Key protein for astaxanthin esterification and application thereof

Abstract

The invention provides a key protein for astaxanthin esterification and application thereof, and belongs to the technical field of bioengineering. The identity of the amino acid sequence of the protein and the amino acid sequence shown in SEQ ID NO: 1 is more than 90%. The present disclosure can improve the yield of esterified astaxanthin.

Description

Technical Field

[0001] This disclosure belongs to the field of bioengineering technology, and specifically relates to a key protein for astaxanthin esterification and its application. Background Technology

[0002] Astaxanthin is a naturally occurring red ketone carotenoid. Only a few types of bacteria, microalgae, fungi, and the only terrestrial plant, calendula, can synthesize astaxanthin de novo. Its chemical name is 3,3'- dihydroxy 4,4'-Diketone β,β' Carotene is a terpene unsaturated compound with the molecular formula C2. 40 H 52 O4. Because the hydroxyl groups at both ends of the astaxanthin molecule are very unstable, it is easily oxidized. In nature, astaxanthin exists alone or in mixtures as free astaxanthin, glycosylated astaxanthin, or astaxanthin esters (monoesters or diesters).

[0003] Currently, esterified astaxanthin is mainly derived from Haematococcus pluvialis, Zofenbrychophora, and the terrestrial plant genus *Adonis*. Due to the lack of effective astaxanthin esterification enzymes, most of the engineered strains constructed so far only synthesize free astaxanthin, and have not yet achieved efficient synthesis of esterified astaxanthin. Summary of the Invention

[0004] This disclosure provides a key protein for astaxanthin esterification and its application, which can improve the yield of esterified astaxanthin. The technical solution is as follows: This disclosure provides a key protein for astaxanthin esterification, wherein the amino acid sequence of the protein is more than 90% identical to the amino acid sequence shown in SEQ ID NO:1.

[0005] In yet another implementation of this disclosure, the gene sequence encoding the protein is derived from *Microcystis aeruginosa*. Nodgat2g Gene sequence.

[0006] In another implementation of this disclosure, the protein is a mutant of the NoDGAT2G protein that retains the His-352 site.

[0007] In another implementation of this disclosure, the mutant is obtained by site-directed mutagenesis of the NoDGAT2G protein.

[0008] Secondly, this disclosure also provides a method for producing esterified astaxanthin, wherein the protein described above is expressed in an astaxanthin-producing host cell to catalyze the production of esterified astaxanthin in the host cell.

[0009] In another implementation of this disclosure, the expression of the aforementioned protein in host cells to catalyze the production of esterified astaxanthin in the host cells includes: constructing an expression vector for esterified astaxanthin based on the protein; and transforming the expression vector into the host cells for culture to catalyze the production of esterified astaxanthin in the host cells.

[0010] In yet another implementation of this disclosure, the expression vector includes components capable of driving [the expression] in a host cell. Nodgat2g The expressive element.

[0011] In yet another implementation of this disclosure, the element includes pYLXP'- Nodgat2g Vector or pYLXP'- Nodgat2g The mutants include single-site site-directed mutants, two-site site-directed mutants, or multi-site site-directed mutants.

[0012] In another implementation of this disclosure, the step of transforming the expression vector into the host cells and culturing them to catalyze the production of esterified astaxanthin by the host cells includes: transforming the expression vector into the host cells to obtain recombinant host cells; culturing the recombinant host cells to express the protein and catalyze the synthesis of esterified astaxanthin, thereby obtaining a culture medium containing esterified astaxanthin; and separating and purifying the culture medium to obtain the esterified astaxanthin.

[0013] In another implementation of this disclosure, the host cell is selected from engineered strains of the following microorganisms: lipophilic yeast or microalgae.

