Crti enzyme mutants, recombinant plasmids, recombinant strains and use thereof

Abstract

The present application belongs to the technical field of genetic engineering, and relates to a CrtI enzyme mutant, a recombinant plasmid, a recombinant strain and application thereof. The CrtI enzyme mutant provided by the present application is obtained by random mutation of a wild-type CrtI gene through error-prone PCR, and the amino acid sequence thereof is shown as SEQ ID NO. 1. The obtained mutant has multiple site variations in the amino acid sequence, thereby significantly improving the catalytic efficiency and substrate affinity of the enzyme. The present application further constructs a recombinant expression vector containing the CrtI enzyme mutant gene, and transforms the recombinant expression vector into a red Phaffia yeast competent cell, and obtains a high-yield astaxanthin recombinant strain through resistance screening and pigment phenotype screening. Compared with the wild-type CrtI enzyme, the CrtI enzyme mutant of the present application significantly improves the conversion rate of beta-carotene to astaxanthin in the red Phaffia yeast.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to CrtI enzyme mutants, recombinant plasmids, recombinant strains and their applications. Background Technology

[0002] Astaxanthin is a carotenoid with extremely strong antioxidant properties. It can be produced through both chemical and biosynthetic processes, but with increasing safety requirements, biosynthesis is increasingly replacing chemical synthesis. Studies have shown that astaxanthin has significant physiological functions in antioxidation, anti-inflammation, immune regulation, anti-aging, and prevention of cardiovascular diseases, thus it has broad application prospects in food, feed, health products, cosmetics, and pharmaceuticals.

[0003] Astaxanthin biosynthesis belongs to the carotenoid metabolic pathway. The phytoene desaturase (CrtI) enzyme is the rate-limiting enzyme in lycopene production. Encoded by the CrtI gene, its activity directly affects the amount of astaxanthin produced downstream. Existing research indicates that the CrtI enzyme activity and substrate affinity in natural Pharf yeast are low, leading to the accumulation of intermediate products and low conversion efficiency, thus limiting the astaxanthin synthesis throughput. Error-prone PCR, as a molecular evolutionary technique involving random mutation, is more targeted than traditional mutagenesis breeding. It can introduce multi-site mutations across the entire genome. By constructing mutant libraries and combining them with phenotypic screening, it is expected to obtain enzyme mutants with higher catalytic efficiency and stability, thereby effectively improving the astaxanthin biosynthesis level in Pharf yeast.

[0004] Red Pfaff yeast ( Xanthophyllomyces dendrorhous Original name Phaffia rhodozyma Pharfogel's yeast is an important astaxanthin-producing microorganism. Compared with another astaxanthin-synthesizing microorganism, Haematococcus pluvialis, it has advantages such as faster growth rate, higher culture density, and no need for light. However, the current astaxanthin yield of Pharfogel's yeast is relatively low, which limits its production and application. The construction of high-yielding strains is urgently needed. Therefore, obtaining a high-yielding astaxanthin Pharfogel's yeast strain is of great significance for achieving efficient, controllable, and industrialized production of natural astaxanthin. Summary of the Invention

[0005] This invention addresses the technical problem of low astaxanthin yield in existing Pharrellis rubrum yeast strains, which cannot meet the requirements for efficient, controllable, and industrialized production of natural astaxanthin. It proposes a CrtI enzyme mutant CrtI-27 modified by error-prone PCR, a recombinant plasmid, and a recombinant Pharrellis rubrum yeast strain XD-UV2-27. Compared with the original strain XD-UV2, this strain exhibits a higher astaxanthin yield, enabling efficient, controllable, and industrialized production of natural astaxanthin, which can be applied in fields such as animal feed.

[0006] To achieve the above objectives, the present invention first provides the following technical solution: a CrtI enzyme mutant, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0007] Furthermore, the present invention also provides the encoding gene of the CrtI enzyme mutant, the nucleotide sequence of which is shown in SEQ ID NO. 2.

[0008] Furthermore, the present invention also provides a recombinant plasmid comprising the coding gene of the CrtI enzyme mutant.

[0009] Furthermore, the present invention also provides a recombinant strain comprising the aforementioned recombinant plasmid.

[0010] Furthermore, the recombinant strain uses Pharfogel's erythrosporum as a host.

[0011] Furthermore, the *Phaeodactylum rubrum* yeast mentioned is selected from *Phaeodactylum rubrum* yeast with accession number CCTCC NO: M20231707. Xanthophyllomyces dendrorhous XD-UV2 strain.

