RU2728240C1 - Method for producing secreted fully functional phospholipase a2 in saccharomyces cerevisiae yeast, a precursor protein for implementing said method (versions) - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: method is developed to obtain in Saccharomyces cerevisiae yeast secreted fully functional phospholipase A2, including phospholipase A2 sequence of strain Streptomyces violaceoruber A-2688, including carrier mutation S31A, S82A and N108Q, and containing at N-end additional dipeptide, consisting of amino acid residues of serine and glycine. Proposed method involves biosynthesis of the precursor of the target protein, which is a version of phospholipase A2, containing an intein insertion between the glycine residue in position 75 and the serine residue in position 76. Invention also includes four precursor proteins which are encoded by sequences SEQ ID N: 1–4, respectively.EFFECT: removing the intein insertion and converting the precursor protein into the active target protein by autocatalytic processing.5 cl, 11 ex, 1 tbl, 2 dwg

Description

The invention relates to the field of biotechnology and concerns the development of a method for producing a fully functional phospholipase A2 secreted in yeast.

Phospholipase A2 (Pla2) catalyzes the hydrolysis of the ester bond in the second position of glycerophospholipids (phospholipids) - compounds that are the main components of the membranes of all living cells and for this reason are contained in many products of animal and plant origin. The variety of molecular forms of phospholipids is determined by the type of phosphate group (choline, ethanolamine, inositol, or series) and the structure of fatty acid residues.

& Dennis, 1982]. Другими областями применения Pla2 являются текстильная промышленность, кормопроизводство, а также синтез искусственных фосфолипидов, востребованных, в частности, в фармакологии и парфюмерии [De Maria et al., 2007; Hoogevest & Wendel, 2014; Liu et al., 2015]. Интенсивное использование Pla2 ожидают в топливно-энергетической промышленности для получения биодизеля [Cesarini et al., 2015].Pla2 is one of the demanded enzymes in the enzyme preparations (FP) market. In the food industry, it is used to improve the quality of mayonnaise, degumming vegetable oils, making bakery products, dairy products, and also in the production of cheese [Baklanov 2008;

& Dennis, 1982]. Other areas of application of Pla2 are the textile industry, feed production, as well as the synthesis of artificial phospholipids, which are in demand, in particular, in pharmacology and perfumery [De Maria et al., 2007; Hoogevest & Wendel, 2014; Liu et al., 2015]. Intensive use of Pla2 is expected in the fuel and energy industry for biodiesel production [Cesarini et al., 2015].

Among the FPs containing Pla2, the following are best known:

• EP Maxapal ^® A2 (DSM) obtained by microbiological synthesis using a recombinant strain of Aspergillus niger fungi,

• FP "DENAZYME PLA2" or "PLA2 Nagase" (Nagase Corporation), which is based on Pla2 Streptomyces violaceoruber, obtained by microbiological synthesis using producers of the genus Streptomyces;

• FP "Lecitase L10" (Novozymes A / S), obtained by purification from the pancreas of pigs.

FP Pla2 is obtained using natural or recombinant producer strains. However, regardless of the origin, commercial Pla2 EPs are characterized by a high cost, which is due to low production resulting from the features of the biosynthesis of this enzyme, in particular, from its ability to bind and hydrolyze phospholipids of cell membranes, causing their destruction, leading to the death of producing cells.

The advantages of natural Pla2 producers are due to their relatively good adaptation to the biosynthesis of this toxic enzyme [Sugiyama et. al. 2002; WO 2004/097012].

One of the adaptation mechanisms is illustrated by fungal Pla2 producers. Most of them synthesize secreted Pla2 in the form of immature pro-enzymes with reduced activity. Their activation is carried out intracellularly at the later stages of secretion under the action of specialized proteinases. As a result, mature, fully active Pla2 emerge from the cells into the culture medium [WO 2004/097012]. Such a strategy for the biosynthesis of toxic proteins, which, in the event of a premature activation, can cause damage to producing cells, is widespread in nature and is also traditional for use in biotechnology.

A related process for the production of toxic proteins is also known. It differs from the previous one in that the producing cells do not provide intracellular maturation and secrete pro-enzymes into the extracellular environment, which remain in an inactive or weakly active state for an indefinitely long time until the moment of activation. In this case, pro-enzymes are called zymogens ¹ , ( ¹ Zymogen [Greek zyme - ferment and genes - generative, emerging] - functionally inactive or weakly active zymogen, which is activated upon post-translational removal of a fragment from the molecule that contributes to maintaining a latent state ( for example, the pepsinogen zymogen is converted into the digestive enzyme pepsin upon splitting into the corresponding peptides)) and their activation is carried out outside the cells under the action of other enzymes, or autocatalytically when external conditions change (for example, pH). In the latter case, we speak of a self-activated zymogen. In particular, animal coagulation factors are produced in the form of inactive zymogens [Stubbs & Bode 1994]. The production of phospholipases A2 in the form of natural or artificial zymogens has not yet been known.