[0014] The beneficial effects of the technical solutions provided in this disclosure are: By expressing the key protein for astaxanthin esterification provided in this embodiment of the invention in astaxanthin-producing host cells, the host cells can be directed to produce esterified astaxanthin. This protein catalyzes the efficient production of esterified astaxanthin from host cells, rather than free astaxanthin, significantly improving the stability of astaxanthin. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of this disclosure, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 It is pYLXP' -Nodgat2g PCR detection diagram of plasmid; Figure 2 It is pYLXP'- Nodgat2g TLC image of plasmid expression in Yersinia lipophila; Figure 3 This is a sequence alignment diagram of DGAT protein with esterification function; Figure 4 This is a diagram showing the location of conserved histidine sites in the three-dimensional structure of different proteins; Figure 5 It is pYLXP' -Nodgat2g PCR detection diagram of -H352G plasmid; Figure 6 It is pYLXP' -Nodgat2g TLC image of -H352G plasmid expression; Figure 7 It is pYLXP'- Nodgat2g PCR detection diagram of plasmids such as -L398N; Figure 8 It is pYLXP'- Nodgat2g TLC image of L398N plasmid expression; Figure 9 It is pYLXP'- Nodgat2g PCR detection diagram of plasmids such as -Q354A; Figure 10 It is pYLXP'- Nodgat2g -TLC detection image after expression of plasmids such as Q354A; Figure 11 It is pYLXP'- Nodgat2g PCR detection diagram of plasmids such as -Q354W; Figure 12 It is pYLXP'- Nodgat2g -TLC detection image after expression of plasmids such as Q354W; Figure 13 It is pYLXP'- Nodgat2g PCR detection diagram of plasmids such as -E99G; Figure 14 It is pYLXP'- Nodgat2g TLC image of E99G plasmid expression; Figure 15 It is pYLXP'- Nodgat2g PCR detection diagram of plasmids such as -V403A+Q425A; Figure 16 It is pYLXP'- Nodgat2g TLC image of plasmids -V403A+Q425A after expression; Figure 17 It is pYLXP'- Nodgat2g PCR detection diagram of plasmids such as -Q174A+Q354A; Figure 18 It is pYLXP'- Nodgat2g TLC image of plasmids -Q174A+Q354A after expression; Figure 19 It is pYLXP'- Nodgat2g PCR detection diagram of plasmids such as -Q354W+Q425A+E99G; Figure 20 It is pYLXP'- Nodgat2g TLC detection image of plasmids such as -Q354W+Q425A+E99G after expression. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this disclosure clearer, the embodiments of this disclosure will be described in further detail below with reference to the accompanying drawings.

[0018] Astaxanthin is a naturally occurring red ketocarboxylic acid. Naturally occurring forms of astaxanthin include free astaxanthin, esterified astaxanthin, and glycosylated astaxanthin. Esterified astaxanthin is more stable than free astaxanthin. Compared to free astaxanthin, at the same dose, the concentration (bioavailability) of esterified astaxanthin in the blood is several times or even ten times higher.

[0019] Currently, esterified astaxanthin is mainly obtained through fermentation of Haematococcus pluvialis. Haematococcus pluvialis is placed in a culture medium under light conditions. After accumulating a certain biomass, the culture is switched to a nitrogen-deficient or other stress-based culture mode, causing rapid accumulation of astaxanthin within the Haematococcus pluvialis cells, mostly as esterified astaxanthin. The esterified astaxanthin is then extracted through leaching and filtration. However, obtaining esterified astaxanthin using this method is time-consuming and costly, limiting its widespread application.

[0020] This disclosure provides a key protein for astaxanthin esterification, the amino acid sequence of which has more than 90% identity with the amino acid sequence shown in SEQ ID NO:1.

[0021] By expressing the protein provided in this embodiment of the present disclosure in astaxanthin-producing host cells, the host cells can be directed to produce esterified astaxanthin. This protein catalyzes the synthesis of esterified astaxanthin, which helps improve the stability of astaxanthin.

[0022] In some examples, the amino acid sequence of the protein is the same as that shown in SEQ ID NO:1. In this case, the amino acid sequence of the protein is 100% identical to that shown in SEQ ID NO:1.

[0023] In other examples, the amino acid sequence of the protein is a mutant of the amino acid sequence shown in SEQ ID NO:1. In this case, the amino acid sequence of the protein is more than or equal to 90% and less than 100% identical to the amino acid sequence shown in SEQ ID NO:1.

[0024] The gene sequence encoding the protein was derived from the marine algae strain IMET1. Nodgat2g Gene sequence, as shown in SEQ ID NO:2 in the table below.

[0025] Among them, Microcystis globulus Nodgat2g The gene sequence and its corresponding amino acid sequence are shown in Table 1 below:

[0026] Optionally, the mutant mentioned above is a mutant of the NoDGAT2G protein that retains the His-352 site.

[0027] Experiments showed that the NoDGAT2G protein, or a mutant of the NoDGAT2G protein retaining the His-352 site, significantly increased the production of esterified astaxanthin in host cells upon transfection. The NoDGAT2G protein catalyzes the esterification reaction of astaxanthin with fatty acids, and the retention of His-352 optimizes its catalytic activity, promoting the accumulation of astaxanthin esters (the lipid-soluble form).

[0028] Optionally, the mutant is obtained by site-directed mutagenesis of the NoDGAT2G protein.

[0029] Site-directed mutagenesis is a molecular biology technique used to precisely introduce mutations (such as base substitutions, insertions, or deletions) at specific locations in a DNA sequence, thereby altering specific amino acids or regulatory sequences of a target protein. Site-directed mutagenesis only modifies the target site and does not affect other parts of the gene.

[0030] In this embodiment, the site-directed mutant was obtained through polymerase chain reaction (PCR) amplification and vector digestion. PCR amplification refers to the introduction of the mutation into the gene sequence encoding the protein using primers containing the mutation site, forming a PCR mutant fragment. Vector digestion involves double-digesting the vector and the PCR mutant fragment with restriction endonucleases to produce compatible ends. The mutant fragment is then ligated to the compatible ends of the vector using T4 DNA ligase to form the mutant.

