RU2409671C1 - Recombinant plasmid for phospholipase gene expression in pichia pastoris yeast, pichia pastoris yeast strain-phospholipase producer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to recombinant plasmid pP10PLC-Bc for phospholipase gene expression in Pichia pastoris yeast. Also, the invention refers to a recombinant Pichia pastoris PLC-Bc-1 yeast strain RNCIM (Russian National Collection of Industrial Microorganisms) No. Y-3359 phospholipase producer.

EFFECT: extended range of products for producing phospholipase.

2 cl, 2 dwg, 4 ex

Description

The invention relates to biotechnology, genetic engineering, and for the production of a multicopy plasmid vector for integration and gene expression.

One of the most common ways to increase the amount of the target product when it is obtained by microbiological synthesis is to introduce the gene responsible for the synthesis of the produced substance into the cell of the recipient strain and to obtain the target product by culturing the constructed recombinant strain. As a rule, for introducing a gene into the cell of a recipient strain, a vector is used that contains, in particular, an expression cassette (promoter, transcription terminator, selective marker) and an integration site.

Known is the multicopy expression vector pNB268 [ATCC 69355] for gene expression on the Yarrowia lipolytica yeast chromosome, containing the XPR2 alkaline protease gene promoter, the LEU2 selective marker, a homologous region for integration into a given XPR locus.

Known (www.invitrogen.com) is the multicopy expression vector pPIC9 for gene expression on the Pichia pastoris chromosome, which contains the strong inducible promoter 5 'AOX1, transcription terminator 3' AOX1, HIS4 selective marker, 3 'AOX1 and ά integration site amylase signal peptide. However, methanol is the inducer of expression of the cloned gene in this case, which complicates its technological use.

For the expression of genes encoding non-toxic proteins for Pichia pastoris proteins, a constitutive expression plasmid vector pGAPZάA (www.invitrogen.com) has been proposed. It contains the GAP promoter, 3 'AOX1 transcription terminator, Zeocin selective marker, 3' AOX1 and ά- integration site amylase signal peptide. However, strains obtained using the pGAPZάA vector, as a rule, do not have high productivity, which is probably due to the low number of copies of the AOX1 integration site on the Pichia pastoris yeast chromosome.

The objective of the invention is the expansion of the arsenal of recombinant plasmids for expression in yeast Pichia pastoris gene encoding phospholipase and recombinant strains of yeast Pichia pastoris producing phospholipase.

The problem was solved by constructing the recombinant plasmid pP10PLC-Bc for expression in the yeast Pichia pastoris of the gene from Bacillus cereus, responsible for the synthesis of phospholipase C enzyme PLC-Bc. The constructed plasmid has a size of 10770 bp and consists of the following elements (figure 2):

1) fragment 5 '18S rRNA: 1-269 bp .;

2) GAP promoter: 270-1026 bp;

3) ά-amylase signal peptide: 1029-1295 bp;

4) PLC-Bc phospholipase gene; 1304-2054 bp;

5) 3 'AOX1 transcription terminator: 2088-2421 bp;

6) HIS4 ORF: 3279-5813 bp;

7) fragment 3 '18S rRNA: 6085-6774 bp .;

8) pUC ori: 7974-8648 bp;

9) ampicillin resistance gene: 9448-10308 bp

The problem was also solved by constructing a strain of Pichia pastoris-PLC-Bc-1 VKPM Y-3359, a producer of phospholipase C PLC-Bc.

To obtain a recombinant plasmid, the pP10 vector is used, which contains the DNA sequence encoding the 18S rRNA region as an integration site, contains a selective bla marker as a selection marker for transformants in E. coli cells, and contains pUC ori as the origin of replication the expression cassette includes the GAP promoter, AOX1 transcription terminator, HIS4 selective marker, and the vector contains an ά-amylase signal peptide as a signal peptide. The pP10 vector (FIG. 1) is constructed as follows.

