Fusion protein CrtZW and application thereof in catalyzing beta-carotene to produce astaxanthin

Abstract

The invention provides a fusion protein CrtZW and application thereof in catalyzing beta-carotene to produce astaxanthin, and relates to the technical field of astaxanthin synthesis, the sequence of the fusion protein CrtZW is shown as SEQ ID NO.4, the fusion protein can catalyze beta-carotene to synthesize astaxanthin in one step, and the fusion protein CrtZW is suitable for large-scale production.

Description

Technical Field

[0001] The invention relates to the technical field of astaxanthin synthesis, and more specifically to a fusion protein CrtZW and application thereof in catalyzing beta-carotene to produce astaxanthin. Background Art

[0002] Astaxanthin is a strong antioxidant that can be used in food, health care, beauty, medicine and other fields. There are two main methods for producing astaxanthin: chemical synthesis and biosynthesis. The sources of astaxanthin in the biosynthesis method include Haematococcus pluvialis, Phaffia rhodozyma, Paracoccus, recombinant Escherichia coli, and recombinant Yarrowia lipolytica.

[0003] Astaxanthin has two chiral carbon atoms and three conformations: SS, SR, and RR. Only SS has biological activity. In the chemical synthesis process, the SS conformation accounts for only 25% of the total and has low biological activity. The biosynthesis process has a long fermentation time, low product concentration, and high subsequent purification costs.

[0004] β-carotene can be converted into astaxanthin through a two-step catalytic reaction: β-carotene is converted into zeaxanthin under the catalysis of CrtZ enzyme, and zeaxanthin is converted into astaxanthin under the catalysis of CrtW enzyme; or β-carotene is converted into canthaxanthin under the catalysis of CrtW enzyme, and canthaxanthin is converted into astaxanthin under the catalysis of CrtZ enzyme. The two-step method of catalyzing β-carotene to astaxanthin has many steps, a long time, and a low yield.

[0005] Therefore, providing a method for efficiently catalyzing β-carotene to produce astaxanthin is a technical problem that those skilled in the art urgently need to solve. Summary of the invention

[0006] In view of this, the present invention provides a fusion protein CrtZW and its application in catalyzing the production of astaxanthin from β-carotene.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A fusion protein CrtZW, the amino acid sequence of which is as shown in SEQ ID NO.4:

[0009] MLWIWNALIVFVTVIGMEVIAALAHKYIMHGWGWGWHLSHHEPRKGAFEVNDLYAVVFAALSILLIYLGSTGMWPLQWIGAGMTAYGLLYFMVHDGLVHQRWPFRYIPRKGYLKRLYMAHRMHHAVRGKEGCVSFGFLYAPPLSKLGGGGSGGGGSGGGGSGGGGSEAAAKEAAAKGGGGSGGGGSGGGGSRQTWIGLTLAGMIVAGW GSLHVYGVYFHRWGTSSLVIVPAIVAVQTWLSVGLFIVAHDAMHGSLAPGRPRLNAAVGRLTLGLYAGFRFDRLKTAHHAHHAAPGTADDPDFYAPAPRAFLPWFLNFFRTYFGWREMAVLTALVLIALFGLGARPANLLTFWAAPALLSALQLFTFGTWLPHRHTDQPFADAHHARSSGYGPVLSLLTCFHFGRHHEHHLTPW, as in SEQ. ID NO.4.

[0010] As an inventive concept identical to the above technical solution, the present invention also seeks to protect the use of the fusion protein CrtZW in one-step catalysis of β-carotene to synthesize astaxanthin.

[0011] It can be seen from the above technical scheme that the present invention obtains the recombinant fusion protein CrtZW by truncating the specific sequences of the CrtZ enzyme and the CrtW enzyme and then connecting them through a link sequence. The fusion protein can catalyze the synthesis of astaxanthin from β-carotene in one step. Compared with the existing technology, it saves the process flow and can be applied to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The attached figure is a secondary structure diagram of the original protein of Pantoea ananatis CrtZ;

[0013] Figure 2 The attached figure is a secondary structure diagram of the original protein of Brevundimonas sp.SD212CrtW;

[0014] Figure 3 The attached figure is a secondary structure diagram of the fused protein CrtZW;

[0015] Figure 4 The attached figure is a graph showing the astaxanthin content of different treatment groups;

[0016] Figure 5 The attached figure shows the astaxanthin concentration under different truncation methods. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] Example 1 Construction of fusion protein

[0019] 1. The original protein sequence of Pantoea ananatis CrtZ was truncated. The original sequence is as follows:

[0020] MLWIWNALIVFVTVIGMEVIAALAHKYIMHGWGWGWHLSHHEPRKGAFEVNDLYAVVFAALSILLIYLGSTGMWPLQWIGAGMTAYGLLYFMVHDGLVHQRWPFRYIPRKGYLKRLYMAHRMHHAVRGKEGCVSFGFLYAPPLSKL For example, SEQ ID NO.1, where the underlined part is the truncated sequence; the original protein structure was constructed using ALPHAFOLD MONOMER V2.0, and the three-dimensional structure of the enzyme was drawn using VMD software. The secondary structure of the enzyme is shown using the Cartoon drawing method, and the purple grid shows the substrate binding pocket, see Figure 1 .

