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    • 专利摘要:
    Plasmid and procedure for the expression of a protein in microalgae. Plasmid for expressing a protein, comprising a gene coding for said protein and the cassette identified by the sequence seq id no: 1, wherein said cassette comprises the hybrid promoter phsp70a/rbcs2 of chlamydomonas, the fusion of the gene encoding the peptide fmdv 2a autohydrolyzate and the gene coding for the enzyme aminoglycoside 3'fostotransterase (aphviii). Method of expressing a protein in microalgae, comprising transforming said microalga with a plasmid and expressing said protein and the fusion of the gene coding for the self-hydrolysable peptide fmdv 2a and the gene coding for the enzyme aminoglycoside 3'phosphotransferase (aphviii), wherein said protein is separated from the aphviii enzyme after autohydolysis of the self-hydrolysable peptide fmdv 2a. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2633751A1
    申请号:ES201600166
    申请日:2016-02-23
    公开日:2017-09-25
    发明作者:Rosa LEÓN BAÑARES；Marta VILA SPÍNOLA
    申请人:Universidad de Huelva；
    IPC主号:
    专利说明:

    FIELD OF THE INVENTION
    The invention is in the technical field of obtaining proteins of interest in a host, wherein said host is a eukaryotic microalgae. Said microalgae may be of a genus of the chlorophyte division. Specifically, the present invention relates to a plasmid and the use of this plasmid in a method of expressing a protein in microalgae. The plasmid comprises a fusion comprising a gene that codes for the FMDV 2A self-curing peptide of Foot and mouth disease virus and the gene that encodes the Streptomyces rimosus 3'phosphotransferase enzyme (APHVIII).
    BACKGROUND OF THE INVENTION
    Microalgae are a heterogeneous group of microorganisms, mostly photosynthetic, of great ecological importance and with enormous biotechnological potential, whose use for large-scale production of carotenoids, polyunsaturated fatty acids and other compounds of high added value is well established. Moreover, in recent years, there has been a growing interest in microalgae as a possible raw material for a third generation of third generation biofuels with a carbon neutral balance. This has increased interest in microalgae genetic engineering as a possible tool to achieve economically viable production of raw materials and to increase the productivity of compounds with high added value. However, genetic manipulation of microalgae has so far been limited to a low number of species.
    It is estimated that the number of microalgae species ranges between 300,000 and one million species, of which approximately 30,000 have been classified and studied and less than 20 have been genetically transformed successfully. Most of the work on genetic manipulation has focused on classical species such as Chlamydomonas reinhardtii or Phaeodactylum tricornutum, which are not always those of greatest interest applied.
    The lack of endogenous promoters and other regulatory regions is one of the main problems for the genetic manipulation of new species of microalgae, but even
    When these elements are available, low efficiency and instability in the expression of transgenes are still common problems.
    The most commonly used selective genes in microalgae manipulation are those that confer resistance to antibiotics such as bleomycin (BLE), streptomycin (MOA) and paromomycin (APHVIII), or herbicides, such as the acetoliase synthase (ALS) gene, which confers resistance to the herbicide silfonylurea, or the BAR gene that confers resistance to the herbicide phosphinothricin (PPT). As for the promoters, their choice is, as indicated, critical for the development of an efficient transformation system. Promoters such as the small subunit of Chlamydomonas rhyphosphate carboxylase (RbcS2) have been used successfully in Chlamydomonas and other chlorophytes such as Chlorel / a ellipsoidea or Volvox cateri.
    The existence of self-processing peptides, such as FMDV virus (foot and mouth desease virus) peptide 2A, has been known for a long time. The peptide, of only 24 amino acids, is spontaneously hydrolyzed by the peptide bond that links the amino acids Gly23 and Pro24. The possibility of using this small FMDV 2A peptide as a strategy to express two genes under the same promoter and then thanks to its capacity for self-processing to obtain the two independent gene products has been used in several eukaryotic systems such as mammalian and plant cells or in therapy gene.
    Its operation in microalgae has recently been demonstrated (Rasala, BA et al. Robust expression and secretion of xylanasae1 in C. reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide. PLoS One 2012; 7 (8): e43349) . This document describes the fusion of the bleomycin resistance gene (BLE) with the Trichoderma reesei xylanase gene using the small self-hydrolyzing peptide (FMDV 2A) and demonstrated the obtention of the two proteins independently, the bleomycin binding protein (BLE) and xylanase. Moreover, they conclude that this fusion allows to obtain transformants with high levels of xylanase. But the selection antibiotic they use, bleomycin, has as its main drawback its high mutagenic power and low transformation efficiencies.
