RU2756330C1 - Komagataella phaffii yeast transformant that produces cronobacter turicensis phytase - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. A transformant of the yeast Komagataella phaffii producing phytase of Cronobacter turicensis is proposed. The transformant contains the DNA sequence of the m-phyCt gene encoding the phytase of Cronobacter turicensis, or one of the variants of such a sequence due to the degeneracy of the genetic code.

EFFECT: resulting phytase retains at least 80% and about 100% activity when exposed to pepsin or trypsin, respectively, for 1 hour at 37°C and a protease/enzyme ratio of 0.1 by weight.

1 cl, 3 dwg, 1 tbl, 3 ex

Description

The invention relates to microbiology and biotechnology and relates to the production of strains of yeast Komagataella phaffii (Pichia pastoris) capable of producing the enzyme phytase.

Phosphorus is an essential chemical essential for the growth and development of animals. Most plant feeds used in animal husbandry, such as cereals and legumes, contain phosphorus mainly in the form of phytate (myo-inositol hexakisphosphate) [Advanced Food Research, 1982, 28, 1-92.]. Monogastric animals are unable to assimilate phytate phosphorus due to the lack of the necessary enzyme in the digestive tract [Can. J. Anim. Sci., 2013, 93, 9-21.].

The presence of phytate in the compound feed also prevents the effective assimilation of calcium, magnesium, zinc, iron and other minerals and leads to the binding of feed proteins into complexes inaccessible for digestion [Current Topics In Nutraceutical Research, 2008, 6 (3), 131-144.] ...

Phytases (myo-inositol hexakisphosphate phosphohydrolases) hydrolyze phytate with the elimination of phosphate groups. These enzymes are successfully used as a feed additive, significantly increasing the absorption of phosphorus [J. Sci. Food Agric., 2015, 95, 878-896.]. They provide the release of not only phytate-bound phosphorus, but also proteins, macro- and micronutrients, increasing the nutritional properties of feed [British Poultry Science, 2004, 45 (1), 101-108.].

Natural sources of phytases are various microorganisms: bacteria, fungi [FEMS Microbiology Reviews, 2005, 29 (1), 3-23.].

In industry, phytases are obtained by microbiological synthesis using producer strains, the industry demand for which is constantly growing [Afr. J. Biotechnol., 2009,8 (17), 4229-4232.]. The most promising producers of phytase are obtained on the basis of recombinant strains of methylotrophic yeast Komagataella phaffii (Pichia pastoris) [J. Mol. Recognit., 2005, 18 (2), 119-138. doi: 10.1002 / jmr.687], which have powerful systems for the expression and secretion of recombinant proteins (including phytases) and for which culture media have been developed and the fermentation process has been developed using a high-density culture. The process of cultivation of methylotrophic yeast is quite simple, since their growth is not blocked by metabolic products [FEMS Microbiol. Rev., 2000, 24: 45-66, doi: 10.1111 / j.1574-6976.2000.tb00532.x].

On the basis of the yeast K. phaffii, phytase producers have been constructed: Citrobacter amalonaticus [BMC Biotechnology, 2015, 15, 88.], Escherichia coli [J. Agric. Food Chem., 2013, 61 (25), 6007-6015., CN 102586294, CN 102943083], Peniophora lycii [Appl. Microbiol. Biotechnol., 2006, 72 (5), 1039-47.], Citrobacter braakii [Acta Microbiologica Sinica, 46 (6), 945-950.], Yersinia rohdei, Yersinia pestis, Y. intermedia [Appl. Microbiol. Biotechnol., 2008, 80 (3), 417-426.] And others.

The genes encoding phytases of various origins are expressed in the yeast K. phaffii with different efficiencies. In the expression of genes encoding phytases from Yersinia rohdei, Yersinia pestis, Y. intermedia, E. coli, or Aspergillus niger, in the yeast K. phaffii, the phytase activity in the culture liquid on the 4th day of cultivation is 313, 19, 270, 219, and 16 units, respectively. / ml. [Appl. Microbiol. Biotechnol., 2008, 80 (3), 417-426.]. At the same time, phytases of various origins differ in properties that are essential for industrial use, such as the optimal working pH range, resistance to the action of proteolytic enzymes, temperature, pH and others [J. Agric. Food Chem., 2013, 61 (25), 6007-6015; Annu. Rev. Anim. Biosci., 2013, 1, 283-309.].