[0021] 2. The original protein sequence of Brevundimonas sp.SD212CrtW was truncated. The original sequence is as follows:

[0022] PRQTWIGLTLAGMIVAGWGSLHVYGVYFHRWGTSSLVIVPAIVAVQTWLSVGLFIVAHDAMHGSLAPGRPRLNAAVGRLTLGLYAGFRFDRLKTAHHAHHAAPGTADDPDF YAPAPRAFLPWFLNFFRTYFGWREMAVLTALVLIALFGLGARPANLLTFWAAPALLSALQLFTFGTWLPHRHTDQPFADAHHARSSGYGPVLSLLTCFHFGRHHEHHLTPW As shown in SEQ ID NO.2, the underlined part is the truncated sequence. The structure was constructed using ALPHAFOLD MONO MER V2.0, and the three-dimensional structure of the enzyme was drawn using VMD software. The secondary structure of the enzyme is shown using the Cartoon drawing method. The purple grid shows the substrate binding pocket. The structure is shown in Figure 2 .

[0023] 3. Connect the above truncated sequences with the link sequence:

[0024] The link sequence is as follows:

[0025] GGGGSGGGGSGGGGSGGGGSEAAAKEAAAKGGGGSGGGGSGGGG S, such as SEQ ID NO.3;

[0026] The sequence of the fused protein CrtZW is as follows:

[0027] MLWIWNALIVFVTVIGMEVIAALAHKYIMHGWGWGWHLSHHEPRK

[0028] GAFEVNDLYAVVFAALSILLIYLGSTGMWPLQWIGAGMTAYGLLYFMVH

[0029] DGLVHQRWPFRYIPRKGYLKRLYMAHRMHHAVRGKEGCVSFGFLYAPPL

[0030] SKLGGGGSGGGGSGGGGSGGGGSEAAAKEAAAKGGGGSGGGGSGGGGS

[0031] RQTWIGLTLAGMIVAGWGSLHVYGVYFHRWGTSSLVIVPAIVAVQTWLS

[0032] VGLFIVAHDAMHGSLAPGRPRLNAAVGRLTLGLYAGFRFDRLKTAHHAH

[0033] HAAPGTADDPDFYAPAPRAFLPWFLNFFRTYFGWREMAVLTALVLIALFG

[0034] LGARPANLLTFWAAPALLSALQLFTFGTWLPHRHTDQPFADAHHARSSGY

[0035] GPVLSLLTCFHFGRHHEHHLTPW, such as SEQ ID NO.4.

[0036] The structure of the fusion protein is Figure 3 As shown in the figure, it can be seen that after being connected with appropriate link sequences, the two proteins can independently form their own structures without much influence on each other.

[0037] Example 2 Comparison of different catalytic methods

[0038] According to the codon preference of Pichia pastoris, the gene was designed and synthesized. Pichia pastoris was used as the chassis cell to express the recombinant Pantoea ananatis CrtZ, Brevundimonas sp.SD212CrtW and CrtZW. The reaction solution was prepared according to different enzyme addition methods, and the catalytic effects of different enzymes and addition methods were compared. 1ml reaction solution was prepared as follows: substrate + enzyme solution + buffer = 400μL + 400μL + 200μL.

[0039] The substrate solution formula is: 0.3 mg / ml beta-carotene Tween 60 solution; the buffer solution formula is: 0.4 M Tris-HCl (pH 7.0), containing 1 mM dithiothreitol, 0.1% v / v Tween 60, 3 mM ATP, 0.5 mM ferrous sulfate, 0.5 mM 2-oxoglutaric acid, and 5 mM ascorbic acid.

[0040] The groups are as follows:

[0041] CrtZ→CrtW: first add CrtZ and react for 3 h, then add CrtW and continue to react for 3 h;

[0042] CrtW→CrtZ: first add CrtW and react for 3 h, then add CrtZ and continue to react for 3 h;

[0043] CrtZ+CrtW: CrtZ and CrtW were added simultaneously and reacted for 3 h;

[0044] CrtZW: Add CrtZW and react for 3 h;

[0045] Statistics of astaxanthin content in each group, see Figure 4 .

[0046] from Figure 4 It can be seen that the final astaxanthin content obtained by adding two enzymes at the same time is significantly lower than the catalytic method of adding two enzymes successively. The catalytic effect of fusion protein is significantly better than the other catalytic methods.

