A recombinant macrobrachium rosenbergii lipocalin-based pigment complex and in vitro preparation method and application thereof

Abstract

The application belongs to the technical field of biological pigment reconstruction and protein engineering, and particularly relates to a pigment complex based on recombinant macrobrachium rosenbergii lipid carrier protein as well as an in-vitro preparation method and application thereof. The formation of the pigment complex relies on the synergistic stabilization of a key residue network of the recombinant macrobrachium rosenbergii lipid carrier protein, a pigment characteristic end group and phosphatidyl ethanolamine. The application has important scientific significance and industrial value for revealing the protein-pigment-lipid ternary interaction rule, constructing a thermal response color-changing system, expanding the application of protein-based pigment materials and providing a technical basis for color biomimetic construction in food and other systems.

Description

Technical Field

[0001] This invention belongs to the field of biopigment reconstructing and protein engineering technology, specifically relating to a pigment complex based on recombinant giant freshwater shrimp lipid transport protein and its in vitro preparation method and application. Background Technology

[0002] Color is one of the core sensory attributes of consumer products such as food, beverages, cosmetics, and functional materials, directly influencing consumer preferences and product market value. With the increasing demand for natural and healthy products, developing pigment systems that are naturally derived, rich in color, and stable has become an important research direction in related fields.

[0003] However, in practical applications, constructing a pigment system that is both naturally sourced, edible, and capable of long-term stability still faces significant technical bottlenecks, which limits the widespread application of natural pigment systems in high-end foods, personalized cosmetics, and functional materials.

[0004] While commonly available natural pigments are safe to source, they generally suffer from limitations such as limited color range, poor stability, and susceptibility to pH, light, and temperature fluctuations. For example, astaxanthin, a common fat-soluble pigment, has poor solubility in water and only exhibits a single orange-red color. In contrast, while synthetic pigments offer vibrant colors and high stability, their potential health risks and increasingly stringent legal regulations are limiting their application.

[0005] Therefore, developing a richer variety of natural pigment molecules and constructing protein-pigment composite systems that are rich in color, highly stable, and safe and reliable are of great research value and industrialization potential for constructing thermo-responsive color-changing systems, expanding the application of protein-based pigment materials, and providing a technical foundation for the biomimetic construction of colors in food and other systems. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a mutant of the lipid transport protein rMrLC.

[0007] This invention provides a mutant of the lipid transporter rMrLC, which, based on the wild-type lipid transporter rMrLC, has one or more of the following amino acid mutation sites: position 57, position 91, or position 93.

[0008] These three amino acid sites (Tyr57, Asp91, and His93) play a crucial role in the binding of the lipid transport protein rMrLC to pigment molecules, collectively constituting the decisive region regulating pigment formation and its spectral properties. Site-directed mutagenesis of these sites can significantly affect the pigment-protein binding pattern, inducing color expressions with different spectral characteristics. Experimental results show that Tyr57, Asp91, and His93 together determine the formation of a significant redshift, allowing for tunable variations in pigment color from blue-green to red.

[0009] In some embodiments, the 57th mutation site includes: Y57F or Y57F; and / or

[0010] The 91st mutation site includes: D91A or D91E; and / or

[0011] The 93rd mutation site includes either H93Y or H93A.

[0012] In some embodiments, the mutation sites of the mutant include Y57F, or Y57F, or D91A, or D91E, or H93Y, or H93A, or Y57H and H93Y, or D91A and H93A, or Y57F and D91A, or Y57F and H93A.

[0013] Through systematic screening and verification, compared with other mutation methods, the above-mentioned mutation sites, by altering the charge, polarity, or spatial configuration of key amino acids, can more precisely regulate the interaction between pigments and proteins, resulting in pigments exhibiting a series of characteristic spectra such as pink, magenta, and purple, thereby achieving targeted regulation of spectral color. The mutants exhibit excellent spectral tunability and color diversity, providing a wealth of molecular tools for the design of lipid-carrying protein-based optical biological probes and the development of functional pigment materials.

[0014] In some embodiments, the amino acid sequence of the wild-type lipid transport protein rMrLC is shown in SEQ ID NO:1.

[0015] In some embodiments, the amino acid sequence of the lipid transporter rMrLC mutant is shown in any one of SEQ ID NO:2~12.

[0016] The present invention also provides biomaterials, comprising at least one of the following: A) to D)

[0017] A) Nucleic acid encoding the rMrLC mutant lipid transport protein as described above;

[0018] B) Expression units containing nucleic acids as described in A);

[0019] C) A recombinant vector, comprising a backbone vector and a nucleic acid as described in A) or an expression unit as described in B);

[0020] D) Host cells, which are transformed or transfected with plasmid vectors as described in C), or whose genomes are integrated with nucleic acids as described in A) or expression units as described in B).

[0021] In some embodiments, the nucleic acid encoding the rMrLC mutant lipid transporter has a nucleic acid sequence as shown in any one of SEQ ID NO:14-23, or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity with the nucleic acid sequence as described above.

[0022] In some embodiments, the nucleic acid encoding the wild-type lipid transport protein rMrLC has a nucleic acid sequence as shown in SEQ ID NO:13, or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity with the nucleic acid sequence as described above.

[0023] The nucleic acid sequences were optimized by removing the N-terminal signal peptide, codon optimization, and adjusting the GC content, significantly improving their expression efficiency in the host system. Specifically, removing the N-terminal signal peptide avoids misidentification and ineffective cleavage of the original signal peptide in the prokaryotic expression system, reducing the risk of product degradation; codon optimization adjusts rare codons according to host preferences, improving translation elongation efficiency and accuracy; and moderate adjustment of the GC content helps stabilize the mRNA secondary structure, reducing the occurrence of premature transcription termination. Experiments show that the nucleic acid sequences shown in any one of SEQ ID NO:14~23 can achieve higher expression levels when used to encode the rMrLC mutant lipid transport protein as described above.

[0024] The expression unit described in this invention includes a promoter, a nucleic acid as described above, and a terminator.

[0025] In some embodiments, regulatory fragments may be included on either side of or between the promoter and terminator. These regulatory fragments may include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites operatively linked to the nucleic acid molecule, such as promoter enhancers and poly(A) signals. It should be noted that the types, numbers, and order of these elements can be adjusted according to expression requirements, and each element can be selected from various alternative forms known in the art. All functional vector constructs formed by different combinations of these elements fall within the scope of protection of this invention.

[0026] The recombinant vectors described in this invention may also include conventional functional elements in the art, including but not limited to promoters, enhancers, terminators, polyA tails, selection marker genes, reporter genes, origins of replication, ribosome entry sites, and other sequences used to enhance expression or achieve regulation. It should be noted that the type, number, and order of the above elements can be adjusted according to expression requirements, and each element can be selected from various alternative forms known in the art. All functional vector constructs formed by different combinations of these elements fall within the protection scope of this invention.

[0027] The recombinant vector can be constructed using recombinant DNA technology, in vitro transcription, or chemical synthesis methods, and can be used to store and amplify the nucleic acid or expression unit in prokaryotic or eukaryotic host cells, or to express the target protein. The present invention does not limit the specific use.

[0028] In some embodiments, the backbone vector is a pET series vector. For example, the pET series vectors include, but are not limited to, pET-28a(+), pET-30a(+), or pET-32a(+). Specifically, the backbone vector is pET-28a(+), which carries a His tag that facilitates subsequent protein purification, and its multiple cloning site and regulatory elements are suitable for the construction and efficient expression of wild-type and mutant rMrLC.

[0029] The host cells described in this invention can be derived from at least one of plants, animals, bacteria, and fungi. These host cells can be used to express, replicate, package, or maintain exogenous genetic material.

[0030] In some embodiments, the host cell is *Escherichia coli*. Exemplarily, the *E. coli* includes the BL21 series and its derivatives, such as BL21(DE3), BL21(DE3)pLysS, or Rosetta(DE3). Specifically, the host cell is preferably *E. coli* BL21(DE3), which carries the λ phage DE3 lysogen, inducing highly efficient expression driven by the T7 promoter, suitable for pET series vector-mediated production of rMrLC mutant proteins.

[0031] Furthermore, the present invention provides a coloring composition comprising, as previously described, a rMrLC mutant lipid transporter, a pigment, and phospholipids, or comprising, or wild-type rMrLC lipid transporter, a pigment, and phospholipids.

[0032] In some embodiments, the pigment includes astaxanthin, carotenoids, canthaxanthin, lutein, zeaxanthin, β-carotene, fucoxanthin, or urchinone. Specifically, the pigment is astaxanthin.

[0033] In some embodiments, the phospholipid includes ceramide, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, or cardiolipin. Specifically, the phospholipid is phosphatidylethanolamine.

