CN114410659B

CN114410659B - Phaeodactylum tricornutum CRTISO5 gene, protein and application thereof in fucoxanthin synthesis

Info

Publication number: CN114410659B
Application number: CN202210089004.6A
Authority: CN
Inventors: 李小波; 曹天骏; 张欢
Original assignee: Westlake University
Current assignee: Westlake University
Priority date: 2022-01-21
Filing date: 2022-01-21
Publication date: 2023-05-02
Anticipated expiration: 2042-01-21
Also published as: CN114410659A

Abstract

The invention discloses CRTISO5 gene and protein of Phaeodactylum tricornutum and application thereof in fucoxanthin synthesis, and the invention discloses a novel function of CRTISO5 gene for the first time, which can improve the fucoxanthin content in Phaeodactylum tricornutum; and, it can be used in photosynthetic organisms (plants, algae, photosynthetic bacteria) to increase the content of fucoxanthin in the photosynthetic organisms, and then increase the light capturing efficiency, and is beneficial to the light capturing efficiency and biomass accumulation, and the protein encoded by the gene can be used for fucoxanthin synthesis outside organisms.

Description

Phaeodactylum tricornutum CRTISO5 gene, protein and application thereof in fucoxanthin synthesis

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a Phaeodactylum tricornutum CRTISO5 gene, a Phaeodactylum tricornutum CRTISO5 protein and application thereof in fucoxanthin synthesis.

Background

Carotenoids are widely found in photosynthetic organisms and have both antioxidant and light energy capturing properties. Because of the diversity of structures, different carotenoids have different application values. Various carotenoids, such as fucoxanthin, astaxanthin, etc., have high market value. In recent years, fucoxanthin has been reported to have an anticancer function in addition to antioxidation.

In addition, fucoxanthin has important application in photosynthetic light energy capture. Blue-green light has strong penetrability in seawater, so that most of marine algae contain fucoxanthin and are used for capturing blue-green band visible light. Fucoxanthin is transferred to other photosynthetic organisms, and can be used for widening light-capturing spectrum and improving photosynthesis efficiency.

Currently, the synthetic pathway of fucoxanthin has not been completely resolved. There is no report about the synthesis of fucoxanthin gene.

Disclosure of Invention

One of the objects of the present invention is to provide a Phaeodactylum tricornutum (Phaeodactylum tricornutum) CRTISO5 gene.

A second object of the present invention is to provide a protein encoded by the above gene.

It is a primary object of the present invention to provide the use of the CRTISO5 gene or protein.

In order to achieve the above purpose, the technical scheme adopted by the invention is summarized as follows:

a CRTISO5 gene has the nucleotide sequence shown in SEQ ID NO.1, which consists of 1986 bases or DNA molecule hybridized with the DNA sequence defined by SEQ ID NO.1 under strict conditions.

The amino acid sequence of the protein (1) encoded by the gene is shown as SEQ ID No. 2. The sequence consists of 661 amino acid residues.

The protein encoded by the CRTISO5 gene may also include a protein derived from (1) having the function of (1) and formed by substitution, deletion or addition of one or more ((e.g., 1 to 30; preferably 1 to 20; more preferably 1 to 10; e.g., 5, 3)) amino acid residues of the amino acid sequence of SEQ ID NO. 2; or a protein derived from (1) having homology of 80% ((preferably 90% or more, such as 95%,98%,99% or more)) or more with the protein sequence defined in (1) and having the function of the protein of (1).

Recombinant microorganisms containing CRTISO5 gene, wherein the recombinant microorganisms comprise algae, fungi or bacteria, and the gene editing vector belongs to the protection scope of the invention.

