Ho1 and PcyA were obtained using the fully automated protein structure homology modeling software (SWISS-MODEL), which can be accessed through the Expasy website (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) string / fold 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] The structural characterization of the constructed chimeric protein model is shown in Figure 2. Table 1 shows the extended geometric structural characteristics of the connecting peptide and its length. The length is calculated from the distance of the flanking α-carbon atom amino acids and ranges 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 demonstrated by the average distance between the active site cavities of the two enzymes Hol and PcyA. The specific method is as follows: the distance between the active site cavities of the two enzymes Hol and PcyA in 5 corresponding structural models is calculated using the Cavity Plus web server (http: / / www.pkumdl.cn:8000 / cavityplus / index.php) and the average value is obtained. The average value range is

[0152] Example 2 Cloning of genes for expressing bifunctional chimeric proteins in E. coli

[0153] The gene construct 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 included the connecting peptide designed in Example 1. The gene construct was then expressed in BL21(DE3) E. coli. The BL21(DE3) E. coli was incubated in Luria-Bertani (LB) medium, and expression of the recombinant chimeric protein was induced using ZY autoinduction medium or 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG).

[0154] To obtain all variants, the tandem ho1-pcyA gene (encoding a chimeric protein) was chemically synthesized using the method developed by Agarwal and his team (Agarwal KL et al, 1970). Oligonucleotides were prepared in solid phase by phosphoramidite chemistry for DNA synthesis (Beaucage SL et al, 1981), and DNA fragments flanked by BamH I-Xho I restriction sites were obtained and inserted into the pET-30a expression vector (Invitrogen Corp. USA) for expression under the control of the T7 promoter.

[0155] A two-round PCR strategy was used to generate the ho1-linker-pcyA variant, using primers containing the linker to be inserted between the ho1 and pcyA genes. Figure 3 provides a simplified schematic of the strategy. As shown in Figure 3, two rounds of PCR were performed. In the first round, three PCR experiments were performed: (A, B) primers or DNA fragments that insert the linker and flanking sequences of the ho1 and pcyA genes, respectively; and (C) primers or DNA fragments encoding the linker, which contain extensions of the ho1 and pcyA overhangs / flanking ends to be filled in, were used. In the second round, the purified products from the three PCR experiments (A, B, and C) were used as templates for the next PCR experiment, using primers flanking the ho1 and pcyA genes and containing restriction enzyme sites for further cloning.

[0156] Table 2 lists the primers used for PCR amplification of the gene constructs. Based on the sequence code in 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-terminus-linker, (5) 5'ho1 C-terminus-linker, and (6) 5'RC linker-pcyA. All primer sequences are in the 5' to 3' direction, and RC represents the reverse complement sequence of the specific primer. The gene constructs with the linkers inserted were sequenced to verify their integrity and sequence.

[0157] 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.

[0158] Table 2 DNA primers used to insert the linker between ho1 and pcyA genes in PCR experiments

[0159] All experimental products were transformed into XL-1-Blue E. coli strains. DNA from individual colonies was purified and analyzed by restriction enzyme digestion. An automated sequencer was then used to sequence the colony DNA that matched the desired pattern for each gene construct, screening a representative clone for each gene construct. Selected clones were transformed into BL21 (DE3) E. coli strains for protein expression. A general cloning method is shown in Figure 4.

[0160] Transformed BL21(DE3) E. coli strains were cultured in LB medium, and expression of the recombinant chimeric protein was induced using either 1 mM IPTG or ZY autoinduction medium. IPTG was used for induction in small-scale screening, while ZY autoinduction medium was used for induction in large-scale screening. First, recombinant BL21(DE3) E. coli strains transformed with the linker-containing gene construct were cultured on LB plates containing 50 μg / mL kanamycin and 1% glucose at 37°C for 16 hours to inhibit expression of the recombinant chimeric protein. Subsequently, the culture was inoculated at an initial optical density of 0.1 and incubated for 20 hours. The culture was then centrifuged, the cells harvested, washed with phosphate buffer (pH 7.0), and centrifuged again before PCB extraction. Expression of the recombinant chimeric protein is shown in Figure 5. SDS-PAGE analysis of the entire recombinant E. coli cell population revealed expression of the recombinant chimeric protein in all experiments, with expression levels ranging from 5% to 30% of the total cellular protein content, sufficient to catalyze the conversion of heme to biliverdin and ultimately to PCBs in the multi-stage reaction.

[0161] For PCB analysis, the cell pellets of 1 L of recombinant Escherichia coli strain expressing a recombinant chimeric protein containing a 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 cells were centrifuged at 8000 × g for 10 minutes, the cell pellet was removed, the supernatant was collected, and chloroform-water extraction was performed. 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 ABL, 1973). The equation "c[mM]=Abs 680nm PCB concentration was calculated using the formula “ / 37.9×dilution factor” (Glazer AN et al, 1973; Ge B et al, 2013). Table 3 shows the results of PCB production by recombinant E. coli cells.

[0162] As an essential component of the recombinant chimeric protein described herein, the linker peptide plays a crucial role in obtaining stable and biologically active PCB molecules. This invention effectively demonstrates the relationship between protein structure and function. Not all linker peptides used to construct recombinant chimeric proteins will generate functional molecules, and thus PCBs. The selected linker peptides can have a certain impact on the expression level of the recombinant chimeric protein. Furthermore, the overall solubility of the recombinant chimeric protein and the folding of the individual domains it contains may be affected by the linker peptide.