    DESCRIPTION OF THE INVENTION
    The problem in view of the closest state of the art can be defined as the contribution of a plasmid and method to transform microalgae with high efficiency
    of transformation and express proteins of interest in said microalgae. The solution to this problem is to provide a plasmid and defined procedure
    by the claims of the present application.
    In a first aspect, the present invention provides a plasmid for expressing aprotein, which comprises a gene that codes for said protein and the pHSP70A cassetteRbcS2-6xHis-PLK-FMDV2A-APHVIII-3'UTR identified by the sequence SEO ID NO: 1,where said cassette comprises:- the hybrid promoter pHSP70AlRbcS2 from Chlamydomonas,-the fusion of the gene that codes for Foot and mouth FMDV 2A self-hydrolyzing peptidedisease virus and the gene encoding the enzyme aminoglycoside 3'phosphotransferase fromStreptomyces rimosus (APHVIII).
    A second aspect is the first aspect of the present invention, wherein said plasmidis identified by the sequence SEO ID NO: 2.
    The plasmid of the first or second aspect of the invention solves the problemsraised in the prior art because it offers a procedure for the expression ofhigh amounts of the protein of interest and better transformation efficiency.
    The plasmid of the first or second aspect of the invention contains the translational fusion of thegene of interest with the paromomycin resistance gene, which lacks the effectsmutagenic of other antibiotics and allows, thanks to the FMDV 2A autohydrolyzable peptide, theindependent obtaining of the protein of interest and the antibiotic resistance protein.
    In addition, the plasmid of the first or second aspect of the invention allows fusion of theFMDV2A peptide by the amino terminus of the aminoglycoside 3'phosphotransferase, wherebyinterference from the small fragment resulting from peptide autolysis is avoidedFMDV2A exerts on the antibiotic activity of the aminoglycoside 3'phosphotransferase.
    The FMDV 2A peptide allows the autohydrolysis of the two proteins once formed, fromof the same common transcript, but uses the APHVI / I gene as a selectable gene, whichencodes the enzyme aminoglycoside 3'phosphotransferase from Streptomyces rimosus and confersAntibiotic resistance paromomycin. Previous attempts to fuse proteins with geneAPHVII 'have not been successful, since this gene seems to lose its functionality when itadd peptides to the fine carboxyl end l.
    The plasmid of the first or second aspect of the invention contains the gene encoding the Streptomyces rimosus aminoglycoside enzyme 3'phosphotransferase (APH VIII), which confers resistance to the antibiotic paromomycin, linked by its amino terminal end with a polycloning region through a self-hydrolyzing peptide, such as the FMDV 2A peptide of the foot and mouth desires virus.
    This allows insertion and expression in a coordinated manner with the antibiotic resistance gene of any gene of interest. Furthermore, preceding the polycloning region we have introduced an epitope, easily detectable by immunochemical techniques, and the entire cassette is under the control of the hybrid promoter RbcS2 / HSP70A from Chlamydomonas (Fig. 1; Fig. 2). The relative position of the promoters with respect to the polilinker and all the elements of the plasmid have been optimized, eliminating unnecessary nucleotides, to improve the expression and thus increase the efficiency of the transformation up to 3 times with respect to other plasmids that have the same promoter. .
    Since the antibiotic resistance gene (APHVIII) and the gene of interest are expressed under the control of the same promoter, screening with increasing amounts of the antibiotic paromomycin provides a simple method to select the transformants with the highest level of expression of the APHVIII gene and consequently of the gene of our interest.
    The present invention avoids the use of the BLE gene, which is resistant to bleomycin, which has proven highly mutagenic and introduces undesirable alterations to isolated transformants. Furthermore, the placement of the polycloning region at the amino terminus solves the problems to fuse protein sequences to the carboxyl end of the 3'phosphotransferase aminoglycoside protein. We have been able to verify that even small peptides such as the FMDV 2A self-processable peptide interfere with the functionality of the 3'phosphotransferase aminoglycoside if they are attached to its carboxyl terminus.
    In a third aspect, the present invention provides a method of expressing a protein in microalgae, comprising:
    (to) transforming said microalgae with a plasmid according to claim 1 or 2,
    (b) expressing said protein and the fusion of the gene that codes for the FMDV 2A self-curing peptide of Foot and mouth disease virus and the gene that encodes the Streptomyces rimosus 3'phosphotransferase enzyme (APHVIII), where said protein
    it is separated from the APHVIII enzyme after autohydrolysis of the FMDV 2A autohydrolyzable peptide.
    A fourth aspect is the process of the third aspect of the invention, wherein said microalgae is a microalgae of a genus of the chlorophyte division.
    Preferably, said microalgae is of the genus Chlamydomonas or Phaeodactylum.
    More preferably, said microalgae is Chlamydomonas reinhardtíi.