[0012] Furthermore, the present invention also provides the application of any one of the CrtI enzyme mutant, the coding gene, the recombinant plasmid, and the recombinant strain in the production of astaxanthin.

[0013] Furthermore, the present invention also provides a method for producing astaxanthin, which utilizes the recombinant strain to produce astaxanthin under fermentation culture conditions.

[0014] Compared with existing technologies, the advantages and positive effects of this invention are as follows: by performing multi-site random mutations on the CrtI gene using error-prone PCR technology, the target limitations of site-directed mutation can be effectively overcome; the modified recombinant strain has increased astaxanthin synthesis flux, reduced β-carotene accumulation, and more concentrated carbon flux; the recombinant strain is genetically stable, has excellent fermentation performance, and is suitable for industrial cultivation; the CrtI enzyme mutant and recombinant strain provided by this invention provide a new and efficient pathway for the green production of natural astaxanthin. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the biosynthetic pathway of astaxanthin in *Phaeff's red yeast* according to the present invention; wherein, CrtI is phytoene dehydrogenase; CrtYB is lycopene β-cyclase / synthetase complex; CrtS / CrtR are β-carotene hydroxylase / ketoylase; Figure 2 This is a schematic diagram of nucleic acid electrophoresis for CrtI gene amplification in this invention; Figure 3This is a schematic diagram of nucleic acid electrophoresis for gene amplification of the CrtI enzyme mutant CrtI-27 of this invention; Figure 4 This is a diagram illustrating the construction and verification of the recombinant expression vector for the CrtI enzyme mutant CrtI-27 of this invention. Figure 5 This is a schematic diagram of the electroporation and positive clone screening of *Phaefflera heptaphylla* according to the present invention; wherein, A. results of streaking culture in YPD plates containing Zeocin antibiotic; B. schematic diagram of gene amplification and nucleic acid electrophoresis of positive clones; Figure 6 This is an amino acid sequence alignment diagram of the wild-type CrtI enzyme of *Phaeodactylum rubrum* and the mutant CrtI-27 modified by error-prone PCR; where a represents the wild type and b represents the mutant. Figure 7 This is a comparison chart of astaxanthin production between the recombinant strain XD-UV2-27 and the original strain XD-UV2; where *p≤0.05; **p≤0.01; ***p≤0.001; Figure 8 This is a graph showing the accumulation curve of astaxanthin during the fermentation process of the recombinant strain XD-UV2-27 of this invention. Detailed Implementation

[0016] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] This invention is based on the astaxanthin biosynthesis pathway of Pharfovia rubescens (e.g. Figure 1 As shown in the figure, a red Paffia yeast CrtI enzyme mutant, recombinant plasmid, recombinant strain and its application in improving astaxanthin production are proposed through error-prone PCR modification. It has good application potential in the fields of biosynthesis of natural astaxanthin, functional foods, feed additives and health products.

[0018] To provide a clearer and more detailed description of the CrtI enzyme mutant, recombinant plasmid, recombinant strain, and their applications provided in the embodiments of the present invention, the following description will be provided in conjunction with specific embodiments.

[0019] In the following examples, molecular biology experimental methods not specifically described were performed in accordance with the specific methods listed in J. Sambrook's "Molecular Cloning: A Laboratory Manual," 3rd edition, or according to the kit and product instructions; the reagents and biological materials mentioned are commercially available unless otherwise specified.

[0020] Example 1: Amplification and template preparation of the CrtI gene in Pharfia redis. DNA amplification enzyme: 2×Phanta Flash Master Mix (Dye Plus); The reaction system is as follows: enzyme 25 μL, DNA template 2 μL, front primer 1.5 μL, back primer 1.5 μL, and ddH2O to bring the total volume to 50 μL.

[0021] The red Pharf yeast preserved in our laboratory Xanthophyllomyces dendrorhous Using strain XD-UV2 (accession number CCTCC NO: M20231707, deposited on September 18, 2023, depositary institution: China Center for Type Culture Collection, Wuhan University) as a template, genomic DNA was extracted using a genomic DNA extraction kit. Genomic DNA was extracted based on publicly available sequences from the GenBank DNA sequence database established by the National Center for Biotechnology Information (NCBI). Phaffia rhodozyma Specific primers were designed based on the crtI gene sequence (accession number MZ748351.1). The extracted Pharrellis redis DNA was used as a template for CrtI gene amplification. The reaction used 2×Phanta Flash Master Mix (Dye Plus) DNA amplification enzyme. The reaction mixture was prepared according to the enzyme's instructions: 25 μL enzyme, 2 μL DNA template, 1.5 μL front primer, 1.5 μL back primer, and ddH2O to a final volume of 50 μL. The annealing temperature was set at 58℃, the extension time at 72℃ for 2 min, and other reaction conditions were performed according to the enzyme's instructions.