The most important point of the invention is that a novel function of CRTISO5 gene is disclosed, CRTISO5 mutant is obtained by gene editing, and compared with wild type Phaeodactylum tricornutum, the mutant has lower fucoxanthin content than the wild type, but a carotenoid which is not existed in the wild type is accumulated, and the carotenoid is named as 7',8' -double dehydrodinoxanthin according to the similarity of the carotenoid and the known compound dinoxanthin. It can be seen that the CRTISO5 gene or protein participates in the synthesis of fucoxanthin, that is, the CRTISO5 gene has the function of increasing the content of fucoxanthin in Phaeodactylum tricornutum, and can be used for increasing the yield of fucoxanthin produced by microorganisms (algae, fungi and bacteria); and, for increasing the content of fucoxanthin in photosynthetic organisms (plants, algae, photosynthetic bacteria) and consequently increasing the light harvesting efficiency, the efficiency of photosynthesis and the accumulation of biomass are advantageous, which can be achieved by means of transgenesis.

In addition, through the identification of the enzymatic function of the CRTISO5 protein, the result shows that the 7',8' -bisdehydrodinoflagellate Huang Sucai can be converted into fucoxanthin only in the presence of the CRTISO5 protein, and therefore, the protein encoded by the CRTISO5 gene can be used for in vitro synthesis of fucoxanthin.

It should be noted that the functions of the gene protected by the present invention include not only the CRTISO5 gene described above, but also homologous genes having a high homology (such as homology of more than 40%, preferably more than 50%, preferably more than 60%, more preferably more than 70%, more preferably more than 80%, more preferably more than 90%, more preferably more than 95%, more preferably more than 98%) with SEQ ID NO. 1.

The invention has the advantages that:

in Phaeodactylum tricornutum, we find a CRTISO5 gene for the first time, and the protein encoded by the gene is responsible for the last step of fucoxanthin synthesis, that is, the invention discloses a novel function of the CRTISO5 gene for the first time, which can improve the fucoxanthin content in Phaeodactylum tricornutum. And, it can be used in photosynthetic organisms (plants, algae, photosynthetic bacteria) to increase the content of fucoxanthin in the photosynthetic organisms, and then increase the light capturing efficiency, and is beneficial to the light capturing efficiency and biomass accumulation, and the protein encoded by the gene can be used for fucoxanthin synthesis outside organisms.

Drawings

FIG. 1 is evidence that the CRTISO5 gene was edited in the CRTISO5 mutant;

FIG. 2 is evidence of reduced fucoxanthin content of crtiso5 mutants;

in the figure, the right image is a photograph of the appearance of the cell, and the left image is a High Performance Liquid Chromatography (HPLC) pigment analysis;

FIG. 3 shows the nuclear magnetic resonance identification of the accumulated pigment, i.e.CRTISO5 protein substrate structure, in CRTISO5 mutant, i.e.the molecular structure of 7',8' -bisdehydrodinoxanthin;

FIG. 4 is a vector used for producing CRTISO5 protein in E.coli;

FIG. 5 shows the results of in vitro synthesis of fucoxanthin using CRTISO5 protein in the examples.

Detailed Description

The present invention will be described in detail with reference to specific examples. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. The test methods in the following examples are conventional methods unless otherwise specified. The reagents and materials employed, unless otherwise indicated, are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botanicals, microorganisms, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination, and bioinformatics, which will be apparent to one of skill in the art. These techniques are fully explained in the published literature, and the methods of DNA extraction, phylogenetic tree construction, gene editing method, gene editing vector construction, gene editing plant acquisition, etc. used in the present invention can be realized by the methods disclosed in the prior art except the methods used in the examples described below.

The terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" as used herein are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-or double-stranded structures. Such nucleic acids or polynucleotides include, but are not limited to, gene coding sequences, antisense sequences, and regulatory sequences of non-coding regions. These terms include a gene. "Gene" or "gene sequence" is used broadly to refer to a functional DNA nucleic acid sequence. Thus, a gene may include introns and exons in genomic sequences, and/or coding sequences in cDNA, and/or cDNA and regulatory sequences thereof. In particular embodiments, for example in relation to isolated nucleic acid sequences, it is preferred that they are cDNA.

Examples

1. Acquisition of CRTISO5 Gene

Screening by using a plurality of technical means such as transcriptome and metabolome to obtain a Phaeodactylum tricornutum CRTISO5 gene, wherein the length of the full-length coding frame nucleotide sequence of the gene is 1986bp, the gene consists of 661 amino acids, the nucleotide sequence is shown as SEQ ID NO.1, and the protein sequence is shown as SEQ ID NO. 2.