    FREE TEXT OF THE LIST OF SEQUENCES
    Below is a translation of the free text in English that appears in the sequence list.
    SEQ ID NO: 1. Partial sequence of plasmid Phyc069; pHSP70A-RbcS2-6xHisPLK-FMDV2A-APHVIII-3 'UTR cassette
    one . .460 HSP70A-RbcS2 506 ..650 RbcS2 intron 1 687 .. 7046xHis 705 .. 752 Polilinker 753..824 FMDV2A 834 .. 1637 APHVIII SEQ ID NO: 2. Complete sequence of plasmid Phyc069 696 .. 1155 HSP70A-RbcS2 1201 .. 1345 RbcS2 intron 1 1382 .. 1399 6xHis 1400 .. 1447 Polilinker 1448 .. 1519 FMDV2A 1529 .. 2332 APHVIII
    BRIEF DESCRIPTION OF THE FIGURES
    Figure 1. Schematic representation of plasmid Phyc069 with indication of the main restriction sites.
    Figure 2. Detail of the main components of plasmid Phyc069.
    Figure 3. Comparison of the efficiency of plasmid pSI1 03 (A) and Phyc069 (B). First row, results using 200 ng of each plasmid. Second row, results using 500 ng of each plasmid. Third row, results using 1 I-Ig of each plasmid.
    Figure 4. Schematic representation of plasmid Phyc069-ARS, with indication of the main restriction sites.
    Figure 5. Results of RT-PCR by immunodetection with commercial monoclonal antibodies anti-alkaline histidine phosphatase (SIGMA), which bind to the ARS2 protein, with a molecular weight of 77425 daltons, and the ARS2-APHVIII fusion protein, with a molecular weight of 106,000 daltons.
    PREFERRED MODES OF EMBODIMENT
    EXAMPLE 1. Transformation of the eukaryotic C. reinhardtii microalgae with the plasmid Phyco69
    The plasmid for the expression of exogenous proteins in eukaryotic microalgae by translational fusion with the selectable marker gene APHVI / I has been denoted Phyc069.
    This example describes the transformation of the microalgae model Chlamydomonas reinhardtii with the plasmid Phyc069 and the transformation frequency observed for this plasmid compared to other plasmids containing the same selective marker gene, such as pS1103.
    The 704 line of Chlamydomonas reinhardtii, which lacks a cell wall, was acquired from the Culture Collection of Algae at the University of Texas (UTEX, Austin, TEX, USA) and kept in Tris-Acetate-Phosphate (T AP) medium, prepared as described in Harris (2009).
    Plasmid Phyc069 and plasmid pSI103 were stored in E. coli with 15% glycerol at 80 ° C for maintenance and propagation. Plasmid pSI103 has been described in Sizova, 1. et al. A Streptomyces rimosus aphVI / I gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene 2001; 277: 221-229.
    Plasmids were obtained from the corresponding bacteria by the classical methods of plasmid DNA isolation based on separation with silica columns.
    C. reinhardtii cells were allowed to grow until the middle of the exponential phase of
    5 growth (about 1.6x106 mL-1 cells), were collected by centrifugation and resuspended in fresh T AP medium to obtain a 100 times concentrated cell suspension.
    Increasing amounts, ranging between 0.2 and 2 g, of each plasmid were added to
    10 different 15 mL tubes with 0.6 mL of the 100 times concentrated cell suspension, 0.1 mL of 20% PEG 8000 and 0.3 gr of glass beads. Each tube was stirred vigorously for 8 seconds (the stirring time may vary depending on the species to be transformed). The transformed cells were resuspended in 50 mL of sterile Tris Acetate phosphate (TAP) medium and incubated overnight in the culture chamber. The whole process was carried out.
    15 in sterility. After this incubation in the absence of an antibiotic, the culture was collected by centrifugation and the precipitated cells were resuspended in 0.8 mL of culture medium and inoculated on plates with solid AP T medium and the corresponding antibiotic, in this case paromomycin (30 J ..lg mr The colonies of transformed microalgae are visible after 5-7 days of culture (Fig. 3).
    The number of transformants obtained for the different plasmid concentrations was that indicated in Table I
    Table 1. Frequency of transformation of C. reinhardtii for different quantities of 25 plasmids
    Plasmid 200 ng500ng1 ~ g2 ~ g
    Phyc069 98215380820
    pSI103 2860140250
    Each transformation was done in triplicate, the indicated values being the average of three repetitions.
    The number of transformants obtained for both plasmids is greater with higher amounts of DNA. In all cases the number of transformants was much higher for
    the plasmid Phyc069, object of this patent, obtaining transformation frequencies 3 times higher than those obtained with the standard plasmid pSI1 03.