The production of new phytases with commercially valuable properties and capable of being efficiently expressed in the yeast K. phaffii is an urgent task.

The technical problem to be solved by the claimed invention is to expand the arsenal of recombinant microorganisms that produce phytase.

The technical result achieved during the implementation of the claimed invention is to obtain a transformant of the yeast Komagataella phaffii - a producer of PhyCt phytase from Cronobacter turicensis.

Obtaining the claimed transformant involves the introduction of the DNA sequence of the m-phyCt gene encoding phytase PhyCt from C. Turicensis (or one of the variants of its DNA sequence due to the degeneracy of the genetic code) into the yeast K. phaffii cells using an expression cassette containing the m-phyCt gene or one of the variants of its sequence, a promoter working in the yeast K. phaffii, a signal peptide for the secretion of the enzyme into the culture fluid, as well as a terminator, a marker gene and, preferably, a site for homologous integration into the chromosome.

The nucleotide sequence of the m-phyCt gene encoding phytase PhyCt from C. turicensis is presented in the international Genbank database under the number MT489463. Due to the "degeneracy" of the genetic code, the same amino acid sequence can be encoded by a large number of nucleotide sequences, therefore, not only the nucleotide sequence itself (m-phyCt gene), but also all its variants determined by the degeneracy of the genetic code can be used for cloning.

Integration of the nucleotide sequence is carried out by either homologous or non-homologous recombination. Transformation of the expression cassette into K. phaffii yeast cells is carried out in particular by the electroporation method [http://tools.thermofisher.com/content/sfs/manuals/pich_man.pdf], by the method using polyethylene glycol or protoplasts [http: // www. thermofisher.com/order/catalog/product/K173001].

The construction of expression cassettes is carried out by standard methods of genetic engineering [Rybchin V.N. Fundamentals of Genetic Engineering. - SPb .: SPbSTU, 1999; Sambrook J., Maniatis T., Fritsch E. Molecular cloning: a laboratory manual. - N.Y .: Cold Spring Harbor Laboratory, 1989.] using genetic elements suitable for working with the yeast K. phaffii. AOX1, DAS, FLD1, ICL1, PHO89, THI11, ADH1, ENO1, GUT1, GAP, TEF1, PGK1, GCW14, G1, G6 or others are used as promoters [Appl. Microbiol. Biotechnol., 2014, 98, 5301-5317.]. Α-factor, PHO1, SUC2, PHA-E, KILM1, pGKL, CLY, CLY-L8, K28 pre-pro-toxin or others are used as signal peptides [Appl. Microbiol. Biotechnol., 2014, 98, 5301-5317.]. Any suitable markers can be used as selectable markers, for example, genes for resistance to antibiotics zeocin, geneticin (G418) or blasticidin C, as well as genes for complementing auxotrophic mutations in the K. phaffii genome, for example, HIS4, MET2, ADE1, ARG4, URA3, URA5 , GUT1 [Yeast, 2005, 22, 249-270.]. As arms for homologous integration, gene sequences AOX1, HIS4 [http://www.thermofisher.com/order/catalog/product/V17520] or other sequences homologous to regions of the K. phaffii yeast chromosome are used.

The resulting transformants are tested for the presence of the target gene in the chromosomal DNA and the ability to exhibit phytase activity.

The invention is illustrated by the following figures.

FIG. 1. Expression cassette

FIG. 2. Electropherogram of the PCR fragment corresponding to the m-phyCt gene

FIG. 3. Resistance of C. turicensis phytase to the action of proteolytic enzymes (pepsin and trypsin).

Example 1 Construction of a K. phaffii yeast transformant producing C. turicensis phytase

When constructing an integrative expression cassette, the "fusion-PCR" method is used [Gene., 1989, 77 (1), 61-68.].

The sequence of the m-phyCt gene, presented in the international GenBank database under the number MT489463 and encoding phytase from C. turicensis, was synthesized by the method described in [J. Microbiol. Methods, 2010, 81 (2), 147-152.]. A DNA sequence is obtained, at the 3 'and 5' ends of which the EcoRI and NotI restriction sites are provided, listed in the sequence listing under the number SEQ ID NO: 1.