Phenotypic analysis of Crtiso5 mutants

Heretofore, no report has been made on the function of the CRTISO5 gene in phaeodactylum tricornutum, and the applicant has conducted the following studies, and has found for the first time that the CRTISO5 gene plays an important role in phaeodactylum tricornutum fucoxanthin synthesis.

Construction of CRTISO5 Gene editing vector

The CRTISO5 gene-edited targeting sequence (sgRNA) was selected using the CRISPOR website (http:// CRISPOR. Tefor. Net /), two primers were designed based on the selected sgRNA (Table 1), after annealing, double-stranded DNA containing cohesive ends was obtained, cloned into the target vector containing the blasticidin-S deaminase gene, and the Phaeodactylum tricornutum U6 promoter was responsible for expression of the sgRNA. The Cas9 gene is controlled by the gamma tubulin promoter.

TABLE 1 CRTISO5 sgRNA primers

2. Acquisition of Phaeodactylum tricornutum conjugal transformation and crtiso5 mutant

Coli containing the above vector and pTA-Mob vector required for conjugal transformation was mixed with wild type Phaeodactylum tricornutum at 1000:1, and after concentration, the f/2 medium plates are coated. Two days later, cells were scraped and plated on f/2 medium plates containing 5. Mu.g/mL blasticidin.

Two weeks later, the clones were picked, resuspended in liquid medium and plated again to obtain subclones. Multiple subclones were picked and colony PCR and sequenced. The CRTISO5 gene in the obtained CRTISO5 mutant was missing 15 base pairs compared to the wild type (fig. 1).

Fucoxanthin phenotyping of crtiso5 mutants and structural identification of accumulated carotenoids

In constant light (80. Mu. Mol.m) ^-2 ·s ^-1 ) Wild type and mutant were cultured under the conditions. Centrifugal collection 10 ⁷ Cells were added with 250 μl of 90% acetone and mixed sonically under dark conditions. After centrifugation, the supernatant was collected and subjected to a pigment analysis by High Performance Liquid Chromatography (HPLC).

HPLC parameters were as follows:

instrument: thermo Ultimate 3000UHPLC;

chromatographic column: the C18 column (5 μm,

250×4.6mm，waters，USA)；/>

temperature: 20 ℃;

flow rate: 1mL per minute;

sample injection volume: 10. Mu.L;

gradient mobile phase: consists of solvent a (methanol: water=90:10) and solvent B (ethyl acetate). At 0: 100% A,0% B;20-22 minutes: 0% A,100% B;23-28 minutes: 100% A;0% B.

HPLC results showed that CRTISO5 mutant had a lower fucoxanthin content than the wild type, and it was seen that the CRTISO5 gene or protein was involved in the synthesis of fucoxanthin, and CRTISO5 mutant exhibited green color due to the low fucoxanthin content, but accumulated a carotenoid which was not present in the wild type (FIG. 2).

The accumulated carotenoids were collected during HPLC, and were subjected to structural analysis using a Bruker AVANCE NEO MHz nmr, the molecular structure of the analysis being shown in fig. 3, and the enzymatic function of CRTISO5 protein was further identified in an in vitro system using the accumulated carotenoids as substrates for CRTISO5 protein, named 7',8' -bisdehydro-dinoxanthin, according to their similarity to the known compound dinoxanthin.

Construction of expression vector and protein acquisition

1. Cloning of Phaeodactylum tricornutum CRTISO5 Gene cDNA (SEQ ID NO. 1)

Wild Phaeodactylum tricornutum RNA was extracted using RNeasy Plant Minikit kit. Using SuperScript ^TM III Reverse Transcriptase kit reverse transcription was performed. And cloning the CRTISO5 gene cDNA by using the obtained total cDNA as a template through PCR.

PCR system (table 2):

TABLE 2.20. Mu.L amplification System

PCR cycle:

1)94℃：5min；

2)94℃：30s；

3)55℃：30s；

4)72℃：2min；

steps 2) -4) are cycled 35 times;

6)72℃：5min。

the PCR primers (Table 3) include not only sequences that overlap with the CRTISO5 cDNA sequence (uppercase), but also sequences homologous to the target vector (lowercase).