[0031] Because the His-352 site is crucial for protein function (such as catalysis and substrate binding), random mutations can lead to protein inactivation. Site-directed mutagenesis, however, ensures that only the target site is altered, without affecting the His-352 site. The target site refers to any site other than His-352. Furthermore, in the NoDGAT2G protein, mutating the His-352 site to other amino acids (such as Ala or Arg) can verify whether this site is essential for the catalytic activity of astaxanthin esterification. Mutants retaining the His-352 site can serve as controls, demonstrating its irreplaceable nature.

[0032] Secondly, this disclosure also provides a method for producing esterified astaxanthin, the method comprising: The protein described above is expressed in host cells that produce astaxanthin to catalyze the production of esterified astaxanthin in the host cells.

[0033] The above production methods have the same beneficial effects as the aforementioned proteins, and will not be repeated here.

[0034] Thirdly, this disclosure also provides another method for producing esterified astaxanthin, the method comprising: S201: Construct an expression vector for esterified astaxanthin based on the protein.

[0035] An expression vector is a vector that adds expression elements (such as promoters and terminators) to the basic skeleton of a cloning vector, enabling the target gene to be expressed.

[0036] For example, the expression vector includes a capability to drive expression in the host cell. Nodgat2g The expressive element.

[0037] In this embodiment, the component includes pYLXP'- Nodgat2g Or pYLXP'- Nodgat2g Mutant.

[0038] In other examples, the element may also be another expression carrier.

[0039] The use of this disclosure Nodgat2g The gene was selected from wild-type Micrococcus microcarpa. Nodgat2g The gene, whose gene sequence is the gene sequence shown in SEQ ID NO:2 in Table 1 above.

[0040] When constructing the expression vector, Yersinia lipophila was selected as the host cell, and pYLXP' plasmid was used as the vector.

[0041] In this embodiment, the sequence of the pYLXP' plasmid can be found in the gene sequence shown in Table 2 SEQ ID NO:3 below.

[0042]

[0043] The pYLXP' plasmid has a strong TEF promoter and an XPR terminator, and also carries a leucine (Leu) selection marker gene.

[0044] Because the pYLXP' plasmid carries the Leu selection marker gene, the expression vector constructed based on this backbone can be stably expressed in leucine auxotroph (leu2-) yeast strains. In other words, once the leucine auxotroph (leu2-) yeast strain expresses the expression vector constructed from the pYLXP' plasmid, the yeast strain can still continue to grow on leucine-free medium (-Leu), thus enabling rapid differentiation between positive clones (containing the pYLXP' plasmid) and negative clones (not containing the pYLXP' plasmid), avoiding cumbersome PCR or enzyme digestion verification.

[0045] Optionally, step S201 may include the following steps: 2011: [Reference to] wild-type *Microcystis globulus* Nodgat2g Gene optimization 。

[0046] The so-called optimization treatment refers to the treatment of acyltransferase genes. Nodgat2g Codon optimization was performed to improve the acyltransferase gene. Nodgat2g Expression efficiency in host cells.

[0047] 2012: The optimized version Nodgat2g The gene was cloned into the pYLXP' plasmid to obtain the expression vector.

[0048] In this embodiment, the expression vector is pYLXP'- Nodgat2g and pYLXP'- Nodgat2g The mutant.

[0049] Among them, pYLXP'- Nodgat2g The mutants include single-site site-directed mutants, two-site site-directed mutants, or multi-site site-directed mutants, which can be seen in the different mutants in the subsequent experiments.

[0050] 2013: The expression vector was transformed into the DH5α strain.

[0051] The DH5α strain, as a widely used cloning strain, is capable of replicating pYLXP'- Nodgat2gOr pYLXP'- Nodgat2g The mutant was obtained to obtain a high-purity, high-concentration expression vector.

[0052] 2014: From transferring to pYLXP'- Nodgat2g Or pYLXP'- Nodgat2g The expression vector was extracted from the mutant DH5α strain.

[0053] S202: The extracted expression vector is transformed into host cells and cultured to catalyze the production of esterified astaxanthin in the host cells.

[0054] In this embodiment, pYLXP'- Nodgat2g The vector-encoded gene, after transcription and translation, can express the NoDGAT2G protein (i.e., the enzyme), while pYLXP'- Nodgat2g The mutant gene encoded by the mutant is transcribed and translated to express a mutant NoDGAT2G protein with specific amino acid substitutions.

[0055] For example, the host cell is selected from engineered strains of the following microorganisms: Yeastra lipolytica, Saccharomyces cerevisiae, Escherichia coli, or microalgae.