[0047] Example 3 Catalytic differences between different links connecting CrtZ and CrtW

[0048] Different link sequences were selected to connect CrtZ and CrtW proteins to form different fusion proteins. Genes were designed and synthesized according to the codon preference of Pichia pastoris, and Pichia pastoris was used as a chassis cell to express the recombinant fusion protein. The link sequences are shown in Table 1;

[0049] Table 1 Comparison of astaxanthin production catalyzed by fusion proteins connected with different link sequences

[0050]

[0051]

[0052] As can be seen from Table 1, after selecting the sequence SEQ ID NO.3 as the link sequence to connect CrtZ and Crt W, the effect of catalyzing astaxanthin synthesis is the best.

[0053] Example 4 Comparison of catalytic effects of different truncation methods

[0054] To avoid the protein being too long and causing incorrect folding after fusion that affects enzyme activity, the two enzymes were truncated separately, leaving only the active site. The different truncated proteins were connected through the link sequence and recombinantly expressed in Pichia pastoris to detect the catalytic differences of each fusion protein;

[0055] The different truncated protein sequences are as follows:

[0056] CrtZ C-terminal truncation

[0057] MLWIWNALIVFVTVIGMEVIAALAHKYIMHGWGWGWHLSHHEPRKGAFEVNDLYAVVFAALSILLIYLGSTGMWPLQWIGAGMTAYGLLYFMVHDGLVHQRWPFRYIPRKGYLKRLYMAHRMHHAVRGKEGCVSFGFLYAPPLSKL, such as SEQ ID NO. 12.

[0058] CrtZ N-terminal truncation

[0059] TVIGMEVIAALAHKYIMHGWGWGWHLSHHEPRKGAFEVNDLYAVVFAALSILLIYLGSTGMWPLQWIGAGMTAYGLLYFMVHDGLVHQRWPFRYIPRKGYLKRLYMAHRMHHAVRGKEGCVSFGFLYAPPLSKLQATLRERHGARAGAARDAQGGEDEPASGK, such as SEQ ID NO.13.

[0060] CrtW C-terminal truncation

[0061] MTAAVAEPRIVPRQTWIGLTLAGMIVAGWGSLHVYGVYFHRWGTSSLVIVPAIVAVQTWLSVGLFIVAHDAMHGSLAPGRPRLNAAVGRLTLGLYAGFRFDRLKTAHHAHHAAPGTADD PDFYAPAPRAFLPWFLNFFRTYFGWREMAVLTALVLIALFGLGARPANLLTFWAAPALLSALQLFTFGTWLPHRHTDQPFADAHHARSSGYGPVLSLLTCFHFGRHHEHHLTPW as SEQ ID NO.14.

[0062] CrtW N-terminal truncation

[0063] RQTWIGLTLAGMIVAGWGSLHVYGVYFHRWGTSSLVIVPAIVAVQTWLSVGLFIVAHDAMHGSLAPGRPRLNAAVGRLTLGLYAGFRFDRLKTAHHAHHAAPGTADDPDFYAPAPRAF LPWFLNFFRTYFGWREMAVLTALVLIALFGLGARPANLLTFWAAPALLSALQLFTFGTWLPHRHTDQPFADAHHARSSGYGPVLSLLTCFHFGRHHEHHLTPWRPWWRLWRGES, as in SEQ ID NO.15.

[0064] Results Figure 5 ,from Figure 5 It can be seen that protein truncation has a significant effect on astaxanthin synthesis, but the N-terminus of CrtZ has a greater negative effect on the structure of the fusion protein, and truncation can significantly reduce the catalytic effect. The catalytic effect is best after the C-terminus of CrtZ is truncated / the CN-terminus of CrtW is truncated at the same time.

[0065] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0066] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A fusion protein CrtZW, characterized in that The amino acid sequence is shown in SEQ ID NO.4: MLWIWNALIVFVTVIGMEVIAALAHKYIMHGWGWGWHLSHHEPRKGAFEVNDLYAVVFAALSILLIYLGSTGMWPLQWIGAGMTAYGLLYFMVHDGLVHQRWPFRYIPRKGYLKRLYMAHRMHHAVRGKEGCVSFGFLYAPPLSKLGGGGSGGGGSGGGGSGGGGSEAAAKEAAAKGGGGSGGGGSGGGGSRQTWIGLTLAGMIVAGW GSLHVYGVYFHRWGTSSLVIVPAIVAVQTWLSVGLFIVAHDAMHGSLAPGRPRLNAAVGRLTLGLYAGFRFDRLKTAHHAHHAAPGTADDPDFYAPAPRAFLPWFLNFFRTYFGWREMAVLTALVLIALFGLGARPANLLTFWAAPALLSALQLFTFGTWLPHRHTDQPFADAHHARSSGYGPVLSLLTCFHFGRHHEHHLTPW, as in SEQ ID NO.

4.

2. Use of the fusion protein CrtZW as claimed in claim 1 in one-step catalysis of β-carotene to synthesize astaxanthin.

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