TABLE 3 CRTISO5 cDNA amplification primers

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The PCR product was cloned into the pMAL-c5x vector by Information homologous recombination. In the vector, CRTISO5 gene is fused with MBP at N end, polyhistidine tag (His-tag) is arranged at C end, and exogenous gene expression is induced by isopropyl-beta-D-thiogalactoside (IPTG). The constructed plasmid was transformed into the expression BL21 (DE 3) strain and positive clones were screened by PCR. The constructed successful vector is shown in FIG. 4.

2. Expression of Phaeodactylum tricornutum CRTISO5 Gene in E.coli

The strain was cultured at 37℃using LB medium containing 100mg/L of ampicillin until the OD600 was 0.6 to 0.8. Isopropyl- β -D-thiogalactoside (IPTG) was added to a final concentration of 0.4mM and the incubation was continued for 12 hours at 16 ℃.

3. Purification of Phaeodactylum tricornutum CRTISO5 protein

After high pressure disruption, the mixture was centrifuged at 13000g at 4℃for 15 minutes. The obtained supernatant was subjected to protein purification using an AKTA system and a Source Q ion exchange column. Protein concentration was performed using a 30kd mwco ultrafiltration tube. Bovine Serum Albumin (BSA) was used as a configuration standard and protein concentration was determined by the bicinchoninic acid method. Split charging to 50 μg per tube, quick freezing with liquid nitrogen, and storing at-80deg.C.

4. Enzymatic functional identification of CRTISO5 protein

The production of fucoxanthin is verified by adding fucoxanthin synthesis precursor 7',8' -didehydromethyl fucoxanthin and purified CRTISO5 protein into an in vitro system. The method comprises the following steps:

1. establishment of enzymatic research system

200. Mu.L of buffer (0.1% TritonX-100, 100mM Tris,10mM MgCl2,1mM DTT,pH 7.4) was added to the purified and dried 7',8' -bisdehydrodinoxanthin and the precursor was dissolved by ultrasonic homogenization.

Three components were then added to the final concentration in brackets:

flavin adenine dinucleotide (FAD, 100 μm), oxidized form;

Na ₂ S ₂ O ₄ (1 mM) to convert FAD to a reduced form;

50 μg of purified CRTISO5 protein.

CRTISO5 protein was omitted in the control reaction. After two hours, 200. Mu.L of acetone and 200. Mu.L of ethyl acetate were added and mixed well to terminate the reaction.

Analysis of CRTISO5 protein product

The carotenoid product is extracted by centrifugation after termination of the reaction: the organic layer of the supernatant was aspirated, dried with nitrogen and dissolved in 50. Mu.L of aqueous methanol.

The solubilizate was analyzed by High Performance Liquid Chromatography (HPLC) to analyze the production of fucoxanthin and the residual of 7',8' -bisdehydrodinoxanthin. HPLC parameters were as follows:

instrument: waters Acquity UPLC;

chromatographic column: ACQUITY UPLC HSS T31.8.8 μm (3 μm,

2.1×150mm)；

temperature: 45 ℃;

flow rate: 0.3mL per minute;

sample injection volume: 3 μL;

gradient mobile phase: consists of solvent A (acetonitrile: methanol: methyl tert-butyl ether=70:20:10) and solvent B (10 mM ammonium acetate). At 0: 60% A,40% B;4 minutes: 75% A,25% B;12 minutes: 100% A.

FIG. 5 shows that 7',8' -bisdehydromethylfucoxanthin is converted to fucoxanthin by adding CRTISO5 protein as compared with control, that is, CRTISO5 protein has the function of synthesizing fucoxanthin in vitro.