[0056] In this embodiment of the disclosure, the host cell is an engineered strain of *Lactobacillus lipolyticus*.

[0057] Optionally, step S202 may include the following steps: 2021: The engineered strain of Yeast lipolyticis was activated.

[0058] Since the engineered strains are all refrigerated before use, they need to be thawed to revive and activate them.

[0059] 2022: The activated strain of the engineered strain was spread on a culture medium plate and cultured to obtain the chassis strain.

[0060] 2023: The expression vector was transformed into the host cells to obtain recombinant host cells.

[0061] 2024: Cultivate the recombinant host cells to express the protein and catalyze the synthesis of esterified astaxanthin, thereby obtaining a culture medium containing esterified astaxanthin.

[0062] Take pYLXP'- Nodgat2g Vector, polyethylene glycol, lithium acetate, single-stranded DNA Stranded DNA (ssDNA) and a certain amount of the substrate bacterial strain were mixed and incubated at 20℃-40℃ for 20-60 min, followed by heat shock for 5-20 min. The bacterial culture was then spread onto leucine-free auxotropic agar plates and incubated at 28℃ for 2 hours. After 3 days, the culture medium was obtained.

[0063] For example, in this embodiment, under aseptic conditions, polyethylene glycol (PEG) (PEG with a molecular weight of 6000, denoted as PEG6000) (50% w / v, 100 mL of solution contains 50 g PEG6000), 1 mol / L lithium acetate solution, and 10 mg / mL denatured salmon sperm DNA (total DNA extracted from a large number of salmon sperm, i.e., the ssDNA mentioned above) were mixed in a 1.5 mL sterile centrifuge tube in a certain proportion. Then, a single yeast colony formed after culturing an engineered strain of *Yeastra lipolytica* was added and the mixture was thoroughly resuspended.

[0064] Add approximately 300 ng of purified pYLXP'- to the transformation system Nodgat2g The vector was incubated at 30°C for 35 minutes, followed by heat shock at 39°C for 10 minutes. After transformation, the cells were resuspended in 100 μL of sterile deionized water and plated on Complete Supplement Mixtureminus Leucine (CSM-LEU) plates. After incubation at 30°C for 48-72 hours, positive clones were selected for subsequent scale-up culture and fermentation verification.

[0065] 2025: Separate the culture medium to obtain the extract.

[0066] The culture medium was centrifuged, vortexed, and extracted to obtain the extract.

[0067] 2026: The extract was separated by thin-layer chromatography and detected by liquid chromatography to obtain esterified astaxanthin.

[0068] The extract was separated by thin-layer chromatography (TLC) and detected by high-performance liquid chromatography (HPLC) to obtain and identify esterified astaxanthin.

[0069] For example, in this embodiment, 5 mL of the fermented culture broth was centrifuged at 4000×g for 10 min to collect the cell precipitate. 5 mL of ethyl acetate and an appropriate amount of 2 mm diameter glass beads were added to the cell precipitate, and the mixture was thoroughly extracted using a vortex mixer at 2500 rpm for 50 minutes, dissolving the carotenoids in the upper organic phase. During the high-speed vortex mixing, the glass beads collided and rubbed violently with the yeast cells. This mechanical motion generated strong shear force, effectively grinding and breaking down the tough cell walls, thereby releasing carotenoids, lipids, proteins, and other substances from within the cells into the surrounding solution.

[0070] Then, thin-layer chromatography was performed to separate and recover the pigments. The oil phase sample obtained from the transformed *Yarrowia lipolytica* fermentation extract was dried under nitrogen, redissolved in 20 μL of chloroform, and spotted onto a silica gel plate (on the starting line). The sample on the silica gel plate was then initially separated. For the first development (i.e., the first separation), a hexane-acetone mixture (3:2, v / v) was used as the mobile phase. After the solvent front reached the top of the plate, a second development was performed using freshly prepared developing solvent of the same proportion, resulting in different types of carotenoid bands on the silica gel plate. After the carotenoid bands developed, silica gel powder from the target band area was carefully scraped off with a clean blade, transferred to pre-weighed filter paper, and finally collected in a 4 mL brown sample vial. 1 mL of ethyl acetate and several 2 mm diameter glass beads were added. The sample was extracted by vortexing at 2500 rpm for 10 minutes, followed by centrifugation at 3500 × g for 10 minutes. The collected supernatant was filtered through a 0.22 μm hydrophobic membrane and then analyzed by high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS).