    EXAMPLE 2. Cloning and expression of the ARS gene of Chlamydomonas reihardtii in the eukaryotic microalgae C. reinhardtii with the plasmid Phyco69
    This example describes the cloning of the ARS gene, which encodes the arylsulfatase enzyme, whose enzymatic activity can be easily tested with a chromogenic substrate in the Phyc069 plasmid, object of this patent and its expression in the model microalgae
    Chlamydomonas reinhardtií.
    A DNA fragment corresponding to the messenger RNA of the ARS gene of Chlamydomonas reinhardtií was obtained. The fragment was designed to contain cut sites for the Xhol and Ndel enzymes and lack stop codon. It was also designed so that when introduced between the Xhol and Ndel cutting sites of the polylinker of the plasmid Phyc069 it will be in the same reading phase as the APHVIII gene.
    Plasmid Phyc069-ARS was used to transform the Chlamydomonas reinhardtii strain 704 microalgae, using the glass bead stirring method as described in the previous example.
    The 704 line of Chlamydomonas reinhardtii, which lacks a cell wall, was acquired from the Culture Collection of Algae at the University of Texas (UTEX, Austin, TEX, USA) and maintained in TAP medium.
    C. reinhardtií cells were allowed to grow to half of the exponential growth phase (about 1.6x106 mL01 cells), were collected by centrifugation and resuspended in fresh TAP medium to obtain a 100-fold concentrated cell suspension.
    Approximately 1 ~ g of the Phyc069-ARS plasmid was added to an eppendorf tube containing 0.6mL of the concentrated cell suspension, 0.1 mL of 8000 PEG and a small amount of glass beads. The tube was vigorously shaken for 8 seconds.
    The transformed cells were resuspended in 50 mL of sterile Tris Acetate phosphate (TAP) medium and incubated overnight in the culture chamber. After this incubation in the absence of an antibiotic, the culture was collected by centrifugation and the precipitated cells were resuspended in 0.8 mL of culture medium and inoculated in plates with solid AP T medium and the corresponding antibiotic, in which 4 reactions of transformation that were plated on plates with paromomycin concentrations of 50, 100, 150 Y
    5 200 ~ lg mL1. Transformed microalgae colonies are visible after 5-7 days of culture. The number of transformants decreased as the antibiotic concentrations were more restrictive, the two transformants that appeared at the highest antibiotic concentration were chosen.
    10 The correct insertion of both the selective gene, APHVIII, and the gene of our interest ARS in the genome was checked by PCR and its correct transcription and translation was verified by RT-PCR from messenger RNA and by immunodetection with commercial anti monoclonal antibodies -poli Histidia-alkaline phosphatase (SIGMA), which bind to the ARS2 protein, with a molecular weight of 77,425 daltons, and the fusion protein
    15 ARS2-APHVIII, with a molecular weight of 106,000 daltons (Fig. 4).
    权利要求:
    Claims (5)
    [1]
    1. A plasmid for expressing a protein, characterized in that it comprises a gene encoding said protein and the cassette pHSP70A-RbcS2-6xHis-PLK-FMOV2A-APHVIII-3'UTR identified by the sequence SEQ ID NO: 1, wherein said cassette comprises:
    5 -the hybrid promoter pHSP70AlRbcS2 of Chlamydomonas, -the fusion of the gene that codes for the self-hydrolyzing peptide FMOV 2A of Foot and mouth disease virus and the gene that codes for the enzyme aminoglycoside 3'phosphotransferase of Streptomyces rimosus (APHVIII).
    [2]
    2. Plasmid according to claim 1, identified by the sequence SEQ ID NO: 2.
    10 3. A method of expressing a protein in microalgae, comprising:
    (to) transforming said microalgae with a plasmid according to claim 1 or 2,
    (b) express said protein and the fusion of the gene that codes for the FMOV 2A self-hydrolyzing peptide of Foot and mouth disease virus and the gene that encodes the Streptomyces rimosus 3'phosphotransferase enzyme (APHVIII), where
    Said protein is separated from the APHVIII enzyme after autohydrolysis of the FMOV 2A autohydrolyzable peptide.
    [4]
    4. Method according to claim 3, characterized in that said microalgae is a microalgae of a genus of the chlorophyte division.
    [5]
    5. Method according to claim 4, characterized in that said microalgae is of the genus Chlamydomonas.
    [6]
    6. Plasmid according to claim 5, characterized in that said microalgae is
    Chlamydomonas reinhardtii.
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    DE29916782U1|1999-09-23|2000-05-04|Entelechon Gmbh|Aminoglycoside O-Transferase Type VIII |
    GB2507030A|2012-08-16|2014-04-23|Spicer Consulting Ltd|Algal genome modification|
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