Sequence listing

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<120> Phaeodactylum tricornutum CRTISO5 gene, protein and application in fucoxanthin synthesis

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ggcgccctgt cagctaaata cggtgacaag gtcctcgtgc tagagtcgca cattaaatgc 360

ggaggatcgg ctcatacatt ctcccgtatg cacaacggtg aaaaatattc cttcgaagtg 420

ggtccttcaa tttttgaagg actcgaccgt ccaagcctga atccccttcg catgattttt 480

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acgggatatt ggcgtttccc tatcggtagt caaagcaaat tcgaagatct gcttatggaa 600

caagcggaag atggccccaa ggctgttgag gaatggaaca tgttacgcaa acgcctcaag 660

acacttggtg gttctacaac tgcagtttcg ttgttgaacc tacgtcaaga tcctggtttt 720

ttagcgacaa cagctggtag tttgcctttt gtggcaacgc atcctgatgt gtttctcgac 780

ttgtcgctta cgtttgattc tctccacaag acggttgata aaattgtgac ggtccctttc 840

ctccgaaact ttatcgatac catgtgcatt ttctgcggct tcccagccaa gggcgcgatg 900

acggcgcaca tgctttatat cttagagcgc ttctttgaag agtcagcttg ctattctgtt 960

ccgattggag gtacatgcga aatgggaaac acattggtac gcggcttgga aaagtttggt 1020

ggcaaaatcc agttgaatgc tcacgtagac gaaattttgg tcgaaaacgg acgtgccgtg 1080

ggtgttcgtc tcaagaacgg aaatgttgtt aaagcaaaca aagccgtggt gagcaatgcc 1140

acgccttttg ataccgtgaa gatgcttgga gaaaaacaag cacttccaga aggtgtcgcg 1200

aaatggaagg aagagcttgg gaaactccca cgtcacggag cgattatgca tttattttta 1260

gctattgatg cgaaggatct ggacctttcg cacattcaag accccgctca tttagtagtt 1320

caagactggg gacgttcttt acaagactcg cagaacttgt gtagcttctt cattcctagt 1380

ttacttgaca agacgttatg tccggaaggc aagcatgtca ttcatgtata ctcttctgga 1440

ggggaaccgt atgagccgtg ggaaaagctc aagccaggga cacaggagta cgacgattac 1500

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gatcgaggta cgtatggaat ggcatgggct gctggtacat cagcgcccca ggccggcctt 1680

cttcagaata ttctcccttt cccattccca aaccttaaga caccagtcga tggtctctta 1740

cgatgcggcg actcctgctt tcccggtatc ggaactccaa gtgcggccgc ctcgggagcg 1800

attgcagcga acacaatgaa ccccgtcggc aagcatttag atttgctgaa agaagccagt 1860

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Met Leu Arg Leu Ala Ala Leu Phe Ala Ala Ile Ala Ala Val Asp Val