[0071] Next, the types of carotenoids were analyzed using LC-MS. A Thermo Scientific U3000 ultra-high performance liquid chromatography system, equipped with a diode array detector and a QExactive high-resolution mass spectrometer, was used for chromatographic separation. A YMCC30 reversed-phase column (4.6 × 250 mm, 5 μm) was used for separation. The mobile phase composition was: phase A was methanol-methyl tert-butyl ether-water (81:15:4, v / v / v), and phase B was methanol-methyl tert-butyl ether-water (7:90:3, v / v / v). The gradient elution program was as follows: phase B was linearly increased from 0% to 100% within 0–60 min, the flow rate was 1.0 mL / min, the column temperature was 30 °C, and the detection wavelength was 450 nm. Atmospheric pressure chemical ionization (APCI) was used for detection in positive ion mode. The main parameters were set as follows: nebulizer gas flow rate 45 arbitrary units, auxiliary gas flow rate 15 arbitrary units, ionization voltage 5 kV, capillary temperature 320℃, and auxiliary gas heating temperature 300℃. The full scan range was set to m / z 500-1400, and the data-dependent secondary mass spectrometry acquisition mode was used. XcaliburQualBrowser software was used for spectral analysis, and the molecular ion peak was extracted using the precise mass number (m / z) of the target compound. The mass error tolerance was set to 5 ppm, and the mass accuracy was retained to four decimal places.

[0072] The following specific examples further illustrate the application of the protein provided in the embodiments of this disclosure in the production of esterified astaxanthin.

[0073] First, verification of the gene function of the key protein used for astaxanthin esterification: When the expression vector pYLXP'- Nodgat2g After being transferred into the DH5α strain, the colonies produced by the DH5α strain were detected by PCR as follows: Figure 1 As shown. Among them, Figure 1 In this context, bp stands for base pair, which is used to represent the length of a DNA fragment. For example, 1000bp = 1000 base pairs. Figure 1 The DL (DNA Ladder / Marker) in this context refers to a mixture of DNA fragments of known length (including 5000bp, 3000bp, 2000bp, etc.). Its function is to "determine" the size of the sample bands; by comparing the position of the sample bands with the marker bands, the size of the DNA fragments in the sample can be determined. Figure 1 The successful construction of the vector was verified by sequencing.

[0074] The expression vector pYLXP' -Nodgat2g Heterologous expression of the gene was validated in the astaxanthin-producing *Yarrowia lipolytica* strain nAst6. TLC analysis revealed that the expression... Nodgat2gAstaxanthin monoesters and diesters appeared in the carotenoid extracts of the strain, while no astaxanthin was expressed. Nodgat2g These bands were not detected in the control strains.

[0075] Combination Figure 2 , Figure 2 NoDGAT2G in the image is the sample source identifier, indicating that these carotenoid components come from extracts containing the NoDGAT2G protein. The vertical bands in the figure represent the separation results, showing different carotenoid components. The higher the band, the faster the different carotenoids migrate in the sample spotted on the silica gel plate; the lower the band, the slower the migration. TLC separation allows for a direct visual representation of the types and relative amounts of carotenoid components in the sample (band brightness reflects concentration; brighter bands indicate higher concentrations). These results indicate that the carotenoids from the marine microalgae strain IMET1... Nodgat2g The *Yersinia lipophila* nAst6 strain exhibited significant esterification and astaxanthinization activity. This indicates that the NoDGAT2G protein (composed of...) is... Nodgat2g The gene (encoded) can catalyze the esterification reaction of astaxanthin in Yersinia lipolytica, generating various astaxanthin monoesters and diesters.

[0076] To further clarify the types of esterified astaxanthin catalyzed by the NoDGAT2G protein, the carotenoid extracts of strains expressing the NoDGAT2G protein were analyzed by liquid chromatography-mass spectrometry (LC-MS). The results showed that the main types of astaxanthin monoesters included astaxanthin-C16:0 (palmitoyl astaxanthin monoester), astaxanthin-C16:1 (palmitoyl oleate astaxanthin monoester), astaxanthin-C16:2 (palmitodienoic acid astaxanthin monoester), astaxanthin-C18:0 (stearic acid astaxanthin monoester), astaxanthin-C18:1 (oleic acid astaxanthin monoester), and astaxanthin-C18:2 (linoleic acid astaxanthin monoester). The main types of astaxanthin diesters include astaxanthin-C18:0-C18:0 (distearate astaxanthin ester), astaxanthin-C18:1-C18:1 (dioleoate astaxanthin ester), astaxanthin-C18:2-C18:2 (dilinoleoate astaxanthin ester), astaxanthin-C18:0-C18:1 (stearic acid-oleic acid astaxanthin ester), astaxanthin-C16:0-C16:0 (dipalmitoate astaxanthin ester), and astaxanthin-C16:1-C16:1 (dipalmitoate astaxanthin ester).