[0034] Experiments have shown that astaxanthin, when combined with the lipid transport protein mutant, can greatly improve its solubility and dispersibility in aqueous solution. Furthermore, the lipid transport protein mutant can induce a red shift in astaxanthin, resulting in a color different from its free state.

[0035] Phospholipids are cofactors that stabilize the spatial conformation of the coloring composition. Compared to other phospholipid molecules, phosphatidylethanolamine (PE), located between two pigment molecules, can more effectively stabilize the characteristic spatial conformation of the coloring composition. This plays an irreplaceable role in maintaining its unique color state and achieving a significant red shift in the absorption spectrum.

[0036] In some embodiments, the maximum visible light absorption peak of the coloring composition is located in the range of 500-600 nm. For example, the maximum visible light absorption peak of the coloring composition is located at 500, 510, 520, 530, 540, 550, 560, 570, 580, 590 or 600 nm.

[0037] In some embodiments, the maximum visible light absorption peak of the blue pigment composition rMrLC-AXT-PE, formed by the co-assembly of wild-type lipid transport protein rMrLC, astaxanthin, and phosphatidylethanolamine, is located at 570 nm.

[0038] In some specific embodiments, the visible light maximum absorption peak of the rose-red pigment composition co-assembled with the lipid transport protein rMrLC mutant Y57F, astaxanthin, and phosphatidylethanolamine is located at 545 nm.

[0039] In some specific embodiments, the visible light maximum absorption peak of the purple pigment composition formed by the co-assembly of the lipid transport protein rMrLC mutant D91A, astaxanthin, and phosphatidylethanolamine is located at 555 nm.

[0040] In other specific embodiments, the maximum visible light absorption peak of the pink pigment composition formed by the co-assembly of the lipid transport protein rMrLC mutant Y57F-D91A, astaxanthin, and phosphatidylethanolamine is located at 530 nm.

[0041] In some embodiments, the coloring composition has a core-shell structure. Specifically, the blue pigment composition is formed by the co-assembly of one molecule of lipid carrier protein, two molecules of astaxanthin, and one molecule of phosphatidylethanolamine; wherein the two molecules of astaxanthin and the one molecule of phosphatidylethanolamine form a "sandwich-like" assembly core, which is wrapped by the lipid carrier protein at its dimer interface.

[0042] In some specific embodiments, the rose-red pigment composition is a complex formed by the co-assembly of the rMrLC mutant Y57F homodimer, two molecules of astaxanthin, and one molecule of phosphatidylethanolamine.

[0043] In some specific embodiments, the blue pigment composition is a co-assembly formed by rMrLC homodimer, two molecules of astaxanthin, and one molecule of phosphatidylethanolamine; in the blue pigment composition, the Tyr57, Asp91, and His93 sites form a stable hydrogen bond network with the terminal 3-hydroxyl and / or 4-keto groups of astaxanthin, thereby facilitating the achievement of a characteristic absorption peak of approximately 570 nm and a blue phenotype.

[0044] In some specific embodiments, the blue pigment composition is blue under aqueous conditions at room temperature, and its maximum absorption peak for visible light is located at 550~590 nm; preferably, the maximum absorption peak for visible light is located at 565~575 nm, and exhibits a red shift of about 100~150 nm, preferably about 130 nm, relative to the absorption peak of free astaxanthin at about 440 nm; the blue pigment composition changes from blue to orange-red after being heated at 60~80 °C for 5~20 min.

[0045] Crystal structure analysis shows that the obtained blue pigment composition preferably has an astaxanthin-phosphatidylethanolamine-astaxanthin core structure sandwiched by open-state rMrLC homodimers, wherein phosphatidylethanolamine is located between two astaxanthin molecules and participates in maintaining its spatial arrangement and conformational stability required for significant redshift.

[0046] Furthermore, the present invention also provides a method for preparing the coloring composition as described above, which is prepared from a rMrLC mutant lipid transporter, a pigment, and phospholipids, or from wild-type rMrLC lipid transporter, a pigment, and phospholipids. The present invention does not limit the preparation method, as long as it enables the effective assembly of the rMrLC mutant lipid transporter, the pigment, and phospholipids, or the effective assembly of wild-type rMrLC lipid transporter, the pigment, and phospholipids.

[0047] Wild-type lipid transport proteins or lipid transport protein mutants can be obtained through various expression systems. For example, when expressed using the pET-28a vector, the target protein exists in the form of inclusion bodies; while when expressed using the pET-22b vector, a soluble protein in an extracellular secretory form can be obtained. This invention provides flexible assembly strategies for different protein forms:

[0048] For proteins in the form of inclusion bodies, pigments can be added during their refolding process;

[0049] Alternatively, the inclusion body protein can be refolded first, and then the pigment can be added;

[0050] For soluble proteins, pigments can be added directly.

[0051] All of the above methods can achieve effective binding of proteins and pigments to form stable coloring compositions.

[0052] In some embodiments, the preparation method includes:

[0053] Obtain wild-type lipid transporter or lipid transporter mutant;

[0054] After being refolded by stepwise dialysis with urea, pigments and phosphatidylethanolamine are added;

[0055] After removing urea, the coloring composition was obtained by dialysis.

[0056] In some embodiments, the wild-type lipid transporter or lipid transporter mutant is obtained through prokaryotic expression, inclusion body purification, and stepwise dialysis refolding.

[0057] In some embodiments, the conditions for prokaryotic expression include: culturing in LB medium at 37 °C until OD. 600 After adjusting the concentration to 0.6-0.7, 0.2 mM IPTG was added to induce expression overnight, allowing rMrLC to accumulate in the form of inclusion bodies.

[0058] In some embodiments, the conditions for purifying the inclusion bodies include:

[0059] Washed with a washing buffer containing 25 mM Tris, 5 mM EDTA, 1 mM PMSF and 2 M urea at pH 8.0; dissolved in a denaturing solution containing 10 M urea, 25 mM Tris, 1 mM PMSF and 4 mM β-mercaptoethanol at pH 8.0; purified by Ni²⁺ affinity chromatography; the purified eluent contained 25 mM Tris-HCl, 8 M urea, 4 mM β-mercaptoethanol and 500 mM imidazole at pH 8.0.

[0060] In some embodiments, the conditions for the stepwise dialysis refolding include:

[0061] The refolding buffer contains 25 mM Tris-HCl, 1.0 mM PMSF, 50 mM glycine, and 5 mM EDTA, with a pH of 8.0.

[0062] The urea concentration was gradually decreased in gradients of 6.0 M, 4.0 M, 3.0 M, 2.0 M, 1.0 M and 0 M, with each gradient requiring dialysis for 24 hours.

[0063] The urea concentration was reduced to 3.0M, and astaxanthin and phosphatidylethanolamine were added.

[0064] In some embodiments, the astaxanthin and phosphatidylethanolamine are preferably pre-prepared as a DMSO stock solution, and the astaxanthin and phosphatidylethanolamine are preferably added in the form of a solution or dispersion.

[0065] In some embodiments, the molar ratio of the recombinant lipotransferase or its mutant protein, astaxanthin, and phosphatidylethanolamine is 1:(3~10):(0.5~3). For example, the molar ratio of the recombinant lipotransferase or its mutant protein, astaxanthin, and phosphatidylethanolamine is 1:3:0.5, 1:5:1, 1:7:2, or 1:9:3. Specifically, the molar ratio of the recombinant lipotransferase or its mutant protein, astaxanthin, and phosphatidylethanolamine is 1:5:1.

[0066] In some embodiments, the dialysis is performed using deionized water or a 25 mM sodium phosphate buffer solution containing 150 mM NaCl at pH 7.5.

[0067] Furthermore, the present invention also provides the application of the rMrLC mutant of the lipid transport protein as described above, the biomaterial as described above, the chromogenic composition as described above, or the chromogenic composition obtained by the preparation method as described above in the study of pigment formation mechanism or the preparation of pigment products.

[0068] Furthermore, the present invention also provides pigment products comprising the rMrLC mutant of the lipid transporter as described above, the biological material as described above, the coloring composition as described above, or the coloring composition obtained by the preparation method as described above.

[0069] In some embodiments, the types of pigment products include thermo-responsive color-changing materials, temperature-responsive indicator materials, protein-based pigment formulations, biomimetic optical materials, food colorants, cosmetic pigments, bioimaging probes, or light-harvesting functional materials.

[0070] Furthermore, the present invention also provides cosmetics, food or health products comprising the rMrLC mutant of the lipid transporter as described above, the biological material as described above, the coloring composition as described above, or the coloring composition obtained by the preparation method as described above.

[0071] Compared with the prior art, the present invention has at least the following beneficial effects:

[0072] (1) Compared with existing studies that only reveal the binding relationship between MrLC and astaxanthin, this invention provides for the first time a technical route for constructing a stable blue pigment complex in an in vitro aqueous system using recombinant rMrLC, astaxanthin and phosphatidylethanolamine, which can directly obtain observable blue products, rather than just staying at the level of in vivo correlation or general ligand binding.