The resulting DNA sequence is inserted into the expression cassette (Fig. 1), which includes the following genetic elements:

- gene m-phyCt, inserted into a single reading frame with the nucleotide sequence of the signal peptide α-factor from Saccharomyces cerevisiae, under the control of the AOX1 promoter;

- transcription terminator TTAOX1;

- yeast selection marker HIS4, complementing the mutation in the HIS4 gene in the yeast K. phaffii;

- region of integration - 3'-fragment of the nucleotide sequence of the AOX1 gene.

The resulting integrative expression cassette is transformed into the K. phaffii Y-2837 strain, which is preliminarily grown in a liquid YP nutrient medium (wt%: yeast extract - 1, peptone - 2, water - the rest) with the addition of glucose (2 wt%) to concentration of 1 × 10 ⁸ cells per 1 ml. The cells are separated by centrifugation, washed in ice-cold sterile water, and then in ice-cold 1M sorbitol solution. The cells are then incubated in 25 mM dithiothreitol solution for 15 minutes and washed in ice-cold 1 M sorbitol solution. Then the cells are resuspended in an ice-cold solution of 1 M sorbitol at a concentration of 1-5 × 10 ⁹ cells per ml. An aliquot of 40 μl of the cell suspension is transferred into a chilled Eppendorf, 400 ng of DNA expression integration cassette is added, and incubated on ice for 5 minutes. The mixture of cells and DNA is transferred into a pre-cooled electroporation cuvette. Electroporation is carried out under the following conditions: 1.5 kV, 400 Ohm, 25 uF. After poration add 1 ml of ice-cold solution of 1M sorbitol.

The selection of transformants is carried out for 5 days at a temperature of 30 ° C on an agar medium of the following composition (wt.%): Na ₂ HPO ₄ - 0.6; KH ₂ PO ₄ 0.3; NaCl - 0.05; NH ₄ Cl - 0.1; MgSO ₄ 7H ₂ O 0.065; agar - 2; glucose - 2; CaCl ₂ - 0.07; biotin, mg - 0.0002; calcium pantothenate - 0.04; folic acid - 0.0002; niacin - 0.04; p-aminobenzoic acid - 0.02; pyridoxine hydrochloride - 0.04; riboflavin - 0.02; thiamine hydrochloride - 0.04; boric acid - 0.05; CuSO ₄ 0.004; KJ 0.01; FeCl ₃ - 0.02; sodium molybdate - 0.02; ZnSO ₄ - 0.04, water - the rest.

The presence of the target gene in the chromosomal DNA of the obtained transformants is determined by PCR using the primers PhyCt-F (5'-gaagtttctgacgaaatgaa-3 '), PhyCt-R (5'-ttatctgttgatgtgagcca-3').

PCR reaction mode: 94 ° С - 3 min - 1 cycle; 30 cycles: 94 ° C - 30 s, 57 ° C - 30 s, 72 ° C - 90 s; 72 ° C - 5 min - 1 cycle.

To control the size of the amplified DNA fragment during electrophoresis, a molecular marker GeneRuler 1 kb DNA Ladder (Fermentas) is used (line 2, Fig. 2, the size of the fragments from bottom to top 10000, 8000, 6000, 5000, 4000, 3500, 3000, 2500, 2000, 1500 , 1000, 750, 500, 250 bp).

The presence of a PCR fragment with a size of 1245 bp. (line 1 of Fig. 2) confirms the presence of the m-phyCt gene encoding phytase from C. turicensis in the chromosome of the obtained transformants.

Example 2. Evaluation of the level of phytase activity of transformants of the yeast Komagataella phaffii

The selection of the most productive transformant is carried out by cultivation in a liquid nutrient medium according to the following scheme.

The seed culture of each of the transformants is separately grown in test tubes (50 ml) with 5 ml of liquid YP nutrient medium supplemented with glucose (2 wt%) at 30 ° C for 24 h on a shaker (250 rpm). The inoculation of the fermentation medium is carried out in a ratio of 1/10 for each transformant separately.