[0077] Combination Figure 3 ,in, Figure 3 The horizontal lines represent amino acid sequence fragments of DGAT2 family proteins from different species (such as ScDGAT2-1, HpDGTT4, NoDGAT2G, etc.), and the numbers (590-850) are amino acid position numbers. Figure 3The vertical axis represents "amino acids at the same position in different proteins." Amino acids marked with a red background are highly conserved (such as the His site in the image), indicating that these sites are extremely crucial to protein function (often serving as enzyme active sites or structural support sites). Through multi-species sequence alignment, conserved amino acids (especially His) can be identified, suggesting that these sites are "functional tags" for DGAT2 family proteins. Mutating these sites may significantly reduce protein activity, which can be verified through site-directed mutagenesis experiments.

[0078] The specific amino acid site formed by histidine (Histidine, His, H) is called the His site. Figure 3 The site corresponding to the red triangle in the middle is the His site, also known as the His-352 site.

[0079] Second, key site validation: Multiple sequence alignment analysis of the DGAT2 protein, which has the function of esterifying astaxanthin, was performed using Molecular Evolutionary Genetics Analysis (MEGA) software. The analysis revealed a highly conserved histidine (His) site in its sequence. Figure 3 (The location marked by the red triangle in the middle).

[0080] Combination Figure 4 , Figure 4 Each letter (AE) in the diagram corresponds to a three-dimensional structural model of a DGAT2 family protein (distinguished by different colors, e.g., green for ScDGAT2-1 and beige for NoDGAT2G), constructed through computer simulation or experimental analysis (e.g., X-ray crystallography, cryo-electron microscopy). The red arrows with His numbers mark the specific locations of conserved histidine residues (His) in the three-dimensional structure (e.g., His644 for ScDGAT2-1 and His352 for NoDGAT2G), indicating that these key amino acids aggregate in three-dimensional space to form the protein's "functional center" (e.g., an enzyme activity pocket). Figure 4 As can be seen, conserved His sites are not only concentrated in the sequence, but also occupy key positions in the three-dimensional structure, which once again verifies that "these sites are the functional core".

[0081] To further verify whether this His site is a key site for catalyzing astaxanthin esterification, we used site-directed mutagenesis to mutate the conserved His site in the sequence to glycine (Gly), and constructed a mutant plasmid (pYLXP') in E. coli DH5α. -Nodgat2g-H352G), and selected the correct transformants for sequencing. Subsequently, these plasmids were transformed into *Yarrowia lipolytica* Ast6 for fermentation verification. Thin-layer chromatography (TLC) analysis revealed that only free astaxanthin was detected in the mutant, while the esterified astaxanthin band completely disappeared, indicating that the mutant lost its ability to catalyze esterification of astaxanthin. This site may directly participate in catalytic reactions or maintain the structural stability of the active site in the protein. Among these, binding... Figure 5 Sequencing confirmed pYLXP' -Nodgat2g The H352G carrier was successfully constructed.

[0082] It should be noted that the eight bands here are not a comparison between the experimental group and the control group, but rather the validation results of eight independent single clones.

[0083] Combination Figure 6 Thin-layer chromatography (TLC) analysis revealed that only free astaxanthin was detectable in the mutant, while the band for esterified astaxanthin completely disappeared, indicating that the mutant lost its ability to catalyze esterification of astaxanthin. This site may directly participate in catalytic reactions or maintain the structural stability of the active site in the protein.

[0084] Third, verification of the catalytic efficiency of the mutant: (1) Design and validation of site mutations based on predictions: Five key amino acid residues were selected for mutation design, including L398N, L398G, V403A, V403R, and Q425A.

[0085] The following mutant plasmid, pYLXP'-, was constructed in Escherichia coli DH5α. Nodgat2g -L398N、pYLXP'- Nodgat2g -L398G、pYLXP'- Nodgat2g -V403A、pYLXP'- Nodgat2g -V403R and pYLXP'- Nodgat2g -Q425A.

[0086] Among them, pYLXP'- Nodgat2g -L398N refers to pYLXP'- Nodgat2g Based on this, the amino acid L (leucine, Leu, L) at position 398 is mutated to amino acid N (asparagine, Asn, N). Other amino acids are similar.

[0087] PCR testing, such as Figure 7 The correct transformants were selected and sequenced. The above mutations were constructed in plasmid form in the *Yarrowia lipolytica* strain, which produces high levels of free astaxanthin, and their ability to catalyze astaxanthin esterification was analyzed by TLC.

[0088] Combination Figure 7 The successful construction of the vector was verified by sequencing.

[0089] Combination Figure 8 , Figure 8 The yellow / orange bands on the thin-layer chromatography plate represent different types of carotenoids. The NoDGAT2G protein (wild-type) is located on the lower side. Nodgat2g The gene-encoded protein), V403R / V403A (a NoDGAT2G protein mutant encoded by a gene point mutant) represent experimental samples transformed with different genes.