[0073] (2) The complex obtained by the present invention is blue at room temperature with a maximum absorption peak of about 570 nm, which is a significant red shift of about 130 nm compared to the absorption peak of about 440 nm of free astaxanthin. After heating at 70 °C for 10 min, it turns orange-red, showing the technical effect of having both color development and thermal responsiveness.

[0074] (3) Crystal structure analysis shows that the complex obtained in this invention is not a simple protein-pigment binary complex, but a co-assembly of a rMrLC homodimer sandwiching an AXT-PE-AXT ternary core; wherein, one PE molecule is located between two AXT molecules and participates in stabilizing its spatial conformation, revealing a previously unknown lipid cofactor action mode.

[0075] (4) The key site mutation experiment showed that Tyr57, Asp91 and His93 jointly determined the formation of a significant red shift; among them, the maximum absorption peak of the Y57A system dropped to about 530 nm, the D91A and H93A systems were both about 555 nm, the Y57F system was about 545 nm, and the three mutants almost no longer showed AXT-related visible absorption peaks, indicating that the formation of the blue pigment in this invention depends on a specific residue network and has obvious structural specificity.

[0076] (5) By performing single or multiple mutations on key sites Tyr57, Asp91 and His93, lipid transport protein-astaxanthin-phospholipid co-assembled compounds with different colors can be prepared.

[0077] (6) The carotenoid comparison experiment showed that replacing astaxanthin with canthaxanthin only yielded a deep pink system of about 530 nm, while zeaxanthin and lutein could not form blue pigments. This indicates that the technical effect of the present invention cannot be obtained by simply combining any carotenoid with rMrLC / phosphatidylethanolamine.

[0078] (7) QM / MM analysis further showed that after removing PE, the distance between the two AXT molecules shrank from about 7.7 Å to about 5.1 Å, the protein backbone Cα RMSD increased to about 1.4 Å, and the AXT polyene chain bent inward, proving that PE has an important structural stabilizing effect on maintaining the conformation of blue pigment and significant red shift. This effect is not something that a person skilled in the art could directly expect from the prior art.

[0079] This invention provides a pigment complex based on recombinant giant freshwater prawn lipid transport protein, its in vitro preparation method, and its applications. By specifically assembling recombinant giant freshwater prawn lipid transport protein (wild-type or mutant), pigment, and phosphatidylethanolamine into a ternary structure, a pigment complex was successfully constructed in an aqueous in vitro system. Its significant redshift and thermoresponsive properties depend on the synergistic stabilizing effect of a specific residue network and phosphatidylethanolamine. This pigment complex can be applied to the study of the formation mechanism of blue pigments in crustaceans, the construction of thermoresponsive color-changing materials, and the development of pigment-protein-phospholipid synergistic assembly systems. It also provides a new technical approach for the construction of blue biomimetic materials in the food and related fields. Attached Figure Description

[0080] Figure 1 This shows the distribution of the frequency of codon usage.

[0081] Figure 2 The result is from adjustments made for GC content;

[0082] Figure 3 This is a schematic diagram of the expression, purification, and stepwise dialysis refolding process of the recombinant giant freshwater shrimp lipid transport protein rMrLC in this invention;

[0083] Figure 4 This is a schematic diagram of the process for completing the in vitro reconstruction of blue pigment after the directional addition of astaxanthin and phosphatidylethanolamine at the 3 M urea node in this invention.

[0084] Figure 5 This is a schematic diagram showing the color change and visible absorption characteristics of the blue pigment complex obtained in this invention.

[0085] Figure 6 This is a schematic diagram illustrating the differences in color and spectrum of the system obtained when different carotenoids are used for comparative reconstruction.

[0086] Figure 7 Color changes of pigment complexes prepared for wild-type and mutant lipid transport proteins;

[0087] Figure 8 Schematic diagram of visible absorption characteristics of pigment complexes prepared for wild-type and mutant lipid transport proteins;

[0088] Figure 9 This is a schematic diagram of the structure of Apo rMrLC;

[0089] Figure 10 A crystal photograph of the wild-type blue pigment complex (rMrLC-AXT-PE);

[0090] Figure 11 A schematic diagram of the crystal structure of the wild-type blue pigment complex (rMrLC-AXT-PE);

[0091] Figure 12 A crystal photograph of the rMrLC-CAN complex;

[0092] Figure 13 This is a schematic diagram of the crystal structure of the rMrLC-CAN complex;

[0093] Figure 14 This is a schematic diagram of the crystal structure of the Y57F-AXT-PE composite.

[0094] Figure 15 This is a magnified view of a portion of the wild-type rMrLC-AXT-PE crystal structure.

[0095] Figure 16 Comparative diagram of the crystal structures of wild-type Apo rMrLC and rMrLC-AXT-PE composite;

[0096] Figure 17 Analysis results of the effect of PE removal on the structure calculated by QM / MM (a. IGMH analysis showed that there are van der Waals forces between PE and the two astaxanthin molecules; b. QM / MM simulation showed that the effect of removing PE molecules on the protein backbone was small; c. QM / MM simulation showed that after removing PE molecules, the distance between the two astaxanthin molecules decreased from 7.7 Å to 5.1 Å, and the two astaxanthin molecules could no longer maintain the antiparallel assembly mode). Detailed Implementation

[0097] This invention provides a pigment complex based on recombinant giant freshwater prawn lipid transport protein, its in vitro preparation method, and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the same result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0098] Unless otherwise defined in this invention, scientific and technical terms related to this invention shall have the meanings understood by one of ordinary skill in the art.

[0099] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0100] When used herein, the term “and / or” includes the meaning of “and,” “or,” and “all or any other combination of elements linked by the term.”

[0101] The term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0102] The terms "vector" and "plasmid" are used interchangeably in this invention to refer to DNA molecules that can be used to carry, replicate, and express exogenous nucleic acid sequences. It should be understood that when "plasmid" is used in the description, it is covered within the scope of "vector" as described in this invention; similarly, when "vector" is mentioned, its specific embodiments include, but are not limited to, plasmid forms, and may also be viral vectors, artificial chromosomes, or other nucleic acid constructs suitable for gene manipulation. This interchangeability of terms does not constitute a limitation on the scope of the technical solution.

[0103] Recombinant Macrobrachium rosenbergii Lipocalin (rMrLC) is a class of proteins that can transport hydrophobic small molecules such as fatty acids, vitamins, and pheromones.

[0104] Phosphatidylethanolamine (PE) is an important phospholipid and one of the main components of biological membranes, often referred to as "brain phospholipid." It plays a crucial role in the structure and function of cell membranes and participates in various cellular processes, such as membrane fusion, cell division, and autophagy.

[0105] Astaxanthin (AXT) is a class of carotenoids widely found in crustaceans, and its free state is usually orange-red. When astaxanthin enters a specific protein microenvironment and forms a stable complex system, a significant visible absorption redshift occurs, resulting in a blue or blue-green pigment complex that imparts a unique body color to crustaceans. This natural phenomenon provides important inspiration for the biomimetic construction of protein-based pigment materials.

[0106] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0107] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0108] This invention discloses a pigment complex based on recombinant giant freshwater prawn lipid transport protein, its in vitro preparation method, and its application, belonging to the field of biopigment reconstructing and protein engineering technology. The method includes: constructing and expressing the MrLC gene with the N-terminal signal peptide removed to obtain rMrLC in inclusion body form, followed by washing, denaturation dissolution, and Ni... 2+ Affinity purification and stepwise dialysis were used to obtain a protein solution. During the stepwise dialysis process, when the urea concentration decreased to 3 M, astaxanthin and phosphatidylethanolamine were added to the protein solution under light-protected conditions. Dialysis and refolding continued, and the protein-astaxanthin-phospholipid co-assembly was completed, resulting in a blue pigment complex that appears blue at room temperature. The obtained complex preferably has an AXT-PE-AXT ternary core structure held by rMrLC homodimers, with its maximum absorption peak located at 550-590 nm, preferably around 570 nm, exhibiting a significant red shift of approximately 130 nm relative to free astaxanthin, and showing a thermal response characteristic of changing from blue to orange-red upon heating. Comparative analysis of key site mutants, carotenoid analogs, and the crystal structures of different complexes showed that the formation of the blue pigment described in this invention depends on the synergistic stabilizing effect of the rMrLC key residue network, the unique terminal groups of astaxanthin, and phosphatidylethanolamine, and cannot be expected to be obtained by simply mixing components.