[0163] For the high-PCB-producing recombinant E. coli strain variants, the average value of PCB production in culture flasks ranged from 2.5 ± 0.21 mg / L culture, where the chimeric proteins 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, the short connecting peptide (extended geometric length is ) and long linker peptides (extended geometric length is ) in the E. coli strain, PCBs were not produced. The short linker peptide and the long linker peptide shortened the distance between the active sites of the Ho1 and PcyA enzymes to and Failure to generate PCBs may be due to improper folding or suboptimal distance between the two enzyme active sites, which is not conducive to the dual enzymatic reaction for PCB generation. Generally speaking, recombinant E. coli strains producing chimeric proteins containing AEK2, AEK3, AEK4, G10, (GGGGS)2, (GGGGS)3, and a (GS)5 linker peptide have higher PCB yields.

[0164] Table 3 PCB production of E. coli strains expressing Ho1-connector peptide-PcyA recombinant chimeric protein

[0165] Example 3 Antioxidant activity of PCBs produced by recombinant Escherichia coli strains

[0166] Antioxidant capacity is a key factor in many human diseases and health impairments. A quick, simple, and inexpensive method for assessing antioxidant activity is the use of 2,2-diphenyl-1-picrylhydrazyl (DPPH). DPPH is a stable free radical that acts as a hydrogen radical scavenger (Kedare SB et al., 2011).

[0167] In the test, the dark blue DPPH free radical extracts a hydrogen atom in a single-electron reaction to produce light yellow DPPH-H. The color change is associated with a decrease in absorbance at 517nm. At the same time, the decrease in absorbance at 517nm is directly proportional to the antioxidant capacity of the sample. DPPH free 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 test sample was mixed with 100 μL of 0.1 mM DPPH, vigorously shaken for 30 minutes, and then the absorbance at 517nm was measured using a microplate 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.

[0168] The PCBs obtained in the E. coli cells described in Example 2 were purified. E. coli cells expressing recombinant chimeric proteins containing the connecting peptides AEK2, AEK3, AEK4, G10, (GGGGS)2, (GGGGS)3 and (GS)5 had a higher PCB yield. After purification, the PCBs obtained from the above E. coli cells were 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 6 shows a schematic diagram of the DPPH free radical scavenging rate curve of PCBs produced by E. coli cells containing the connecting peptide (GGGGS)2. Within the PCB concentration range of 1.9 to 1000 μg / mL, the free radical scavenging rate was 12.5% ​​to 92.3%, indicating that the PCBs have excellent antioxidant activity and meet the requirements of industrial and biopharmaceutical applications.

[0169] Example 4 Correlation between recombinant chimeric protein expression level and PCB yield

[0170] The gene construct was transformed into Escherichia coli to produce a recombinant chimeric protein containing a (GGGGS)2 linker peptide. The gene construct was used to analyze the relationship between the expression level of the recombinant chimeric protein and PCB yield, and the results showed that the gene construct was one of the gene constructs with high PCB yield.

[0171] Figure 7 shows the expression levels of the recombinant chimeric protein when the recombinant E. coli strain was cultured in ZY autoinduction medium. Cell growth was stopped at two different time points: after 12 hours of incubation (variant 1), when the culture had an optical density of 1.8; and after 20 hours of incubation (variant 2), when the culture had an optical density of 5.92. The expression levels of the recombinant chimeric protein at these two growth stages were 13% and 36%, respectively. PCBs were purified from a 1L culture, yielding 0.015 mM (0.009 mg / mL) and 0.053 mM (0.031 mg / mL) of PCBs under each condition, respectively. Testing revealed a clear linear correlation between the expression level of the recombinant chimeric protein and the PCB yield of the E. coli cells. In this experiment, the same gene expression variant (Ho1-(GGGGS)2 linker peptide-PcyA) was used to generate functional chimeric proteins. Therefore, the impact of protein folding on PCB yield was minimized during comparison.

[0172] Example 5 Correlation between the solubility of recombinant chimeric proteins and PCB production

[0173] The main goal 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 have been detected in bacterial cells (Hartl FU et al, 2002). Usually, only folded and soluble proteins are functional, while proteins do not acquire 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).

[0174] The recombinant E. coli strains were cultured in ZY autoinduction medium for 20 hours to induce expression of the recombinant chimeric protein. Then, 1 L of culture was taken from each strain and centrifuged to collect the cells. Half of the cells were used to purify PCBs (refer to Example 2 for the purification method), and the other half were used to study the solubility of the chimeric protein. The E. coli cells were then disrupted by ultrasound, and the soluble and insoluble components were separated by centrifugation. The recombinant chimeric protein content in the soluble component of the disrupted cells was compared with the recombinant chimeric protein content in the insoluble component of the disrupted cells by SDS-PAGE analysis. All recombinant chimeric proteins had excellent solubility, exceeding 75%, as shown in Figure 8. However, it was observed that the PCB yields of different recombinant E. coli strains varied. Together with the results obtained in Example 4, this indicates that the folding structure of the resulting 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.

[0175] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

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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