[0090] Compared to wild-type NoDGAT2G, TLC results from mutants V403A and Q425A showed that the bands for astaxanthin monoesters and diesters remained, and the Q425A mutant exhibited a significantly deeper color in both monoester and diester bands, indicating potentially enhanced catalytic activity. This result suggests that V403 and Q425 residues may play an important role in the catalytic function of NoDGAT2G. The substitution of V403 and Q425 residues may optimize the catalytic activity or substrate binding capacity of NoDGAT2G, expanding the substrate binding pocket volume. In particular, the increased color of the esterification product band in the Q425A mutant suggests that this mutation may enhance the enzyme's affinity for the substrate or its catalytic efficiency. These findings provide important clues for further research into the catalytic mechanism of the NoDGAT2G protein.

[0091] (2) Design and validation of point mutations based on protein pocket volume expansion: The following five amino acid residues were mutated to Ala (alanine): Phe447 (Phe = phenylalanine), Pro422 (Pho = proline), Gln354 (Gln = glutamine), Met385 (Met = methionine), Gln174 (Gln = glutamine), and Leu420 (Leu = leucine). Corresponding mutants were constructed in *E. coli* DH5α, and the PCR results are shown below. Figure 9 As shown. Combined with Figure 9 The successful construction of the vector was verified by sequencing.

[0092] Subsequently, thin-layer chromatography (TLC) analysis was performed; the results are shown below. Figure 10 . Figure 10The yellow / orange bands on the thin-layer chromatography plate represent different types of carotenoids. Below, Q354A, M385A, etc., are different point mutants, representing experimental samples with different genes introduced. The results showed that the catalytic activity of mutant Q354A was significantly enhanced, with both astaxanthin monoester and diester bands clearly visible, and the monoester band being darker in color than the wild-type NoDGAT2G. This result indicates that the Q354A mutation may optimize the structure of the binding pocket, thereby improving the enzyme's catalytic efficiency. Furthermore, based on previous experimental results, we screened four mutants that retained esterification function: Q354A, V403A, Q425A, and Q174A. The successful construction of these mutants suggests that Q354, V403, Q425, and G174 may be key sites for enhancing the ability of NoDGAT2G to esterify astaxanthin. Based on the results of the mutation validation assay, we selected three additional mutations that might improve catalytic efficiency: Q354W / V403F / Q425W for experimental verification. The PCR results are as follows: Figure 11 As shown. Combined with Figure 11 Sequencing confirmed successful vector construction. TLC analysis showed that the astaxanthin monoester and diester bands of the mutant Q354W were still present. Figure 12 This indicates that it retains its esterification function.

[0093] (3) Design and validation of distant mutants: The following mutant plasmid was constructed in Escherichia coli DH5α: pYLXP'- Nodgat2g -E99G,pYLXP'- Nodgat2g -A268W, pYLXP'- Nodgat2g -D323M,pYLXP'- Nodgat2g -C359L and pYLXP'- Nodgat2g -P501Q, PCR detection as follows Figure 13 .

[0094] Combination Figure 13 The successful construction of the vector was verified by sequencing. Then, the correct transformants were selected for sequencing. Subsequently, these plasmids were transformed into a high-astaxanthin-producing *Yarrowia lipolytica* chassis strain for fermentation verification. TLC analysis results are as follows: Figure 14 As shown.

[0095] Figure 14 The yellow / orange bands on the thin-layer chromatography plate represent different types of carotenoids. The bands on the lower side, such as E99G, A268W, D323M, and C359L, are different point mutants, representing experimental samples with different genes introduced. From... Figure 14As can be seen, the E99G, A268W, D323M, and C359L mutants retained esterification function. Among them, the monoester and diester bands of the D323M mutant were significantly darker than those of the wild-type control, indicating that its catalytic efficiency may be improved. This result suggests that the D323M mutation may enhance catalytic activity by optimizing substrate binding or transition state stability.

[0096] (4) Design and validation of combinatorial mutations: The mutants specifically constructed in E. coli include: single mutations and stacking mutations: pYLXP'- Nodgat2g- Q354A+V403A、pYLXP'- Nodgat2g- Q354A+Q425A、pYLXP'- Nodgat2g- Q425A+V403A、pYLXP'- Nodgat2g- Q354A+V403A+Q425A, among which some mutants have PCR results as follows: Figure 15 As shown. Combined with Figure 15 The successful construction of the vector was verified by sequencing.

[0097] Transformants with the correct bands were then selected and sequenced for verification before being transformed into a high-astaxanthin-producing *Yarrowia lipolytica* chassis strain for fermentation. Thin-layer chromatography (TLC) analysis was performed, and the results are as follows: Figure 16 As shown. Figure 16 The yellow / orange bands on the thin-layer chromatography plate represent different types of carotenoids. The Q354A+V403A bands on the lower side represent different gene point mutants, indicating experimental samples with different genes introduced. Experimental results showed that compared to wild-type NoDGAT2G, except... Nodgat2g The astaxanthin monoester and diester bands of mutants other than -Q354A+Q425A still exist, but their esterification function is not significantly improved.