[0109] The amino acid sequences of the wild-type and mutant lipid transport proteins of the giant freshwater prawn in this invention are as follows:

[0110] >WT protein sequence(SEQ ID NO:1)

[0111] MDEVPDFVIKGKCPAVDEQRLWQQQLPRHSSFGGVWYQQAISTNPYQLLKKCVRIQYDYNGKGFDVKTAGITPEGNQLKRKGVLTPMPLGDPHLMINLENSFPAPLVILDTDYNNYACMYSCMDYNYGYHSDFAFFFARAPEAYDKYIAKCRAAFDSIGVDSNRLIKTEQGRGCDYEQLTKLIKDEL*

[0112] >Y57F (SEQ ID NO:2)

[0113] MDEVPDFVIKGKCPAVDEQRLWQQQLPRHSSFGGVWYQQAISTNPYQLLKKCVRIQFDYNGKGFDVKTAGITPEGNQLKRKGVLTPMPLGDPHLMINLENSFPAPLVILDTDYNNYACMYSCMDYNYGYHSDFAFFFARAPEAYDKYIAKCRAAFDSIGVDSNRLIKTEQGRGCDYEQLTKLIKDEL*

[0114] >D91E(SEQ ID NO:3)

[0115] MDEVPDFVIKGKCPAVDEQRLWQQQLPRHSSFGGVWYQQAISTNPYQLLKKCVRIQYDYNGKGFDVKTAGITPEGNQLKRKGVLTPMPLGEPHLMINLENSFPAPLVILDTDYNNYACMYSCMDYNYGYHSDFAFFFARAPEAYDKYIAKCRAAFDSIGVDSNRLIKTEQGRGCDYEQLTKLIKDEL*

[0116] >D91A(SEQ ID NO:4)

[0117] MDEVPDFVIKGKCPAVDEQRLWQQQLPRHSSFGGVWYQQAISTNPYQLLKKCVRIQYDYNGKGFDVKTAGITPEGNQLKRKGVLTPMPLGAPHLMINLENSFPAPLVILDTDYNNYACMYSCMDYNYGYHSDFAFFFARAPEAYDKYIAKCRAAFDSIGVDSNRLIKTEQGRGCDYEQLTKLIKDEL*

[0118] >H93A(SEQ ID NO:5)

[0119] MDEVPDFVIKGKCPAVDEQRLWQQQLPRHSSFGGVWYQQAISTNPYQLLKKCVRIQYDYNGKGFDVKTAGITPEGNQLKRKGVLTPMPLGDPALMINLENSFPAPLVILDTDYNNYACMYSCMDYNYGYHSDFAFFFARAPEAYDKYIAKCRAAFDSIGVDSNRLIKTEQGRGCDYEQLTKLIKDEL*

[0120] >Y57H(SEQ ID NO:6)

[0121] MDEVPDFVIKGKCPAVDEQRLWQQQLPRHSSFGGVWYQQAISTNPYQLLKKCVRIQHDYNGKGFDVKTAGITPEGNQLKRKGVLTPMPLGDPHLMINLENSFPAPLVILDTDYNNYACMYSCMDYNYGYHSDFAFFFARAPEAYDKYIAKCRAAFDSIGVDSNRLIKTEQGRGCDYEQLTKLIKDEL*

[0122] >H93Y(SEQ ID NO:7)

[0123] MDEVPDFVIKGKCPAVDEQRLWQQQLPRHSSFGGVWYQQAISTNPYQLLKKCVRIQYDYNGKGFDVKTAGITPEGNQLKRKGVLTPMPLGDPYLMINLENSFPAPLVILDTDYNNYACMYSCMDYNYGYHSDFAFFFARAPEAYDKYIAKCRAAFDSIGVDSNRLIKTEQGRGCDYEQLTKLIKDEL*

[0124] >Y57H-H93Y(SEQ ID NO:8)

[0125] MDEVPDFVIKGKCPAVDEQRLWQQQLPRHSSFGGVWYQQAISTNPYQLLKKCVRIQHDYNGKGFDVKTAGITPEGNQLKRKGVLTPMPLGDPYLMINLENSFPAPLVILDTDYNNYACMYSCMDYNYGYHSDFAFFFARAPEAYDKYIAKCRAAFDSIGVDSNRLIKTEQGRGCDYEQLTKLIKDEL*

[0126] >D91A-H93A(SEQ ID NO:9)

[0127] MDEVPDFVIKGKCPAVDEQRLWQQQLPRHSSFGGVWYQQAISTNPYQLLKKCVRIQYDYNGKGFDVKTAGITPEGNQLKRKGVLTPMPLGAPALMINLENSFPAPLVILDTDYNNYACMYSCMDYNYGYHSDFAFFFARAPEAYDKYIAKCRAAFDSIGVDSNRLIKTEQGRGCDYEQLTKLIKDEL*

[0128] >Y57F-D91A(SEQ ID NO:10)

[0129] MDEVPDFVIKGKCPAVDEQRLWQQQLPRHSSFGGVWYQQAISTNPYQLLKKCVRIQFDYNGKGFDVKTAGITPEGNQLKRKGVLTPMPLGAPHLMINLENSFPAPLVILDTDYNNYACMYSCMDYNYGYHSDFAFFFARAPEAYDKYIAKCRAAFDSIGVDSNRLIKTEQGRGCDYEQLTKLIKDEL*

[0130] >Y57F-H93A(SEQ ID NO:11)

[0131] MDEVPDFVIKGKCPAVDEQRLWQQQLPRHSSFGGVWYQQAISTNPYQLLKKCVRIQFDYNGKGFDVKTAGITPEGNQLKRKGVLTPMPLGDPALMINLENSFPAPLVILDTDYNNYACMYSCMDYNYGYHSDFAFFFARAPEAYDKYIAKCRAAFDSIGVDSNRLIKTEQGRGCDYEQLTKLIKDEL*

[0132] >Y57A-D91A-H93A(SEQ ID NO:12)

[0133] MDEVPDFVIKGKCPAVDEQRLWQQQLPRHSSFGGVWYQQAISTNPYQLLKKCVRIQFDYNGKGFDVKTAGITPEGNQLKRKGVLTPMPLGAPALMINLENSFPAPLVILDTDYNNYACMYSCMDYNYGYHSDFAFFFARAPEAYDKYIAKCRAAFDSIGVDSNRLIKTEQGRGCDYEQLTKLIKDEL*

[0134] The nucleic acid sequences of the wild-type and mutant of the lipocalin of Macrobrachium rosenbergii in the present invention are as follows:

[0135] >WT (SEQ ID NO:13)

[0136] ATGGACGAAGTCCCCGATTTCGTCATCAAAGGCAAATGTCCTGCGGTCGACGAGCAAAGGCTTTGGCAACAACAGCTGCCGAGGCATTCAAGCTTTGGAGGAGTTTGGTATCAACAAGCTATCTCAACGAACCCATATCAGCTGCTGAAGAAATGTGTCCGGATCCAGTATGATTACAACGGTAAAGGCTTCGACGTCAAGACTGCCGGTATCACCCCCGAAGGGAACCAGCTCAAGAGGAAGGGTGTCCTCACGCCCATGCCCCTCGGTGACCCTCATCTCATGATCAACCTCGAAAACTCCTTCCCAGCGCCCCTCGTAATCCTCGACACCGACTACAACAACTACGCCTGCATGTACTCCTGCATGGATTACAACTACGGTTATCACTCAGACTTCGCCTTCTTCTTCGCCCGAGCTCCCGAGGCCTACGACAAGTACATCGCCAAGTGCAGAGCAGCCTTCGACAGCATAGGAGTCGATTCCAACAGATTAATCAAGACCGAGCAAGGCAGAGGCTGCGATTACGAGCAACTGACGAAGCTCATCAAAGATGAACTTTAA

[0137] >Y57F (SEQ ID NO:14)

[0138] ATGGACGAAGTCCCCGATTTCGTCATCAAAGGCAAATGTCCTGCGGTCGACGAGCAAAGGCTTTGGCAACAACAGCTGCCGAGGCATTCAAGCTTTGGAGGAGTTTGGTATCAACAAGCTATCTCAACGAACCCATATCAGCTGCTGAAGAAATGTGTCCGGATCCAGttcGATTACAACGGTAAAGGCTTCGACGTCAAGACTGCCGGTATCACCCCCGAAGGGAACCAGCTCAAGAGGAAGGGTGTCCTCACGCCCATGCCCCTCGGTGACCCTCATCTCATGATCAACCTCGAAAACTCCTTCCCAGCGCCCCTCGTAATCCTCGACACCGACTACAACAACTACGCCTGCATGTACTCCTGCATGGATTACAACTACGGTTATCACTCAGACTTCGCCTTCTTCTTCGCCCGAGCTCCCGAGGCCTACGACAAGTACATCGCCAAGTGCAGAGCAGCCTTCGACAGCATAGGAGTCGATTCCAACAGATTAATCAAGACCGAGCAAGGCAGAGGCTGCGATTACGAGCAACTGACGAAGCTCATCAAAGATGAACTTTAA

[0139] >D91E(SEQ ID NO:15)