1 5 10 15

Thr Ala Phe Thr Pro Ala Thr Lys Pro Phe Leu Thr Ala Ser His Pro

20 25 30

Tyr Gly Leu Arg Ser Thr Thr Asn Glu Asn Val Ala Gln Thr Glu Asn

35 40 45

Thr Ser Arg Glu Lys Val Met Thr Phe Ser Tyr Asp Met Ser Leu Glu

50 55 60

Pro Lys Tyr Glu Lys Pro Thr Tyr Pro Gly Thr Gly Asn Gly Leu Ser

65 70 75 80

Gly Asp Ser Gly Leu Tyr Asp Val Ile Val Ile Gly Ser Gly Met Gly

85 90 95

Gly Leu Ala Cys Gly Ala Leu Ser Ala Lys Tyr Gly Asp Lys Val Leu

100 105 110

Val Leu Glu Ser His Ile Lys Cys Gly Gly Ser Ala His Thr Phe Ser

115 120 125

Arg Met His Asn Gly Glu Lys Tyr Ser Phe Glu Val Gly Pro Ser Ile

130 135 140

Phe Glu Gly Leu Asp Arg Pro Ser Leu Asn Pro Leu Arg Met Ile Phe

145 150 155 160

Asp Val Leu Glu Glu Glu Met Pro Val Lys Thr Tyr Thr Gly Leu Gly

165 170 175

Tyr Trp Thr Pro Thr Gly Tyr Trp Arg Phe Pro Ile Gly Ser Gln Ser

180 185 190

Lys Phe Glu Asp Leu Leu Met Glu Gln Ala Glu Asp Gly Pro Lys Ala

195 200 205

Val Glu Glu Trp Asn Met Leu Arg Lys Arg Leu Lys Thr Leu Gly Gly

210 215 220

Ser Thr Thr Ala Val Ser Leu Leu Asn Leu Arg Gln Asp Pro Gly Phe

225 230 235 240

Leu Ala Thr Thr Ala Gly Ser Leu Pro Phe Val Ala Thr His Pro Asp

245 250 255

Val Phe Leu Asp Leu Ser Leu Thr Phe Asp Ser Leu His Lys Thr Val

260 265 270

Asp Lys Ile Val Thr Val Pro Phe Leu Arg Asn Phe Ile Asp Thr Met

275 280 285

Cys Ile Phe Cys Gly Phe Pro Ala Lys Gly Ala Met Thr Ala His Met

290 295 300

Leu Tyr Ile Leu Glu Arg Phe Phe Glu Glu Ser Ala Cys Tyr Ser Val

305 310 315 320

Pro Ile Gly Gly Thr Cys Glu Met Gly Asn Thr Leu Val Arg Gly Leu

325 330 335

Glu Lys Phe Gly Gly Lys Ile Gln Leu Asn Ala His Val Asp Glu Ile

340 345 350

Leu Val Glu Asn Gly Arg Ala Val Gly Val Arg Leu Lys Asn Gly Asn

355 360 365

Val Val Lys Ala Asn Lys Ala Val Val Ser Asn Ala Thr Pro Phe Asp

370 375 380

Thr Val Lys Met Leu Gly Glu Lys Gln Ala Leu Pro Glu Gly Val Ala

385 390 395 400

Lys Trp Lys Glu Glu Leu Gly Lys Leu Pro Arg His Gly Ala Ile Met

405 410 415

His Leu Phe Leu Ala Ile Asp Ala Lys Asp Leu Asp Leu Ser His Ile

420 425 430

Gln Asp Pro Ala His Leu Val Val Gln Asp Trp Gly Arg Ser Leu Gln

435 440 445

Asp Ser Gln Asn Leu Cys Ser Phe Phe Ile Pro Ser Leu Leu Asp Lys

450 455 460

Thr Leu Cys Pro Glu Gly Lys His Val Ile His Val Tyr Ser Ser Gly

465 470 475 480

Gly Glu Pro Tyr Glu Pro Trp Glu Lys Leu Lys Pro Gly Thr Gln Glu

485 490 495

Tyr Asp Asp Tyr Lys Asn Glu Arg Ala Lys Val Leu Trp Glu Ala Val

500 505 510

Glu Arg Cys Ile Pro Asp Val Arg Asp Arg Leu Glu Phe Ser Ile Val

515 520 525

Gly Ser Pro Leu Ala His Glu Ala Phe Leu Arg Arg Asp Arg Gly Thr

530 535 540

Tyr Gly Met Ala Trp Ala Ala Gly Thr Ser Ala Pro Gln Ala Gly Leu

545 550 555 560

Leu Gln Asn Ile Leu Pro Phe Pro Phe Pro Asn Leu Lys Thr Pro Val

565 570 575

Asp Gly Leu Leu Arg Cys Gly Asp Ser Cys Phe Pro Gly Ile Gly Thr

580 585 590

Pro Ser Ala Ala Ala Ser Gly Ala Ile Ala Ala Asn Thr Met Asn Pro

595 600 605

Val Gly Lys His Leu Asp Leu Leu Lys Glu Ala Ser Gln Arg Asp Pro

610 615 620

Met Tyr Lys Phe Leu Asp Pro Gly Val Phe Gly Ser Ile Tyr Arg Pro

625 630 635 640

Phe Val Glu Ser Leu Thr Pro Ser Thr Glu Leu Gln Val Glu Ser Ile

645 650 655

Gln Asn Thr Ala Asp

660

Claims

1. Phaeodactylum tricornutum (pers.) KuntzeCRTISO5The use of a gene-encoded protein for the in vitro synthesis of fucoxanthin, characterized in that it comprises the following steps ofCRTISO5The nucleotide sequence of the gene is shown as SEQ ID NO.1, and the organism is Phaeodactylum tricornutum.