Another way of natural adaptation of organisms to Pla2 biosynthesis is the change in the phospholipid composition of cell membranes and the exclusion from them of the components to which the produced enzyme exhibits the greatest affinity. As a result of adaptation, the toxic effect of Pla2 on the host cell decreases and, as a consequence, it becomes possible to increase the production of the enzyme [Sugiyama et al., 2002].

Even with adaptation, the use of natural strains for Pla2 production comes with economic constraints. As a rule, to achieve a high yield of the enzyme, the producer strain must be cultivated using special equipment and complex media containing, among other things, a fat component [Valero, 2012]. In this case, the host organism, as a rule, synthesizes many isoforms not only of the target Pla2, but also of other lipases that change the specificity and selectivity of EP and can worsen its properties, and deep purification of the target enzyme seems to be economically unjustified.

In this regard, the optimal way to increase the production of high-quality EP is the development of recombinant technologies that provide the expression of the target Pla2 in the cells of a specially selected recipient microorganism.

We will distinguish between recombinant derivatives of natural enzymes, the amino acid sequence of which is identical to natural prototypes and does not contain purposefully introduced artificial modifications, and recombinant derivatives of natural enzymes, the amino acid sequence of which was artificially modified as a result of genetically engineered manipulations and is not identical to natural prototypes. We will call the first RPM (-), and the second RPM (+) natural enzymes.

We will also distinguish between the specific activity of Pla2, detected under special conditions, for example, at constant pH, in the presence of a high concentration of calcium ions Ca ²⁺ on a lecithin substrate, solubilized using a surfactant solvent "Triton X-100", as well as the specific activity detected on egg yolk that does not contain any extraneous additives. The first will be called "special", and the second - "own" activity Pla2. Some Pla2 have high "special" and low "intrinsic" activity (Cheperegin et al. 2019). At the same time, the effect of Pla2 on cells in the process of biosynthesis and the prospects for industrial application are determined by the level of “intrinsic” rather than “special” activity, and a fully functional RPM (+) Pla2 should have indicators of “special” and “intrinsic” activity similar to indicators of a natural enzyme.

Currently, yeast maintains the status of efficient and advanced eukaryotic gene expression systems [Gasser et al. 2013; Singh et al. 2016; Zahrl et al. 2017; Raveendran et al. 2018]. They have a low level of production of their own secreted proteins, but at the same time they are able to provide effective secretion into the culture medium of target recombinant products.

It is known that, lacking its own A2 phospholipases, yeast is capable of producing RPM (-) natural enzymes. In particular, it is known about the production of RPM (-) Pla2 in yeast of the Indian cobra Naja naja naja [Lefkowitz et al. 1999] and RPM (-) Pla2 bacterial strain 2917 S. violaceoruber [Liu et al. 2015]. However, these RPM (-) Pla2 recorded a low "special" activity, 50-80 U / mg [Lefkowitz et al. 1999] and 170 U / mg [Liu et al., 2015], which is significantly lower than the indicators of highly active Pla2 synthesized by natural producers, strain S. violaceoruber A-2688 - 1000 U / mg [Sugiyama et. al. 2002; http://wayback.archive-it.org/7993/20171031055659/ https://www.fda.gov/downloads/Food/IngredientsPackadngLabeling/GRAS/NoticeInventon ^, /UCM263914.pdf] or mushroom strains - 648 U / mg and above [WO 2004/097012], which form the basis of commercial preparations. In addition, it has been shown that RPM (-) Pla2 of bacterial strain 2917 S. violaceoruber has a close to zero indicator of "intrinsic" activity [Cheperegin et al. 2019]. In this regard, even in spite of the high production of 30 and 200 mg / l, respectively, the indicated RPM (-) Pla2 should be classified as low-activity enzymes, and their potential industrial application should be classified as compromise technologies.

It is known about the efficient expression in yeast of highly active RPM (+) Pla2 [Cheperegin et al. 2019]. However, due to the changes introduced, these RPM (+) acquire high "intrinsic" activity only in the presence of additional amounts of calcium ions Ca ²⁺ [Cheperegin et al. 2019]. In the absence of additional amounts of calcium, these RPM (+) Pla2 are low-activity enzymes that require trade-off technologies for their use.

No examples of the development of yeast-based producers of RPM (-) or RPM (+) Pla2 with high "intrinsic" activity have been found in open sources of information. At the same time, it is they who are in demand for industrial production.

Natural Pla2 of S. violaceoruber bacteria is a unique enzyme that does not have “polluting” lipase activity, and therefore its use reduces losses during degumming of oils [Liu et al., 2015]. From information sources, two natural Pla2 S. violaceoruber are known: highly active Pla2 strain A-2688 [Sugiyama et. al., 2002], hereinafter called Pla22 [Cheperegin et al. 2019], and low-activity Pla2 strain 2917 [Liu et al., 2015]. Information on expression in yeast has been disclosed for low-activity Pla2 [Liu et al., 2015], as well as for RPM (+) Pla22 [Cheperegin et al. 2019], on the basis of which a non-glycosylated variant of Pla22ng was developed, carrying mutations Ser31Ala, Ser82Ala and Asn108Gln in the sites of N-glycosylation [Cheperegin et al. 2019].