[0140] ATGGACGAAGTCCCCGATTTCGTCATCAAAGGCAAATGTCCTGCGGTCGACGAGCAAAGGCTTTGGCAACAACAGCTGCCGAGGCATTCAAGCTTTGGAGGAGTTTGGTATCAACAAGCTATCTCAACGAACCCATATCAGCTGCTGAAGAAATGTGTCCGGATCCAGTATGATTACAACGGTAAAGGCTTCGACGTCAAGACTGCCGGTATCACCCCCGAAGGGAACCAGCTCAAGAGGAAGGGTGTCCTCACGCCCATGCCCCTCGGTgaaCCTCATCTCATGATCAACCTCGAAAACTCCTTCCCAGCGCCCCTCGTAATCCTCGACACCGACTACAACAACTACGCCTGCATGTACTCCTGCATGGATTACAACTACGGTTATCACTCAGACTTCGCCTTCTTCTTCGCCCGAGCTCCCGAGGCCTACGACAAGTACATCGCCAAGTGCAGAGCAGCCTTCGACAGCATAGGAGTCGATTCCAACAGATTAATCAAGACCGAGCAAGGCAGAGGCTGCGATTACGAGCAACTGACGAAGCTCATCAAAGATGAACTTTAA

[0141] >D91A(SEQ ID NO:16)

[0142] ATGGACGAAGTCCCCGATTTCGTCATCAAAGGCAAATGTCCTGCGGTCGACGAGCAAAGGCTTTGGCAACAACAGCTGCCGAGGCATTCAAGCTTTGGAGGAGTTTGGTATCAACAAGCTATCTCAACGAACCCATATCAGCTGCTGAAGAAATGTGTCCGGATCCAGTATGATTACAACGGTAAAGGCTTCGACGTCAAGACTGCCGGTATCACCCCCGAAGGGAACCAGCTCAAGAGGAAGGGTGTCCTCACGCCCATGCCCCTCGGTgccCCTCATCTCATGATCAACCTCGAAAACTCCTTCCCAGCGCCCCTCGTAATCCTCGACACCGACTACAACAACTACGCCTGCATGTACTCCTGCATGGATTACAACTACGGTTATCACTCAGACTTCGCCTTCTTCTTCGCCCGAGCTCCCGAGGCCTACGACAAGTACATCGCCAAGTGCAGAGCAGCCTTCGACAGCATAGGAGTCGATTCCAACAGATTAATCAAGACCGAGCAAGGCAGAGGCTGCGATTACGAGCAACTGACGAAGCTCATCAAAGATGAACTTTAA

[0143] >H93A(SEQ ID NO:17)

[0144] ATGGACGAAGTCCCCGATTTCGTCATCAAAGGCAAATGTCCTGCGGTCGACGAGCAAAGGCTTTGGCAACAACAGCTGCCGAGGCATTCAAGCTTTGGAGGAGTTTGGTATCAACAAGCTATCTCAACGAACCCATATCAGCTGCTGAAGAAATGTGTCCGGATCCAGTATGATTACAACGGTAAAGGCTTCGACGTCAAGACTGCCGGTATCACCCCCGAAGGGAACCAGCTCAAGAGGAAGGGTGTCCTCACGCCCATGCCCCTCGGTGACCCTgccCTCATGATCAACCTCGAAAACTCCTTCCCAGCGCCCCTCGTAATCCTCGACACCGACTACAACAACTACGCCTGCATGTACTCCTGCATGGATTACAACTACGGTTATCACTCAGACTTCGCCTTCTTCTTCGCCCGAGCTCCCGAGGCCTACGACAAGTACATCGCCAAGTGCAGAGCAGCCTTCGACAGCATAGGAGTCGATTCCAACAGATTAATCAAGACCGAGCAAGGCAGAGGCTGCGATTACGAGCAACTGACGAAGCTCATCAAAGATGAACTTTAA

[0145] > Y57H(SEQ ID NO:18)

[0146] ATGGACGAAGTCCCCGATTTCGTCATCAAAGGCAAATGTCCTGCGGTCGACGAGCAAAGGCTTTGGCAACAACAGCTGCCGAGGCATTCAAGCTTTGGAGGAGTTTGGTATCAACAAGCTATCTCAACGAACCCATATCAGCTGCTGAAGAAATGTGTCCGGATCCAGcacGATTACAACGGTAAAGGCTTCGACGTCAAGACTGCCGGTATCACCCCCGAAGGGAACCAGCTCAAGAGGAAGGGTGTCCTCACGCCCATGCCCCTCGGTGACCCTCATCTCATGATCAACCTCGAAAACTCCTTCCCAGCGCCCCTCGTAATCCTCGACACCGACTACAACAACTACGCCTGCATGTACTCCTGCATGGATTACAACTACGGTTATCACTCAGACTTCGCCTTCTTCTTCGCCCGAGCTCCCGAGGCCTACGACAAGTACATCGCCAAGTGCAGAGCAGCCTTCGACAGCATAGGAGTCGATTCCAACAGATTAATCAAGACCGAGCAAGGCAGAGGCTGCGATTACGAGCAACTGACGAAGCTCATCAAAGATGAACTTTAA

[0147] >H93Y(SEQ ID NO:19)

[0148] ATGGACGAAGTCCCCGATTTCGTCATCAAAGGCAAATGTCCTGCGGTCGACGAGCAAAGGCTTTGGCAACAACAGCTGCCGAGGCATTCAAGCTTTGGAGGAGTTTGGTATCAACAAGCTATCTCAACGAACCCATATCAGCTGCTGAAGAAATGTGTCCGGATCCAGTATGATTACAACGGTAAAGGCTTCGACGTCAAGACTGCCGGTATCACCCCCGAAGGGAACCAGCTCAAGAGGAAGGGTGTCCTCACGCCCATGCCCCTCGGTGACCCTtacCTCATGATCAACCTCGAAAACTCCTTCCCAGCGCCCCTCGTAATCCTCGACACCGACTACAACAACTACGCCTGCATGTACTCCTGCATGGATTACAACTACGGTTATCACTCAGACTTCGCCTTCTTCTTCGCCCGAGCTCCCGAGGCCTACGACAAGTACATCGCCAAGTGCAGAGCAGCCTTCGACAGCATAGGAGTCGATTCCAACAGATTAATCAAGACCGAGCAAGGCAGAGGCTGCGATTACGAGCAACTGACGAAGCTCATCAAAGATGAACTTTAA

[0149] > Y57H-H93Y(SEQ ID NO:20)

[0150] ATGGACGAAGTCCCCGATTTCGTCATCAAAGGCAAATGTCCTGCGGTCGACGAGCAAAGGCTTTGGCAACAACAGCTGCCGAGGCATTCAAGCTTTGGAGGAGTTTGGTATCAACAAGCTATCTCAACGAACCCATATCAGCTGCTGAAGAAATGTGTCCGGATCCAGcacGATTACAACGGTAAAGGCTTCGACGTCAAGACTGCCGGTATCACCCCCGAAGGGAACCAGCTCAAGAGGAAGGGTGTCCTCACGCCCATGCCCCTCGGTGACCCTtacCTCATGATCAACCTCGAAAACTCCTTCCCAGCGCCCCTCGTAATCCTCGACACCGACTACAACAACTACGCCTGCATGTACTCCTGCATGGATTACAACTACGGTTATCACTCAGACTTCGCCTTCTTCTTCGCCCGAGCTCCCGAGGCCTACGACAAGTACATCGCCAAGTGCAGAGCAGCCTTCGACAGCATAGGAGTCGATTCCAACAGATTAATCAAGACCGAGCAAGGCAGAGGCTGCGATTACGAGCAACTGACGAAGCTCATCAAAGATGAACTTTAA

[0151] >D91A-H93A(SEQ ID NO:21)

[0152] ATGGACGAAGTCCCCGATTTCGTCATCAAAGGCAAATGTCCTGCGGTCGACGAGCAAAGGCTTTGGCAACAACAGCTGCCGAGGCATTCAAGCTTTGGAGGAGTTTGGTATCAACAAGCTATCTCAACGAACCCATATCAGCTGCTGAAGAAATGTGTCCGGATCCAGTATGATTACAACGGTAAAGGCTTCGACGTCAAGACTGCCGGTATCACCCCCGAAGGGAACCAGCTCAAGAGGAAGGGTGTCCTCACGCCCATGCCCCTCGGTgccCCTgccCTCATGATCAACCTCGAAAACTCCTTCCCAGCGCCCCTCGTAATCCTCGACACCGACTACAACAACTACGCCTGCATGTACTCCTGCATGGATTACAACTACGGTTATCACTCAGACTTCGCCTTCTTCTTCGCCCGAGCTCCCGAGGCCTACGACAAGTACATCGCCAAGTGCAGAGCAGCCTTCGACAGCATAGGAGTCGATTCCAACAGATTAATCAAGACCGAGCAAGGCAGAGGCTGCGATTACGAGCAACTGACGAAGCTCATCAAAGATGAACTTTAA