DNA fragments encoding the GAP promoter and the 18S rRNA region are amplified by polymerase chain reaction. The amplified sequence of the GAP promoter at the blunt ends is ligated into the pPIC9 vector (Invitrogen), from which the 5 'AOX1 promoter sequence is preliminarily cut from the BglII and BamHI sites. Get the vector pPIC9G.

The amplified sequence of the gene encoding the 18S rRNA region at the blunt ends is ligated into the pGEM5Zf (-) vector (Promega), previously digested with restriction enzymes at the EcoRV and HincII sites, to obtain the pNS4 vector, which is then digested with restriction enzyme at the Eco147I site and ligated with a DNA fragment, code expression cassette from pPIC9G, which is pre-digested with restriction enzyme PaeI and blunt.

The constructed pP10 vector is further used to produce recombinant plasmids designed to express the target protein genes in Pichia pastoris yeast.

An important genetic characteristic of the pP10 vector is a set of DNA fragments of certain sizes, which is formed during the polymerase chain reaction carried out using specially selected selective primers designed based on its nucleotide sequence.

This characteristic can be used to identify strains derived from this vector.

Recombinant plasmids are constructed by inserting a heterologous gene into the pP10 vector at NotI or SnaB1 sites using conventional methods.

The Pichia pastoris-PLC-Bc-1 strain producing phospholipase C PLC-Bc is obtained by transforming Pichia pastoris GS115 (his4) VKPM Y2837 cells with the recombinant plasmid pP10-PLC-Bc. Transformants were selected on minimal M9 medium [T. Maniatis et.al., Molecular Cloning, A laboratory Manual, Cold Spring Harbor Laboratory, 390, 1982] for growth ability without a histidine source.

The phospholipase activity was determined from the selected transformants according to the modified Kurioka method [Biotecnology Letters 26, 1475-1479, 2004] and a clone with the maximum phospholipase activity was selected.

The strain Pichia pastoris PLC-Bc-1 - producer of phospholipase C PLC-Bc is deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) and has a registration number VKPM Y-3359.

Cells are stored in glycerol at a temperature of -70 ° C or in freeze-dried form at a temperature of + 4 ° C.

The strain is characterized by the following features.

Morphological signs.

With growth on Malt agar [The yeast of toponomic study. Ed. N.J.W. Kreger-van Rij, Amsterdam, Elsevier Sci. Publ. B.V., 1984, p.421] within 48 hours at a temperature of 30 ° C, the cells form smooth, round colonies with a matte surface of a light cream color.

With growth on Malt extract [The yeast of toponomic study. Ed. N.J.W. Kreger-van Rij, Amsterdam, Elsevier Sci. Publ.B.V., 1984, p. 421] within 48 hours at a temperature of 30 ° C, the cells form an intense even suspension. The culture has a characteristic smell of yeast.

On the Malt agar medium, after 3 days of cultivation, the culture at a temperature of 30 ° C forms round, slightly oval-shaped cells of 5-10 μm in size, some of the cells on the surface of the kidney or connected to daughter cells.

Cultural signs.

Cells grow well on a complete organic YEPD medium of the following composition (wt.%): Peptone - 2, yeast extract - 1, glucose - 2 (or glycerin - 1), water - the rest, as well as minimal medium M9 and other synthetic media for yeast containing glucose or glycerin as a carbon source.

The cells of the strain are characterized by weak growth on media containing methanol as a carbon source.

Physiological and biochemical characteristics.

Cells grow at temperatures from 4 to 37 ° C. The optimum temperature for growing is 30 ° C.

The optimum pH for growth is 4.5-6.5.

As a carbon source, cells use glucose, glycerin, methanol. As a source of nitrogen - mineral salts in ammonium form, amino acids, urea.

Cells are capable of aerobic and anaerobic growth.

The strain does not need histidine for its growth.

The invention is illustrated by the following drawings.

Figure 1 Scheme of the vector pP10.

Figure 2 Scheme of the recombinant plasmid pP10PLC-Bc.

Example 1. Construction of the vector pP10.