Fermentation is carried out at 30 ° C on a shaker (250 rpm) in YP nutrient medium supplemented with 1% glucose for 24 hours. Phytase gene expression is induced by adding methanol to a final concentration of 1% every 24 hours for 2 days. The productivity of each of the obtained K. phaffii transformants is assessed by the activity of phytase accumulated in the culture fluid. For this, the cells of the transformant are precipitated by centrifugation, and the supernatant is used to determine the phytase activity by the modified Fiske-Subarrow method [J. Microbiol. Methods, 1999, 39 (1), 17-22.].

The phytase activity values presented in the table indicate a rather high level of productivity of the obtained K. Phaffii transformants for C. turicensis phytase, as well as the effective expression of PhyCt phytase in the yeast K. phaffii.

The most productive transformant selected according to the results of fermentation No. 10, which synthesizes phytase in an amount of 382 U / ml, is deposited in the Bioresource Center All-Russian Collection of Industrial Microorganisms (BRC VKPM) NRC "Kurchatov Institute" - GosNIIgenetics (117545 Moscow, 1st Dorozhniy proezd, d. . 1) as Komagataella phaffii Cron10 VKPM Y-4840.

The strain is characterized by the following features

Cultural and morphological characteristics

When cultured at a temperature of 28 ° C for 48 hours on agar YP medium of the following composition (wt%: peptone - 2, yeast extract - 1, agar - 2, water - the rest) with the addition of glucose (2 wt%), cells have an oval shape, 3-4 µm in diameter. The cells bud, while budding is true, versatile. True mycelium is not formed. Sporulation occurs when the culture is incubated on an agar medium of the following composition (wt%): potassium chloride - 1.0, sodium acetate - 0.5, glucose - 1.0, agar - 2.0, water - the rest. Asci have a tetrahedral shape, include 4 ascospores.

On agar medium YP with the addition of glucose (2 wt%), colonies of light beige color with a smooth edge, matte surface, lenticular profile, and pasty consistency. When growing in a liquid YP medium with the addition of glucose (2 wt%), at 28 ° C for 24 h of cultivation, the liquid is turbid, the precipitate is white, no coagulation is observed, and no parietal films are formed.

Physiological and biochemical signs

Able to grow in both aerobic and anaerobic conditions.

It is capable of using methanol, ethanol, glucose, glycerin, lactate, succinate as the only source of carbon; it is unable to assimilate maltose, sucrose, acetate, starch, lactose.

When cultivated in the presence of methanol, it is able to synthesize phytase.

Example 3. Resistance of PhyCt phytase, synthesized by the claimed transformant, to the action of proteases (pepsin and trypsin)

At the end of the fermentation process, carried out as described in example 2, the cells of the claimed transformant are precipitated by centrifugation, and the supernatant is used to isolate and purify phytase by cation exchange chromatography [Biotechnology. 2019, 35 (4), 33-41.]. The purified enzyme is incubated in 0.25 M glycine-HCl buffer (pH 2.0) with pepsin or in 0.25 M Tris-HCl buffer (pH 7.0) with trypsin at 37 ° C. The protease / enzyme ratio is 0.1 (by weight). Protease resistance is assessed by measuring the residual relative phytase activity by a modified Fiske-Subarrow method [J. Microbiol. Methods, 1999, 39 (1), 17-22.] After incubation of the enzyme for 10, 30 and 60 minutes.

FIG. 3 shows the relative residual activity of PhyCt phytase under the action of proteases (pepsin and trypsin) for 1 hour at 37 ° C and a protease / enzyme ratio of 0.1 (by weight). The initial value of phytase activity is taken as 100%.

The presented results show that the residual phytase activity of PhyCt phytase under the action of trypsin is almost 100%, and under the action of pepsin - 80%, which is not inferior to the corresponding indicators under the same conditions (time and temperature) for known commercial phytases or exceeds them.

For example, the most trypsin-resistant phytase from Y. intermedia retains 90% of its activity [Biochem. Biophys. Res. Commun., 2006, 350 (4), 884-889.].

Commercial phytase from A. niger under the influence of trypsin loses 60% of its activity already at a trypsin / phytase ratio of 0.01 (by weight). [Arch Biochem Biophys. 1999, 365 (2): 262-7.], And under the influence of pepsin, depending on the ratio of pepsin / enzyme, loses 49-61% of the activity [In: International Phytase Summit. 2012, AB Vista, 96-107].