[0153] >Y57F-D91A(SEQ ID NO:22)

[0154] ATGGACGAAGTCCCCGATTTCGTCATCAAAGGCAAATGTCCTGCGGTCGACGAGCAAAGGCTTTGGCAACAACAGCTGCCGAGGCATTCAAGCTTTGGAGGAGTTTGGTATCAACAAGCTATCTCAACGAACCCATATCAGCTGCTGAAGAAATGTGTCCGGATCCAGttcGATTACAACGGTAAAGGCTTCGACGTCAAGACTGCCGGTATCACCCCCGAAGGGAACCAGCTCAAGAGGAAGGGTGTCCTCACGCCCATGCCCCTCGGTgccCCTCATCTCATGATCAACCTCGAAAACTCCTTCCCAGCGCCCCTCGTAATCCTCGACACCGACTACAACAACTACGCCTGCATGTACTCCTGCATGGATTACAACTACGGTTATCACTCAGACTTCGCCTTCTTCTTCGCCCGAGCTCCCGAGGCCTACGACAAGTACATCGCCAAGTGCAGAGCAGCCTTCGACAGCATAGGAGTCGATTCCAACAGATTAATCAAGACCGAGCAAGGCAGAGGCTGCGATTACGAGCAACTGACGAAGCTCATCAAAGATGAACTTTAA

[0155] >Y57F-H93A(SEQ ID NO:23)

[0156] ATGGACGAAGTCCCCGATTTCGTCATCAAAGGCAAATGTCCTGCGGTCGACGAGCAAAGGCTTTGGCAACAACAGCTGCCGAGGCATTCAAGCTTTGGAGGAGTTTGGTATCAACAAGCTATCTCAACGAACCCATATCAGCTGCTGAAGAAATGTGTCCGGATCCAGttcGATTACAACGGTAAAGGCTTCGACGTCAAGACTGCCGGTATCACCCCCGAAGGGAACCAGCTCAAGAGGAAGGGTGTCCTCACGCCCATGCCCCTCGGTGACCCTgccCTCATGATCAACCTCGAAAACTCCTTCCCAGCGCCCCTCGTAATCCTCGACACCGACTACAACAACTACGCCTGCATGTACTCCTGCATGGATTACAACTACGGTTATCACTCAGACTTCGCCTTCTTCTTCGCCCGAGCTCCCGAGGCCTACGACAAGTACATCGCCAAGTGCAGAGCAGCCTTCGACAGCATAGGAGTCGATTCCAACAGATTAATCAAGACCGAGCAAGGCAGAGGCTGCGATTACGAGCAACTGACGAAGCTCATCAAAGATGAACTTTAA

[0157] >Y57A-D91A-H93A(SEQ ID NO:24)

[0158] ATGGACGAAGTCCCCGATTTCGTCATCAAAGGCAAATGTCCTGCGGTCGACGAGCAAAGGCTTTGGCAACAACAGCTGCCGAGGCATTCAAGCTTTGGAGGAGTTTGGTATCAACAAGCTATCTCAACGAACCCATATCAG CTGCTGAAGAAATGTGTCCGGATCCAGttcGATTACAACGGTAAAGGCTTCGACGTCAAGACTGCCGGTATCACCCCCGAAGGGAACCAGCTCAAGAGGAAGGGTGTCCTCACGCCCATGCCCCTCGGTgccCCTgccCTC ATGATCAACCTCGAAAACTCCTTCCCAGCGCCCCTCGTAATCCTCGACACCGACTACAACAACTACGCCTGCATGTACTCCTGCATGGATTACAACTACGGTTATCACTCAGACTTCGCCTTCTTCTTCGCCCGAGCTCCC GAGGCCTACGACAAGTACATCGCCAAGTGCAGAGCAGCCTTCGACAGCATAGGAGTCGATTCCAACAGATTAATCAAGACCGAGCAAGGCAGAGGCTGCGATTACGAGCAACTGACGAAGCTCATCAAAGATGAACTTTAA

[0159] Unless otherwise stated, all reagents used in this invention can be commercially available reagents commonly used in the field; wherein, astaxanthin is preferably HPLC grade astaxanthin with a purity of not less than 99%, freshly prepared in DMSO before use, and operated under light-protected conditions.

[0160] The present invention will be further illustrated below with reference to the embodiments:

[0161] Example 1: Amino acid sequence optimization design of macrobrachium rosenbergii lipid transport protein (rMrLC)

[0162] (1) Sequence optimization information:

[0163] Expression system: Escherichia coli.

[0164] (2) Codon usage preference adjustment:

[0165] In the target expression host, a codon fitness index (CAI) of 1.0 is considered the perfect ideal expression effect; when CAI > 0.75, the gene expression level is considered excellent. The optimized codon fitness index is 0.85, and the original codon fitness index is 0.37.

[0166] (3) Adjustment of GC content:

[0167] The ideal percentage range for GC content is 35% to 65%. This report uses a 30bp window for GC content statistics. Optimized GC content: 51.24%.

[0168] (4) Original Sequence

[0169] ATGGACGAAGTCCCCGATTTCGTCATCAAAGGCAAATGTCCTGCGGTCGACGAGCAAAGGCTTTGGCAACAACAGCTGCCGAGGCATTCAAGCTTTGGAGGAGTTTGGTATCAACAAGCTATCTCAACGAACCCATATCAG CTGCTGAAGAAATGTGTCCGGATCCAGTATGATTACAACGGTAAAGGCTTCGACGTCAAGACTGCCGGTATCACCCCCGAAGGGAACCAGCTCAAGAGGAAGGGTGTCCTCACGCCCATGCCCCTCGGTGACCCTCATCTC ATGATCAACCTCGAAAACTCCTTCCCAGCGCCCCTCGTAATCCTCGACACCGACTACAACAACTACGCCTGCATGTACTCCTGCATGGATTACAACTACGGTTATCACTCAGACTTCGCCTTCTTCTTCGCCCGAGCTCCC GAGGCCTACGACAAGTACATCGCCAAGTGCAGAGCAGCCTTCGACAGCATAGGAGTCGATTCCAACAGATTAATCAAGACCGAGCAAGGCAGAGGCTGCGATTACGAGCAACTGACGAAGCTCATCAAAGATGAACTTTAA

[0170] (5) Optimized DNA Sequence

[0171] ATGGATGAAGTTCCGGACTTCGTTATCAAAGGTAAATGTCCGGCTGTGGATGAACAGCGTCTGTGGCAGCAGCAGTTGCCACGTCACTCCAGCTTCGGTGGCGTTTGGTATCAGCAGGCGATCTCCACCAATCCGTACCAG CTGCTGAAGAAGTGTGTTCGTATCCAGTACGACTACAACGGTAAAGGTTTCGATGTTAAGACTGCGGGTATCACTCCGGAAGGTAACCAGCTGAAACGTAAAGGTGTTCTGACTCCGATGCCGCTTGGTGATCCGCACCTG ATGATCAACCTGGAGAACAGCTTTCCGGCACCGCTGGTAATTCTGGACACCGACTACAACAACTACGCTTGCATGTACAGCTGCATGGACTACAACTACGGCTACCACTCCGACTTCGCGTTCTTCTTCGCTCGTGCTCCG GAAGCGTACGACAAATACATCGCGAAATGTCGTGCTGCGTTCGACTCCATCGGTGTAGACTCTAACCGTTTGATCAAGACCGAACAGGGTCGTGGTGCGATTACGAACAGCTGACCAAACTGATCAAAGATGAACTGTAA

[0172] (6) Protein Sequence

[0173] MDEVPDFVIKGKCPAVDEQRLWQQQLPRHSSFGGVWYQQAISTNPYQLLKKCVRIQYDYNGKGFDVKTAGITPEGNQLKRKGVLTPMPLGDPHLMINLENSFPAPLVILDTDYNNYACMYSCMDYNYGYHSDFAFFFARAPEAYDKYIAKCRAAFDSIGVDSNRLIKTEQGRGCDYEQLTKLIKDEL*

[0174] Example 2: Expression, purification, and refolding of recombinant rMrLC

[0175] The *Macrobrachium rosenbergii* lipid transport protein *MrLC* gene, with its N-terminal signal peptide removed, was cloned into the pET-28a(+) vector, resulting in an N-terminal 6×His tag for the target protein. The recombinant plasmid was transformed into *Escherichia coli* BL21(DE3) host bacteria and cultured in LB medium at 37 °C until OD500.600 After adjusting the concentration to 0.6–0.7, 0.2 mM IPTG was added to induce expression overnight. Under these conditions, rMrLC accumulated in the form of inclusion bodies.

