A fusion protein CrtZW and its application in catalyzing the production of astaxanthin from β-carotene

Abstract

The present invention provides a fusion protein CrtZW and its application in catalyzing the production of astaxanthin from β-carotene, which relates to the technical field of astaxanthin synthesis. The sequence of the fusion protein CrtZW is shown in SEQ ID NO.4. This fusion protein can catalyze the synthesis of astaxanthin from β-carotene in one step and is suitable for large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of astaxanthin synthesis, and more particularly to a fusion protein CrtZW and its application in catalyzing the production of astaxanthin from β-carotene. Background Art

[0002] Astaxanthin is a super antioxidant and can be applied to the fields of food, health care, beauty, medicine, etc. The production of astaxanthin mainly includes two methods: chemical synthesis method and biosynthesis method. The sources of astaxanthin in the biosynthesis method include Haematococcus pluvialis, Phaffia rhodozyma, Paracoccus, recombinant Escherichia coli, recombinant Yarrowia lipolytica, etc.

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

[0004] β-carotene can be converted into astaxanthin through two-step catalytic reactions: β-carotene is catalyzed by CrtZ enzyme to form zeaxanthin, and zeaxanthin is catalyzed by CrtW enzyme to form astaxanthin; or β-carotene is catalyzed by CrtW enzyme to form canthaxanthin, and canthaxanthin is catalyzed by CrtZ enzyme to form astaxanthin. The two-step catalytic conversion of β-carotene to astaxanthin has many steps, a long time, and a low yield.

[0005] Therefore, providing a method for efficiently catalyzing the production of astaxanthin from β-carotene 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] To achieve the above object, the present invention adopts the following technical scheme:

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

[0009] MLWIWNALIVFVTVIGMEVIAALAHKYIMHGWGWGWHLSHHEPRKGAFEVNDLYAVVFAALSILLIYLGSTGMWPLQWIGAGMTAYGLLYFMVHDGLVHQRWPFRYIPRKGYLKRLYMAHRMHHAVRGKEGCVSFGFLYAPPLSKLGGGGSGGGGSGGGGSGGGGSEAAAKEAAAKGGGGSGGGGSGGGGSRQTWIGLTLAGMIVAGWGSLHVYGVYFHRWGTSSLVIVPAIVAVQTWLSVGLFIVAHDAMHGSLAPGRPRLNAAVGRLTLGLYAGFRFDRLKTAHHAHHAAPGTADDPDFYAPAPRAFLPWFLNFFRTYFGWREMAVLTALVLIALFGLGARPANLLTFWAAPALLSALQLFTFGTWLPHRHTDQPFADAHHARSSGYGPVLSLLTCFHFGRHHEHHLTPW, such as SEQ ID NO.4.

[0010] As an invention concept identical to the above technical solution, the present invention also claims the application of the fusion protein CrtZW in the one-step catalysis of the synthesis of astaxanthin from β-carotene.

[0011] As can be seen from the above technical solution, the present invention truncates the specific sequences of the CrtZ enzyme and the CrtW enzyme, and then connects them through the link sequence to obtain the recombinant fusion protein CrtZW. This fusion protein can achieve the one-step catalysis of the synthesis of astaxanthin from β-carotene. Compared with the prior art, it saves the process flow and can be applied to industrial production. Brief Description of the Drawings

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

[0013] Figure 2 The drawing is the secondary structure diagram of the original protein of Brevundimonas sp. SD212 CrtW;

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

[0015] Figure 4 The drawing is the astaxanthin content diagram of different treatment groups;

[0016] Figure 5 The drawing is the astaxanthin concentration diagram under different truncation methods. Detailed implementation manners

[0017] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0018] Construction of the fusion protein in Embodiment 1

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

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

[0021] 2. Truncate the original protein sequence of Brevundimonas sp. SD212 CrtW. The original sequence is as follows:

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

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

[0024] The link sequence is as follows:

[0025] GGGGSGGGGSGGGGSGGGGSEAAAKEAAAKGGGGSGGGGSGGGG S, as shown in 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, as shown in SEQ ID NO.4.

[0036] The structure of the fusion protein is as Figure 3 shown. It can be seen from the figure that after being connected by a suitable link sequence, both proteins can independently form their own structures with little mutual influence.

[0037] Comparison of different catalytic methods in Example 2

[0038] Genes were designed and synthesized according to the codon preference of Pichia pastoris, and Pichia pastoris was used as the chassis cell to express recombinant Pantoea ananatis CrtZ, Brevundimonas sp. SD212 CrtW and CrtZW. Reaction solutions were prepared with different enzyme addition methods, and the catalytic effects of different enzymes and addition methods were compared. The configuration of 1 ml of reaction solution was as follows: substrate + enzyme solution + buffer = 400 μL + 400 μL + 200 μL.