[0022] After the reaction was completed, the PCR products were examined using agarose gel electrophoresis. The electrophoresis results of the CrtI gene amplification nucleic acid were as follows: Figure 2 As shown, a DNA amplification band with a molecular weight of 1749 bp was obtained. The tested PCR product was further purified using the Cycle Pure Kit PCR purification kit. The specific operation steps were performed according to the Cycle Pure Kit PCR purification kit instruction manual. The purified DNA fragment is the PCR product of the CrtI gene.

[0023] Example 2: Error-prone PCR modification of the CrtI gene Error-prone PCR DNA amplification enzyme: RandomMut DNA polymerase; The reaction system consisted of the following components: RandomMutbuffer (10×) 2 μL, Mutation enhancer (10×) 2 μL, dNTPs (2.5 mM each) 2 μL, DNA template 2 μL, front primer 0.2 μL, back primer 0.2 μL, RandomMut DNA polymerase 0.4 μL, enzyme 0.4 μL, and ddH2O to a final volume of 20 μL.

[0024] Using the amplified CrtI gene as a template, random mutations were performed on the CrtI gene using an error-prone PCR kit. The reaction used the error-prone PCR DNA amplification enzyme RandomMut DNA polymerase. The reaction system was prepared according to the enzyme's instructions, specifically: RandomMutbuffer (10×) 2 μL, Mutation enhancer (10×) 2 μL, dNTPs (2.5 mM each) 2 μL, DNA template 2 μL, front primer 0.2 μL, back primer 0.2 μL, RandomMut DNA polymerase 0.4 μL, enzyme 0.4 μL, and ddH2O to a final volume of 20 μL. The annealing temperature was set at 55℃, the extension time at 72℃ for 10 min, and other reaction conditions were performed according to the kit's instructions.

[0025] After the reaction was completed, the PCR products were examined using agarose gel electrophoresis. The electrophoresis results of the CrtI gene mutant amplification nucleic acid were as follows: Figure 3 As shown, a DNA amplification band with a molecular weight of 1749 bp was obtained. The tested PCR product was further purified using the Cycle Pure Kit PCR purification kit. The specific operation steps were performed according to the Cycle Pure Kit PCR purification kit instruction manual. The purified DNA fragment recovered was the PCR product of the CrtI gene mutant.

[0026] Example 3: Construction of the recombinant expression vector for the CrtI enzyme mutant CrtI-27 The purified CrtI mutant gene fragment CrtI-27 was seamlessly ligated into the expression vector pUG6. The plasmid backbone, CrtIop, and 2×FuseIn Mix were mixed thoroughly and assembled at 50°C for 30 min. The reaction volume was 6 μL (2×FuseIn Mix: 3 μL; plasmid backbone and target sequence added at a molar ratio of 1:2, approximately 100–300 ng, 3 μL). The mixture was transformed into *E. coli* DH5α using the heat shock method and cultured at 37°C for 12–16 h. Single colony selection was performed, and positive clones were verified (e.g., ...). Figure 4The recombinant expression vector of the CrtI mutant gene was constructed.

[0027] Example 4: Electroporation and Recombinant Strain Screening of Pharfia redis Add 1 μl of Kpn I to the extracted recombinant expression vector, followed by 5 μl of enzyme reaction buffer. Incubate at 37°C for 6 h. Linearize the constructed recombinant expression vector by Kpn I enzyme digestion. After recovering the digestion product, concentrate it to below 10 μL. Transform the linearized recombinant expression vector into Phaeopharyngeal yeast competent cells by electroporation. The specific transformation method is as follows: wash twice with 20 mL of ice-cold sucrose-buffered saline (STM) buffer, resuspend the cells in 80 μL of STM buffer, mix the concentrated linearized recombinant expression vector sample with the yeast competent cells, transfer to a pulsed electroporation cuvette, and electroporate under the following conditions: 1000 Ω, 800 V, 50 μF. Immediately add 500 μL of ice-cold YPD medium to the transformation solution, mix well, and incubate at 22°C for 2.5 h. Spread the cells onto YPD plates containing Zeocin antibiotic and incubate at 22°C for 3-5 days.