[0176] After centrifugation to collect bacterial cells, the cells were resuspended in a disruption buffer containing 25 mM Tris and 100 mM NaCl. The cells were disrupted by sonication at 200 W for 20 min under ice bath conditions. Inclusion bodies were collected by centrifugation after sonication. The inclusion bodies were then resuspended in a washing buffer containing 25 mM Tris, 5 mM EDTA, 1 mM PMSF, and 2 M urea at pH 8.0. After washing three times, the precipitate was collected by centrifugation. The precipitate was then placed in a solution containing 10 M urea, 25 mM Tris, 1 mM PMSF, and 4 mM β-mercaptoethanol at pH 8.0 for denaturation and dissolution, and sonication was used to promote complete dissolution of the inclusion bodies.

[0177] Ni is used under denaturing conditions 2+ The His-tagged target protein was purified by affinity chromatography. The eluent contained 25 mM Tris-HCl, 8 M urea, 4 mM β-mercaptoethanol, and 500 mM imidazole at pH 8.0. Subsequently, stepwise dialysis was performed at 4 °C. The dialysis buffer consisted of 25 mM Tris-HCl, 1.0 mM PMSF, 50 mM glycine, and 5 mM EDTA at pH 8.0, with urea concentrations of 6.0 M, 4.0 M, 3.0 M, 2.0 M, 1.0 M, and 0 M, respectively, for 24 h at each gradient. After dialysis, the protein was further dialyzed into 25 mM sodium phosphate buffer (pH 7.5) containing 150 mM NaCl. If necessary, the protein was concentrated using a 10 kDa MWCO ultrafiltration concentrator and further purified by Superdex 200 gel filtration to obtain the rMrLC protein solution for later use.

[0178] Example 3: In vitro reconstruction of the blue pigment complex

[0179] Under light-protected conditions, the rMrLC obtained in Example 2 was placed in a stepwise dialysis refolding system. When dialysis reached a urea concentration of 3 M, astaxanthin and phosphatidylethanolamine were directionally added to the protein solution. The astaxanthin and phosphatidylethanolamine were preferably added slowly dropwise in the form of DMSO stock solution; preferably, they were added at a molar ratio of rMrLC monomer:astaxanthin:phosphatidylethanolamine of 1:5:1, so that astaxanthin and phosphatidylethanolamine could participate in co-assembly before the protein refolding was complete.

[0180] After adding astaxanthin and phosphatidylethanolamine, subsequent dialysis refolding at 3.0 M, 2.0 M, 1.0 M, and 0 M was performed under light-protected conditions. After complete removal of urea, final dialysis was performed with deionized water or 25 mM sodium phosphate buffer (pH 7.5, containing 150 mM NaCl) to remove unbound small molecules and free ligands. Surprisingly, the resulting system exhibited a stable blue color at room temperature and a significant red-shifted absorption peak in the visible light region, indicating that the directional introduction of astaxanthin and phosphatidylethanolamine at the 3 M urea node can drive the ternary co-assembly of proteins, carotenoids, and phospholipids.

[0181] After the above steps, the resulting system is blue at room temperature, indicating that by directionally introducing astaxanthin and phosphatidylethanolamine at the 3 M urea node, the synergistic co-assembly of proteins, carotenoids and phospholipids can be achieved during the rMrLC refolding process, thereby constructing a blue pigment complex and providing experimental evidence for the controllable construction of blue in aqueous systems.

[0182] Example 4 Characterization of the blue pigment complex

[0183] The visible absorption spectrum of the blue pigment complex obtained in Example 3 was detected using a conventional UV-Vis spectrophotometer. The results showed that its maximum absorption peak was located at approximately 570 nm; compared with the absorption peak of free astaxanthin at approximately 440 nm, a significant redshift of approximately 130 nm was observed, indicating that the method can construct a blue pigment system with a strong visible light redshift characteristic.

[0184] After heating the blue pigment complex obtained in Example 3 at 70 °C for 10 min, the sample changed from blue to orange-red. After centrifugation at 8000 g for 5 min, the supernatant was orange while the precipitate was white, indicating that the complex has obvious thermal response color change behavior.

[0185] Comparative Example 1: Canthaxanthin replaced astaxanthin

[0186] Using the same conditions as in Example 3, canthaxanthin was used instead of astaxanthin, and canthaxanthin and phosphatidylethanolamine were added at the 3 M urea node. The results showed that the resulting system was a deep pink color with a maximum absorption peak of approximately 530 nm, and the blue pigment shown in Example 3 was not formed.

[0187] Comparative Example 2: Replacing Astaxanthin with Zeaxanthin

[0188] Using the same conditions as in Example 3, zeaxanthin was substituted for astaxanthin, and zeaxanthin and phosphatidylethanolamine were added at the 3 M urea node. The results showed that no new absorption peak corresponding to the blue pigment appeared in the visible region, and the absorption characteristics around 390 nm were mainly maintained, with no blue pigment formed.

[0189] Comparative Example 3: Replacing Astaxanthin with Lutein

[0190] Using the same conditions as in Example 3, lutein was substituted for astaxanthin, and lutein and phosphatidylethanolamine were added at the 3 M urea node. The results showed that the lutein-rMrLC system exhibited only a slight change in absorption peak, from approximately 384 nm to approximately 387 nm, and no blue pigment was formed.

[0191] Table 1 lists the color and spectral characteristics of the systems obtained in Example 3 and Comparative Examples 1-3.

[0192]

[0193] The above comparative results show that among the tested carotenoids, only astaxanthin can synergistically form a complex with a significant blue phenotype and a characteristic absorption peak of approximately 570 nm with rMrLC and phosphatidylethanolamine; although canthaxanthin has a 4-keto group, it only forms a deep pink system at approximately 530 nm; zeaxanthin and lutein cannot form the blue pigment of this invention. These results indicate that the terminal 3-hydroxy / 4-keto bifunctional configuration of astaxanthin and its synergistic effect with proteins and phospholipids are crucial for the formation of the blue pigment.

[0194] To further illustrate that the formation of the blue pigment in this invention is not a simple assembly of components, but rather depends on a specific network of residues and a ternary assembly mechanism, the following experiments were conducted.

[0195] Example 5: The effect of key site mutations on pigment formation

[0196] To investigate the effect of key amino acid residues in the lipid transport protein (rMrLC) of giant freshwater prawn on the coloration of pigment complexes, this study constructed a series of site-directed mutants and performed in vitro ternary assembly with astaxanthin and phosphatidylethanolamine, measuring the visible light absorption spectrum and apparent color of each complex.

[0197] Single-point mutants Y57A, D91A, H93A, and Y57F, double-point mutants D91A / H93A, Y57F / D91A, Y57F / H93A, and Y57H / H93Y, and triple mutant 3A Y57A / D91A / H93A were prepared using the same method as in Example 2. The expression, purification, and renaturation conditions for each mutant were consistent with those for wild-type rMrLC. Subsequently, they were reconstituted with astaxanthin and phosphatidylethanolamine under the same conditions as in Example 3, and their visible light absorption spectra were measured.

[0198] Experimental results show that the wild-type rMrLC complex exhibits a characteristic absorption peak at 570 nm, corresponding to a blue appearance. When a mutation occurs at a key site, the absorption spectrum of the complex undergoes a blue shift to varying degrees, accompanied by a regular change in color.

[0199] In the mutant at tyrosine 57 (Y57), the maximum absorption peak of both the Y57F and Y57H mutants was blue-shifted to 545 nm, which is about 25 nm compared to the wild type, and the color of the complex changed from blue to magenta.

[0200] In the mutants at aspartic acid 91 (D91) and histidine 93 (H93), the maximum absorption peaks of D91A, D91E, H93Y, H93A and the double mutant D91A-H93A are all located at 555 nm, with a blue shift of about 15 nm, and the complexes all exhibit a purple appearance.

[0201] Further combined mutation results showed that the maximum absorption peak of the Y57F-D91A, Y57F-H93A and Y57H-H93Y double mutant complexes was further blue-shifted to 530 nm, which is about 40 nm blue-shifted compared to the wild type, and the complex color turned pink.

[0202] The above results indicate that the Y57, D91, and H93 sites in rMrLC are key residues determining the color of the pigment complex. By regulating these residues through site-directed mutagenesis, a continuous hue variation from blue (570 nm) to magenta (545 nm), purple (555 nm), and pink (530 nm) can be achieved in vitro. These results provide experimental evidence for elucidating the mechanism of crustacean body color formation and developing protein-based multicolor pigment materials.

[0203] Table 2 Comparison of color and spectral differences between rMrLC wild-type and mutant protein systems

[0204]

[0205] Table 3 shows that the maximum absorption peak of the wild-type system is approximately 570 nm; the maximum absorption peak of the D91A and H93A systems both decrease to approximately 555 nm; the Y57F system is approximately 545 nm; the Y57A system further decreases to approximately 530 nm; while the triple mutant 3A system almost no longer shows any visible absorption peaks related to astaxanthin. Tables 2 and 3 summarize the main spectral changes of each system. These results indicate that Tyr57, Asp91, and His93 jointly participate in constructing the stable microenvironment at the astaxanthin terminus, with Tyr57 contributing the most significantly; simultaneously, the aromatic ring and phenolic hydroxyl groups of Tyr57 both participate in color shift regulation.