A known commercial phytase from Escherichia coli, when exposed to pepsin (at different protease / enzyme ratios), exhibits a residual activity of 93-97% [In: International Phytase Summit. 2012, AB Vista, 96-107], but when exposed to trypsin (even with a protease / enzyme ratio of 0.025) its residual activity is 80% [Arch Biochem Biophys. 1999, 365 (2): 262-7.].

It is known that recombinant phytase from E. coli, expressed in K. phaffii (P. pastoris), loses about 70% of its activity when exposed to pepsin already at a protease / phytase ratio of 0.002 (by weight) [J Agric Food Chem 2013, 61 (25): 6007-6015.].

The high resistance of the PhyCt phytase produced by the claimed transformant to the action of proteolytic enzymes makes it attractive for industrial use, in particular, for use as a feed additive.

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Sequence listing

<110> Federal State Budgetary Institution "State Scientific

Research Institute of Genetics and Selection of Industrial Microorganisms

National Research Center "Kurchatov Institute" (NRC

"Kurchatov Institute" - GosNIIgenetics).

State Research Institute for Genetics and Selection of Industrial

Microorganisms of National Research Center "Kurchatov Institute" (NRC

"Kurchatov Institute" - GOSNIIGENETIKA).

<120> The transformant yeast Komagataella phaffii, producing phytase Cronobacter

turicensis

<130> 20-04

<160> 1

<210> 1

<211> 1263

<212> DNA

<213> Artificial Sequence

<220>

<223> genem-phyCt,phytase coding PhyCt bacteriaCronobacter turicensis

<400> 1

aagaattcga agtttctgac gaaatgaagt tggaaagagt tgttatcgtt tctagacacg 60

gtgttagagc tccaactaag ttcactccat tgatgcaaga agttactcca tacccatggc 120

cacaatggga cgttccattg ggttggttga ctactagagg tggtgaattg gttactgaat 180

tgggtagata ccaaaagtct gttttcgttg acaagggtat cttggaaggt aacgaatgtc 240

catctccaga acaagttgct gttatcgctg acactgacca aagaactaga aagactggtg 300

aagctttctt ggctggtttc gctccaggtt gtcaaaacaa ggttcactac caaaaggaac 360

acgacaagaa ggacccattg ttcaacccgg ttaagacggg tgtgtgtagc ttcaacgttt 420

ctaagactca agaagctatc ttgactagag ctggtggtaa cgttgaaaga tacactcaaa 480

gatacgactc tgctttcaga actttggaac aagttttgaa cttctctcaa tctgctgctt 540

gtaagtctac tagacaacca ggctgcactc tcccaggtgc tctcccatct gaattgaaga 600

cttcttctga caacgtttct ttgtctggtg cttggtcttt gtcttctatg ttgactgaaa 660

tcttcttgtt gcaagaagct caaggtatgc cagaagttgc ttggggtaga atccacggtg 720

aaaaggaatg gactgaattg ttgtctttgc acaacgctca attcgacttg ttgcaaagaa 780

ctccagaagt tgctagaact agagctactc cattgttgga cttgatctct agagctttgg 840

tttctaacgg ttctactgaa aaccactacg gtatcaagtt gccagtttct ttgttgttca 900

tcgctggtca cgacactaac ttggctaact tgtctggtgc tttcgacttg aactggtctt 960

tgccaggtca accagacaac actccaccag gcggcgaatt ggtcttcgag agatggaaga 1020

gagtttctga caacactgac tggattcaag tttctttcgt ttaccagact ctccaacaaa 1080

tgcgtgagtt caagccatct tcttcttctc catctccaca taagatagtt ttgactctcc 1140

catcttgtca ggacaagaac ccagaaggta tgtgcagctt gaagggtttc aatgacatcg 1200

ttcaaagagt tagaatccca caatgtgctg ttatggctca catcaacaga taagcggccg 1260

ctt 1263

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Claims (1)

Yeast transformant Komagataella phaffii, producing Cronobacter turicensis phytase and containing the DNA sequence of the m-phyCt gene encoding Cronobacter turicensis phytase, or one of the variants of such a sequence due to the degeneracy of the genetic code.

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