[0098] To further investigate the effects of mutant combinations on esterification function, the following double mutant combinations were also constructed: Nodgat2g -Q174A+Q354A、 Nodgat2g -Q174A+Q354W, Nodgat2g -Q174A+V403A、 Nodgat2g -Q174A+Q425A、 Nodgat2g -Q354W+V403A、 Nodgat2g -Q354W+Q425A, PCR results are as follows Figure 17 As shown. Combined with Figure 17 The successful construction of the vector was verified by sequencing.

[0099] Subsequently, transformants with the correct bands were selected and sequenced for verification before being transformed into a high-astaxanthin-producing *Yarrowia lipolytica* chassis strain for fermentation. TLC analysis results showed... Figure 18 As shown.

[0100] Figure 18 The yellow / orange bands on the thin-layer chromatography plate represent different types of carotenoids. The Q174A+Q425A bands on the lower side represent different gene point mutants, indicating experimental samples transformed with different genes. All double-mutant combinations retained esterification function, and Nodgat2g -Q174A+Q425A and Nodgat2g The Q354W+Q425A diester band was significantly darker than the control. This suggests that the mutant combinations of Q174A+Q425A and Q354W+Q425A may be more beneficial in improving the catalytic efficiency of NoDGAT2G.

[0101] Based on the above findings, we further investigated the identified high-efficiency mutants. Nodgat2g -Q354W+Q425A and Nodgat2g Based on -Q174A+Q425A, D323M and E99G mutations were superimposed to explore the synergistic effect of multiple-point mutations on catalytic efficiency. PCR results are as follows: Figure 19 Combining Figure 19 The successful construction of the vector was verified by sequencing.

[0102] Transformants with the correct bands were then sequenced and verified before being transformed into a high-astaxanthin-producing *Yarrowia lipolytica* chassis strain for fermentation. However, TLC analysis results showed... Figure 20 As shown. Figure 20 The yellow / orange bands on the thin-layer chromatography plate represent different types of carotenoids. The Q354W+Q425A and other sites on the lower side represent different point mutants, indicating experimental samples with different genes introduced. Although all triple mutant combinations (such as Q174A+Q425A+D323M, Q354W+Q425A+E99G, etc.) retained esterification function, their catalytic efficiency was not significantly higher than that of the double mutant Q174A+Q425A. This indicates that the double mutant Q174A+Q425 is a key residue affecting catalytic efficiency; their mutations produced the most significant optimization effect on catalytic efficiency, while other sites (such as D323, Q354, E99) may play a relatively minor or structural role in this function.

[0103] The above description is merely an optional embodiment of this disclosure and is not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.

Claims

1. A key protein for esterification of astaxanthin, characterized in that, The amino acid sequence of the protein is more than 90% identical to the amino acid sequence shown in SEQ ID NO:

1.

2. The protein according to claim 1, characterized in that, The gene sequence encoding the protein is derived from Nannochloropsis Nodgat2g gene sequence.

3. The protein of claim 1, wherein The protein in question is a mutant of the NoDGAT2G protein that retains the His-352 site.

4. The protein of claim 3, wherein, The mutant was obtained by site-directed mutagenesis of the NoDGAT2G protein.

5. A method for producing esterified astaxanthin, characterized by, The protein of any one of claims 1-4 is expressed in a host cell that produces astaxanthin to catalyze the production of esterified astaxanthin in the host cell.

6. The production method according to claim 5, characterized by, The expression of the protein according to any one of claims 1-4 in the host cell to catalyze the production of esterified astaxanthin in the host cell comprises: Based on the protein, an expression vector for esterified astaxanthin was constructed; The expression vector was transformed into the host cells and cultured to catalyze the production of esterified astaxanthin in the host cells.

7. The production method according to claim 6, characterized in that, The expression vector comprises elements capable of driving Nodgat2g expression in a host cell.

8. The production method according to claim 7, characterized in that, The element comprises pYLXP' Nodgat2g The vector or pYLXP' Nodgat2g Mutants of the vector; The mutants mentioned therein include single-site site-directed mutants, two-site site-directed mutants, or multi-site site-directed mutants.

9. The production method according to claim 6, characterized in that, The step of transforming the expression vector into the host cells and culturing them to catalyze the production of esterified astaxanthin by the host cells includes: The expression vector was transformed into the host cells to obtain recombinant host cells; The recombinant host cells are cultured to express the protein and catalyze the synthesis of esterified astaxanthin, resulting in a culture medium containing esterified astaxanthin. The esterified astaxanthin was obtained by separating and purifying the culture medium.

10. The production method according to claim 6, characterized in that, The host cells are selected from engineered strains of the following microorganisms: Yeastra lipolytica, Saccharomyces cerevisiae, Escherichia coli, Schizochytrium, or microalgae.

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