[0039] Among them, the substrate solution formula was: 0.3 mg / ml β-carotene Tween60 solution; the buffer formula was: 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-ketoglutaric acid, 5 mM ascorbic acid.

[0040] The grouping was as follows:

[0041] CrtZ→CrtW: After adding CrtZ and reacting for 3 h, then adding CrtW and continuing to react for 3 h;

[0042] CrtW→CrtZ: After adding CrtW and reacting for 3 h, then adding CrtZ and continuing to react for 3 h;

[0043] CrtZ + CrtW: Adding CrtZ and CrtW simultaneously and reacting for 3 h;

[0044] CrtZW: Adding CrtZW and reacting for 3 h;

[0045] The astaxanthin content of each group was counted, as shown in Figure 4 .

[0046] From Figure 4 it can be seen that the final astaxanthin content obtained by reacting with the simultaneous addition of 2 enzymes is significantly lower than the catalytic methods of adding 2 enzymes successively. And the catalytic effect of the fusion protein is significantly better than other catalytic methods.

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

[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 the chassis cell to express the recombinant fusion protein. The link sequences are shown in Table 1;

[0049] Table 1 Comparison of the catalytic production of astaxanthin by fusion proteins linked 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 CrtW, the catalytic effect of 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 forming incorrect folding after fusion, which affects the enzyme activity, the two enzymes were truncated respectively, and only the active sites were retained. The differently truncated proteins were connected by link sequences and recombinantly expressed in Pichia pastoris, and the catalytic differences of each fusion protein were detected;

[0055] The sequences of different truncated proteins are as follows:

[0056] C-terminal truncation of CrtZ

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

[0058] N-terminal truncation of CrtZ

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

[0060] C-terminal truncation of CrtW

[0061] MTAAVAEPRIVPRQTWIGLTLAGMIVAGWGSLHVYGVYFHRWGTSSLVIVPAIVAVQTWLSVGLFIVAHDAMHGSLAPGRPRLNAAVGRLTLGLYAGFRFDRLKTAHHAHHAAPGTADDPDFYAPAPRAFLPWFLNFFRTYFGWREMAVLTALVLIALFGLGARPANLLTFWAAPALLSALQLFTFGTWLPHRHTDQPFADAHHARSSGYGPVLSLLTCFHFGRHHEHHLTPW, such as SEQ ID NO.14.

[0062] N-terminal truncation of CrtW

[0063] RQTWIGLTLAGMIVAGWGSLHVYGVYFHRWGTSSLVIVPAIVAVQTWLSVGLFIVAHDAMHGSLAPGRPRLNAAVGRLTLGLYAGFRFDRLKTAHHAHHAAPGTADDPDFYAPAPRAFLPWFLNFFRTYFGWREMAVLTALVLIALFGLGARPANLLTFWAAPALLSALQLFTFGTWLPHRHTDQPFADAHHARSSGYGPVLSLLTCFHFGRHHEHHLTPWRPWWRLWRGES, such as SEQ ID NO.15.

[0064] The results are shown in Figure 5 , from Figure 5 it can be seen that protein truncation has an obvious effect on astaxanthin synthesis, but the N-terminal of CrtZ has a greater negative impact on the structure of the fusion protein, and the catalytic effect can be significantly reduced after truncation. The catalytic effect is the best after truncation of the C-terminal of CrtZ / simultaneous truncation of the N-terminal of CrtW.

[0065] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. The same or similar parts among the various embodiments can be referred 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 obvious to those skilled in the art. The general principles defined herein can 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 these embodiments shown herein, but will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A fusion protein CrtZW, characterized in that Its amino acid sequence is as SEQ ID Shown in NO.4: MLWIWNALIVFVTVIGMEVIAALAHKYIMHGWGWHLSHHEPRKGAFEVNDLYAVVFAALSILLIYLGSTGMWPLQWIGAGMTAYGLLYFMVHDGLVHQRWPFRYIPRKGYLKRLYMAHRMHHAVRGKEGCVSFGFLYAPPLSKLGGGGSGGGGSGGGGSGGGGSEAAAKEAAAKGGGGSGGGGSGGGGSRQTWIGLTLAGM IVAGWGSLHVYGVYFHRWGTSSLVIVPAIVAVQTWLSVGLFIVAHDAMHGSLAPGRPRLNAAVGRLTLGLYAGFRFDRLKTAHHAHHAAPGTADDPDFYAPAPRAFLPWFLNFFRTYFGWREMAVLTALVLIALFGLGARPANLLTFWAAPALLSALQLFTFGTWLPHRHTDQPFADAHHARSSGYGPVLSLLTCFHFGRHHEHHLTPW.

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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