[0028] Select colonies that have deepened to orange-red color and streak them onto a YPD agar plate containing Zeocin antibiotic. Verify positive clones. Figure 5 .

[0029] Example 5: Gene sequence determination and amino acid sequence alignment of recombinant strain XD-UV2-27 Dissolve 0.01 g Westase in 2 mL Mcllvain Buffer to prepare a 0.5% Westase lysis buffer, and filter through a 0.22 μm polyethersulfone membrane. Take 20 μL of Westase lysis buffer, add a small amount of the test bacteria, and incubate at 37 °C for 4 h.

[0030] The CrtI gene was amplified using the bacterial culture diluted 1-fold after the above treatment as a template. The reaction used DNA amplification enzyme 2 × Phanta Flash Master Mix (Dye Plus); the reaction system was prepared according to the enzyme's instructions, specifically: 25 μL enzyme, 2 μL DNA template, 1.5 μL front primer, 1.5 μL back primer, and ddH2O to a final volume of 50 μL. The annealing temperature was set at 58℃, the extension time at 72℃ for 2 min, and other reaction conditions were performed according to the enzyme's instructions.

[0031] After the reaction was completed, the PCR products were examined using agarose gel electrophoresis. The electrophoretic bands were excised and sent to Sangon Biotech (Shanghai) Co., Ltd. for gene sequencing and alignment. Simultaneously, the amino acid sequences were compared. The amino acid sequence of the CrtI enzyme mutant CrtI-27 is shown in SEQ ID NO. 1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO. 2. The amino acid sequence of the wild-type CrtI enzyme is shown in SEQ ID NO. 3. The alignment results are as follows: Figure 6 As shown.

[0032] Depend on Figure 6 It can be seen that, compared with the wild-type CrtI enzyme, the amino acid sequence of the mutant CrtI-27 has the following mutations: the D amino acid at position 8 is mutated to the G amino acid; the Y amino acid at position 141 is mutated to the C amino acid; and the V amino acid at position 562 is mutated to the I amino acid.

[0033] Example 6: Astaxanthin Production Determination of Recombinant Strain XD-UV2-27 The original strain XD-UV2 and the recombinant mutant strain XD-UV2-27 were fermented under the same conditions for 5 days, and their astaxanthin yields were measured respectively. Figure 7 As shown.

[0034] Depend on Figure 7 It can be seen that the astaxanthin yield of the original strain XD-UV2 was 1.45 mg / g, and the astaxanthin yield of the recombinant strain XD-UV2-27 was 3.70 mg / g, which was 2.55 times higher than that of the wild-type strain.

[0035] Example 7: Determination of astaxanthin accumulation curve during fermentation of recombinant strain XD-UV2-27 The recombinant strain XD-UV2-27 was continuously fermented for 7 days under the following conditions: fermentation medium (sucrose 35 g / L, yeast extract 3 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, CaCl2 0.1 g / L) with a volume of 25 mL, temperature 22℃, rotation speed 220 r / min, and inoculum size of 3%. Astaxanthin production was measured every 24 h, and an astaxanthin accumulation curve was plotted. Figure 8 As shown.

[0036] Depend on Figure 8 It can be seen that the astaxanthin production of the recombinant strain XD-UV2-27 stabilized on the fourth day and then showed no significant upward trend.

[0037] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, evolutions, or improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A CrtI enzyme mutant, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. The encoding gene of the CrtI enzyme mutant according to claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

2.

3. A recombinant plasmid, characterized in that, Includes the encoding gene of the CrtI enzyme mutant as described in claim 2.

4. A recombinant bacterial strain, characterized in that, Includes the recombinant plasmid as described in claim 3.

5. The recombinant strain according to claim 4, characterized in that, The recombinant strain used Pharfogel's rubrum as a host.

6. The recombinant strain according to claim 5, characterized in that, The red phloxera yeast mentioned was selected from the red phloxera yeast with accession number CCTCC NO: M20231707. Xanthophyllomyces dendrorhous XD-UV2 strain.

7. The use of any one of the CrtI enzyme mutant of claim 1, the encoding gene of claim 2, the recombinant plasmid of claim 3, and the recombinant strains of claims 4-6 in the production of astaxanthin.

8. A method for producing astaxanthin, characterized in that, Astaxanthin is produced using the recombinant strain according to any one of claims 4-6 under fermentation culture conditions.

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