[0206] Table 3. Analysis of color and spectral differences between rMrLC wild-type and mutant protein systems.

[0207]

[0208] Example 6: Crystal structures of different pigment complexes and their effects on pigment formation

[0209] X-ray crystallography was used to resolve the structures of apo rMrLC, the wild-type blue pigment complex, the Y57F-AXT complex, and the rMrLC-CAN complex (rMrLC-canthaxanthin). The results showed that the apo rMrLC structure had a resolution of 2.93 Å and exhibited a relatively closed conformation; the wild-type blue pigment complex had a resolution of 1.88 Å, showing an open-state rMrLC homodimer sandwiching an AXT-PE-AXT ternary core structure, with one PE molecule located between two AXT molecules; the Y57F-AXT complex had a resolution of 2.99 Å, with an overall framework similar to the wild-type, but the maximum absorption peak decreased to approximately 545 nm after the loss of the phenolic hydroxyl group at position 57; the rMrLC–CAN complex had a resolution of 1.76 Å, with only a single CAN molecule observed crossing the dimer interface, and no central lipid was observed. Table 4 summarizes the main characteristics of each structural system.

[0210] Table 4. Main characteristics of each structural system

[0211]

[0212] Table 5 Crystallographic Statistics

[0213]

[0214]

[0215] Example 7: Structural stability analysis of the pigment complex

[0216] A magnified view of the wild-type rMrLC-AXT-PE crystal structure is shown below. Figure 15 As shown, two astaxanthin molecules are arranged in antiparallel alignment, with PE located between the two astaxanthin molecules. Together, they form a "sandwich-like" structure, which is encapsulated at the rMrLC dimer interface.

[0217] Comparative analysis of the crystal structures of wild-type Apo rMrLC and rMrLC-AXT-PE complex is as follows: Figure 16 As shown. The results indicate that the binding of pigments and phospholipid molecules induced a conformational rearrangement of the protein. Specifically, the addition of astaxanthin and phosphatidylethanolamine caused the apo rMrLC protein dimer to open (laterally extend), while a structural transformation occurred. Figure 16 (The parts highlighted in yellow and blue).

[0218] This embodiment also investigated the stabilizing effect of phosphatidylethanolamine (PE) on the spatial conformation of two astaxanthin molecules in the lipid transport protein-astaxanthin complex through theoretical calculations and molecular simulations.

[0219] 1. Experimental Methods

[0220] (1) The interaction type between PE and the two astaxanthin molecules was analyzed using the independent gradient model (IGMH).

[0221] (2) A control system with PE molecules removed was constructed using quantum mechanical / molecular mechanical (QM / MM) simulation. Under the condition of keeping the protein backbone and solvent environment unchanged, the structures of two astaxanthin molecules containing PE (original system) and without PE (control system) were optimized respectively, and their geometric conformation changes were compared.

[0222] 2. Experimental Results

[0223] (1) IGMH analysis results showed that there was a clear van der Waals interaction region between the PE molecule and the two astaxanthin molecules, indicating that PE forms a tight intermolecular contact with astaxanthin through non-covalent interaction, providing an additional binding interface for the stable assembly of the complex.

[0224] (2) QM / MM simulation results show that the overall conformation of the protein backbone did not change significantly after the removal of PE molecules, indicating that the role of PE is mainly concentrated in the local pigment binding region and has little impact on the overall protein structure.

[0225] (3) Further comparison of the relative positions of the two astaxanthin molecules revealed that in the original system containing PE, the two astaxanthin molecules were stably assembled in an antiparallel manner with a centroid distance of 7.7 Å; however, after removing PE, the distance between the two astaxanthin molecules was reduced to 5.1 Å, and the original antiparallel arrangement was destroyed, making it impossible to maintain its characteristic spatial conformation.

[0226] QM / MM analysis based on the structure of the wild-type blue pigment complex showed that, with PE retained, the optimized model essentially coincided with the crystal structure; when PE was removed, the protein backbone Cα RMSD increased to approximately 1.4 Å, the distance between the two AXT molecules decreased from approximately 7.7 Å to approximately 5.1 Å, accompanied by inward bending of the polyene chain. These results indicate that PE is not a dispensable excipient, but rather an important structural factor maintaining the spatial conformation of AXT and its significant redshift.

[0227] The above results indicate that phosphatidylethanolamine (PE), as a key cofactor, stabilizes the antiparallel assembly of the two astaxanthin molecules through van der Waals interactions, and is an essential structural element for maintaining the characteristic spatial conformation of the pigment complex. The presence of PE not only limits the relative distance between the astaxanthin molecules but also ensures their specific alignment, thus providing a structural basis for the complex to exhibit a significant red shift in absorption spectra and blue coloration.

[0228] The above examples, comparative examples, and experimental cases demonstrate that the present invention can construct a blue pigment complex in an aqueous in vitro system through the specific ternary assembly of rMrLC, astaxanthin, and phosphatidylethanolamine. Its significant redshift and thermoresponsive characteristics depend on the synergistic stabilizing effect of a specific residue network and PE. Further research revealed that by introducing the rMrLC mutant and maintaining the specific ternary assembly with astaxanthin and PE, pigment complexes of various colors, including purple, blue, and magenta, can be constructed in an aqueous system, demonstrating the flexibility and universality of the method in color regulation. These results provide an important model for a deeper understanding of the body color formation mechanism in crustaceans and for revealing the ternary interaction rules of proteins, pigments, and lipids. They also lay a solid technical foundation for the development of novel natural protein-based pigment materials and thermoresponsive color-changing systems.

[0229] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A mutant of the lipid transport protein rMrLC, characterized in that, The mutation sites of the mutant include Y57F, or Y57F, or D91A, or D91E, or H93Y, or H93A, or Y57H and H93Y, or D91A and H93A, or Y57F and D91A, or Y57F and H93A.

2. The rMrLC mutant of the lipid transport protein according to claim 1, characterized in that, The amino acid sequence of the wild-type lipid transport protein rMrLC is shown in SEQ ID NO:

1.

3. A biomaterial, characterized in that, Includes at least one of the following: A) to D) A) Nucleic acid encoding the rMrLC mutant lipid transport protein as described in claim 1 or 2; B) Expression units containing nucleic acids as described in A); C) A recombinant vector, comprising a backbone vector and a nucleic acid as described in A) or an expression unit as described in B); D) Host cells, which are transformed or transfected with plasmid vectors as described in C), or whose genomes are integrated with nucleic acids as described in A) or expression units as described in B).

4. The biomaterial according to claim 3, characterized in that, The nucleic acid encoding the rMrLC mutant lipid transporter has a nucleic acid sequence as shown in any one of SEQ ID NO:14~23, and the nucleic acid encoding the wild-type rMrLC lipid transporter has a nucleic acid sequence as shown in SEQ ID NO:

13.

5. A coloring composition, characterized in that, Includes the rMrLC mutant lipid transporter as described in claim 1 or 2, pigments, and phospholipids; This may include wild-type lipid transporter rMrLC, pigments, and phospholipids; The pigments include astaxanthin, carotenoids, canthaxanthin, lutein, zeaxanthin, beta-carotene, fucoxanthin, or urchinone; The phospholipids include ceramide, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, or cardiolipin.

6. The method for preparing the coloring composition according to claim 5, characterized in that, It was prepared from a mutant of the lipid transporter rMrLC, pigments and phospholipids, or from the wild-type lipid transporter rMrLC, pigments and phospholipids.

7. The application of the rMrLC mutant of the lipid transport protein according to claim 1 or 2, the biomaterial according to claim 3 or 4, the chromogenic composition according to claim 5, or the chromogenic composition obtained by the preparation method according to claim 6 in the study of pigment formation mechanism or the preparation of pigment products.

8. A pigment product, characterized in that, Includes the rMrLC mutant of the lipid transporter as described in claim 1 or 2, the biomaterial as described in claim 3 or 4, the chromogenic composition as described in claim 5, or the chromogenic composition obtained by the preparation method described in claim 6.

9. The pigment product according to claim 8, characterized in that, The types of pigment products include thermo-responsive color-changing materials, temperature-responsive indicator materials, protein-based pigment preparations, biomimetic optical materials, food colorants, cosmetic pigments, and bioimaging probes or light-harvesting functional materials.

10. Cosmetics, food, or health products, characterized in that, Includes the rMrLC mutant of the lipid transporter as described in claim 1 or 2, the biomaterial as described in claim 3 or 4, the chromogenic composition as described in claim 5, or the chromogenic composition obtained by the preparation method described in claim 6.

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