Vector pPIC9 (Invitrogen, No. K171001) was digested with restriction enzymes BglII and BamHI in 2X Tango ^™ (yellow) buffer (Fermentas) to remove the sequence encoding the 5 ′ AOX1 promoter. The reaction is carried out for 1 hour at 37 ° C and stopped by heating at 65 ° C for 10 minutes The BglII - BamHI fragment of the pIPIC vector (7052 bp) is isolated by electroelution from a 1% agarose gel and dephosphorylated with bacterial alkaline phosphatase.

From the strain Pichia pastoris GS115 (his4) VKPM Y2837 by the phenolic method [T. Maniatis et. al., Molecular Cloning, A laboratory Manual, Cold Spring Harbor Laboratory, v.3, 1982] isolate chromosomal DNA, which serves as a matrix for the synthesis of the GAP promoter. The sequence encoding the GAP promoter is amplified by PCR. For PCR synthesis, primers are used that are designed based on the nucleotide sequence encoding the GAP promoter (23-SI, 29-SI):

At the 5'-end of each primer there is a restriction site for the endonuclease forming sticky ends: BamHI for the forward primer and BglII for the reverse (in bold in the above sequences).

To carry out the polymerase chain reaction, 100 μl of the reaction mixture containing 0.5 ng of the DNA template, 1 μM of 23-SI primer, 1 μM of 29-SI primer, 2.5 units are used. Tag polymerase in 10 μl of 10 × Tag buffer, 1.5 mM MgCl ₂ , 0.8 mM dNTP. The reaction is carried out according to the following scheme: 94 ° C - denaturing (1 min), 59 ° C - annealing (1 min), 72 ° C - polymerization (1 min). A total of 30 cycles of amplification. The amplified DNA fragment is purified using DNA extraction KIT (Fermentas).

The PCR amplification thus obtained is hydrolyzed with BglII and BamHI restriction endonucleases, purified on an agarose gel and ligated with the BglII - BamHI fragment of the pPIC9 vector (7052 bp) using phage T4 DNA ligase.

Ligase mixture transform competent E. coli C600 cells prepared the day before by treatment with calcium chloride. After the standard transformation procedure (0 ° С - 40 min, 42 ° С - 2 min, 0 ° С - 5 min), the cells are diluted 10 times with LB medium (T. Maniatis, E. Fritsch, J. Sembrook. Molecular cloning. M .: Mir, 1984, 84), grown for one hour and plated on LB agar containing ampicillin at a concentration of 50 μg / ml. Crops are incubated at 37 ° C.

The next day, the grown ampicillin-resistant colonies were tested using the above primers and positive clones were selected from which plasmid DNA was isolated by a standard method (T. Maniatis, E. Fritsch, J. Sembrook. Molecular Cloning. M .: Mir, 1984, 89). Isolated plasmid DNAs are hydrolyzed with BglII and BamHI restriction endonucleases and checked for inserts corresponding to the sequence encoding the GAP promoter. Of the clones whose plasmid DNA contains the sequence encoding the GAP promoter, the pPIC9G vector is isolated.

Chromosomal DNA isolated from Pichia pastoris strain GS 115 (his4) VKPM Y2837, as described above, serves as a template for PCR synthesis of a fragment of a sequence encoding an 18S rRNA region. For amplification, primers are used that are designed based on the nucleotide sequence encoding the 18S rRNA region (NS1, NS2):

direct - 5'-GTAGTCTATGCTTGTCTC-3 'and

the reverse is 5'-CTTCCGTCAATTCCTTTAAG-3 '.

The conditions under which the polymerase chain reaction is carried out are described above.

The obtained PCR amplification was purified on an agarose gel and ligated (using phageT4 DNA ligase) with the vector pGEM5Zf (-), which was preliminarily digested with restrictase enzymes at EcoRV and HincII sites.

Ligase mixture transform competent E. coli C600 cells, as described above. An ampicillin-resistant clone is selected, a plasmid is isolated from it, it is checked for the presence of inserts by PCR using the primers described above. Get a new plasmid vector pNS4.

The pPIC9G vector is cleaved with a PaeI restriction endonuclease, the sticky ends are blunt with Pfu polymerase, and a 3889 bp fragment is excised from the agarose gel. The excised fragment was purified using DNA extraction KIT and ligated with the pNS4 vector previously cleaved with the Eco 147I restriction endonuclease.

Ligase mixture transform competent E. coli C600 cells, as described above. Ampicillin-resistant clones are selected, plasmid DNA is isolated from them, and it is checked for the presence of an inserted fragment by PCR using all of the primers described above. The pP10 vector is obtained (FIG. 1).

Based on the nucleotide sequence of the pP10 vector, primers are designed that are used for the genetic characterization of the resulting vector:

direct (N1) - 5'-GTAGTCATATGCTTGTCTC-3 'and

reverse (N2) - 5'-AAAGATCTTTGATAGTTGTTCAATTGATT-3 ';

straight (N3) - 5'-AGGCCCCGTGGCCGGGGGACT-3 'and

reverse (N4) - 5'-CTTCCGTCAATTCCTTTAAG-3 '.

The polymerase reaction is carried out as described above, using pairs of N1, N2 and N3, N4 as primers.

The size of the resulting PCR fragments is determined gel electrophoretically using a dye (ethidium bromide) [T. Maniatis, E. Fritsch, J. Sembrook. Molecular Cloning M.: Mir, 1984, 84].

When using primers N1 and N2 during the polymerase chain reaction, a fragment of 2300 pairs of nucleotides is produced, and when using primers N3 and N4, a fragment of 380 pairs of nucleotides is produced.

The size of the resulting DNA fragments is an individual characteristic of the claimed vector and allows identification of strains obtained using the pP10 vector.

Example 2. Construction of a recombinant plasmid pP10PLC-Bc.

Chromosomal DNA is isolated from the Bacillus cereus ATCC 14579 strain by the phenolic method, which serves as a template for the synthesis of the phospholipase C gene PLC-Bc.

The sequence encoding the PLC-Bc phospholipase C gene is amplified by PCR. For PCR synthesis, primers are used that are designed based on the nucleotide sequence encoding the PLC-Bc phospholipase C gene (Plcl, Plc2). At the 5'-end of each primer, a restriction site for the endonuclease Eco 47III was inserted for the forward primer and Not 1 for the reverse primer (in bold sequences):

To carry out the polymerase chain reaction, 100 μl of the reaction mixture containing 0.5 ng of the DNA template, 1 μM Plc1 primer, 1 μM Plc2 primer, 2.5 units are used. Tag polymerase 10 μl of 10 × Tag buffer, 1.5 mM MgCl ₂ , 0.8 mM dNTP. The reaction is carried out according to the following scheme: 94 ° C - denaturing (1 min), 59 ° C - annealing (1 min), 72 ° C - polymerization (1 min). A total of 30 cycles of amplification. The amplified DNA fragment is purified using DNA extraction KIT.

The PCR amplification thus obtained is hydrolyzed with restriction endonucleases Eco 47III and Notl (Fermentas), purified on an agarose gel and ligated with the pP10 vector digested with restriction enzymes at SnaB 1-Not 1 sites using T4 phage DNA ligase.

Ligase mixture transform competent E. coli C600 cells, as described in example 1.

The selection of transformants, the isolation of plasmid DNA and hydrolysis is carried out as described in example 1, using primers P1c1 and P1c2 and restriction endonucleases Eco 47III and Not 1. A new recombinant plasmid pP10PLC-Bc is obtained (Fig. 4).

Plasmid pP10PLC-Bc is digested with restriction enzyme Bgl II, electroeluted in a 1% agarose gel, and used to transform yeast cells, as described in Example 3.

Example 3. Obtaining a yeast strain Pichia pastoris PLC-Bc-1 VKPM Y-3359 - producer of phospholipase C PLC-Bc.

To obtain the yeast strain Pichia pastoris PLC-Bc-1, a producer of phospholipase C PLC-Bc, yeast cells of strain GS115 (his4) VKPM Y-2837 are transformed with plasmid pP10PLC-Bc.

1 ml of an overnight culture of Pichia pastoris Y-2837 yeast cells is grown in 100 ml of YEPD medium at 30 ° C until the culture reaches an optical density corresponding to 2 units. absorption at a wavelength of 600 nm. The cells are washed twice with sterile water, after which they are suspended in 0.3 ml of a 100 mM lithium acetate solution and incubated at 30 ° C for 30 minutes. To 50 μl of the obtained cell suspension, 1 μg of plasmid DNA, 50 μg of salmon semen DNA pre-denatured by heating (10 min at 100 ° C), and 0.3 ml of a solution of 100 mM lithium acetate containing 40% polyethylene glycol 4000 are added. Next, the sample is incubated. 30 minutes at 30 ° C and 20 minutes at 42 ° C, placed for 15 seconds in an ice bath and centrifuged for 10 seconds at 10,000 rpm. Cells are suspended in 1 ml of sterile water and plated on solid M9 medium with the addition of 0.3 vol.% Trace elements. Clones of transformants are grown for 3 days.

The obtained transformants are seeded in liquid YEPD medium and grown at 30 ° C until the stationary phase of growth for 2 days. Next, the cells are precipitated by centrifugation at 10,000 rpm for 5 minutes, 100 μl of the supernatant are taken and phospholipase activity is determined by the accumulation of para-nitrophenol detected by the Kurioki method in the reaction medium. One unit of enzyme activity corresponds to the amount of enzyme that decomposes 1 μmol of paranitrophenylphosphatidylcholine in 1 minute. The enzyme activity is measured using a spectrophotometer at a wavelength of 410 nm.

The transformant that showed the highest activity of phospholipase C PLC-Bc in the culture fluid was stored and deposited as Pichia pastoris VKPM Y-3359.

To prove the presence of the pP10 vector in the genome of the obtained strain of Pichia pastoris PLC-Bc-1 yeast, a genetic characterization based on obtaining DNA fragments of a given size is used. Fragments are produced during the polymerase chain reaction using primers N1, N2, N3 and N4 (example 1).

When using primers N1 and N2, a DNA fragment of 2300 nucleotide sizes is produced; when using primers N3 and N4, a DNA fragment of 380 nucleotide sizes is produced.

Example 4. Synthesis of phospholipase With PLC-Bc strain of Pichia pastoris PLC-Bc-1 VKPM Y-3359.

The inoculum culture of the producer strain PLC-Bc-1 VKPM Y-3359 was grown in 5 ml of YEPD liquid medium at 30 ° C. overnight. The resulting overnight culture inoculated an Erlenmeyer flask, 750 ml, containing 100 ml of YEPD medium, in a ratio of 1: 100. The culture is incubated at 30 ° C and vigorous shaking until the stationary phase is reached within 2 days. Cells are pelleted by centrifugation at 10,000 rpm for 5 minutes, the supernatant is transferred to another container, an aliquot of 100 μl is taken and phospholipase activity is determined as described in Example 3.

Phospholipase activity is 200 u / ml of culture fluid.

Claims (11)

1. Recombinant plasmid pP10PLC-Bc for expression of the phospholipase C PLC-Bc gene in Pichia pastoris yeast having a size of 10770 bp and consisting of elements: 1. fragment 5 '18S rRNA: 1-269 bp .; 2. GAP promoter: 270-1026 bp; 3. ά-amylase signal peptide; 1029-1295 bp; 4. phospholipase C gene PLC-BC: 1304-2054 bp .; 5. termination transcription 3 'AOX1: 2088-2421 bp .; 6. HIS4 ORF: 3279-5813 bp .; 7. fragment 3 '18S rRNA: 6085-6774 bp .; 8. pUC ori: 7974-8648 bp; 9. ampicillin resistance gene: 9448-10308 bp 2. The strain of the yeast Pichia pastoris PLC-Bc-1 VKPM Y-3359 - producer of phospholipase C PLC-Bc.

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