The sequences of the Pla22 and Pla22ng variants of phospholipase A2 are used as the basis for the development of the claimed invention. The preparation of natural highly active phospholipase A2 strain S. violaceoruber A-2688 [Sugiyama et. al., 2002], is produced in the form of a commercial preparation "Pla2 Nagase".

The objective of the claimed group of inventions is to develop a method for producing secreted fully functional phospholipase A2 in the yeast Saccharomyces cerevisiae.

The problem was solved by:

- development of a method for obtaining a secreted fully functional phospholipase A2, carried out by obtaining and introducing into cells of the yeast Saccharomyces cerevisiae a genetic construct encoding a precursor protein, including a sequence of native, containing N-glycosylation sites, or mutant, not containing N-non-glycosylation sites, phospholipase A2 strain S. violaceoruber A-2688 containing an intein insertion between the glycine residue at position 75 and the serine residue at position 76, as well as having an additional dipeptide at the N-terminus consisting of serine and glycine amino acid residues, followed by biosynthesis of this precursor protein and its autocatalytic processing with the formation of the target product;

- obtaining a genetic construct encoding a precursor protein containing an insertion of the intein PRP8 of the Penicillium chrysogenum VKPM F-3 strain as part of the native phospholipase A2 of the Streptomyces violaceoruber A-2688 strain, to implement the proposed method;

- obtaining a genetic construct encoding a precursor protein containing an insertion of the intein PRP8 of the Penicillium chrysogenum VKPM F-3 strain carrying the Cys11Tyr mutation, as part of the native phospholipase A2 of the Streptomyces violaceoruber A-2688 strain, for the implementation of the claimed method;

- obtaining a genetic construct encoding a precursor protein containing an insertion of the PRP8 intein of the Neosartorya aurata NRRL 4378 strain as part of the A2 phospholipase of the Streptomyces violaceoruber A-2688 strain carrying the Ser31Ala, Ser82Ala and Asn108Gln mutations, to implement the claimed method;

- obtaining a genetic construct encoding a precursor protein containing an insertion of the intein PRP8 of the Penicillium chrysogenum VKPM F-3 strain carrying the Cys11Tyr mutation as part of the phospholipase A2 of the Streptomyces violaceoruber A-2688 strain carrying the Ser31Ala, Ser82Gla and Asn8 mutations.

Method for producing secreted fully functional phospholipase A2

The solution to the problem includes the following steps:

- using the methods of molecular biology and genetic engineering, clone the structural gene of the PRP8 intein of the P. chrysogenum (Int4) strain, the PRP8 intein of the P. chrysogenum strain carrying the Cys11Tyr mutation (Int4Y), and the PRP8 intein of the N. aurata NRRL 4378 (Int5) strain;

- using a structural gene encoding RPM (-) Pla22 [Cheperegin et al. 2019], genetic constructs SEQ ID N1 and SEQ ID N2 are obtained, encoding a precursor protein for obtaining phospholipase A2 by autocatalytic processing, which is a glycosylated phospholipase A2 of the S. violaceoruber A-2688 strain containing an insertion of intein Int4 or intein Int4Y, respectively, between a glycine residue at position 75 and a serine residue at position 76 and having an additional dipeptide at the N-terminus, consisting of the amino acid residues of serine and glycine;

- using the structural gene encoding RPM (+) Pla22ng [Cheperegin et al. 2019], the genetic constructs SEQ ID N3 and SEQ ID N4 are obtained, encoding a precursor protein for obtaining phospholipase A2 by autocatalytic processing, which is a non-glycosylated phospholipase A2 of the S. violaceoruber A-2688 strain carrying mutations S31A, S82A and N108Q and containing an insertion Int5 or intein Int4Y, respectively, between the glycine residue at position 75 and the serine residue at position 76 and having an additional dipeptide at the N-terminus consisting of the amino acid residues of serine and glycine;

- the obtained genetic constructs SEQ ID N1, SEQ ID N2, SEQ ID N3 and SEQ ID N4 are introduced into the cells of the yeast S. cerevisiae. To do this, use recipient strains, vectors, promoters, leader regions, which are identical or similar to those described earlier [Cheperegin et al. 2019, RU 2460795 and RU 2522479] and are not essential features of the invention. As a result, producing strains of the claimed precursor proteins are obtained;

- carry out the cultivation of each of the obtained strains of yeast containing genetic constructs SEQ ID N1, SEQ ID N2, SEQ ID N3 and SEQ ID N4, for which the conditions are used to ensure the expression of these constructs, identical or similar to those described earlier [Cheperegin et al. 2019, RU 2460795 and RU 2522479], which are not essential features of the invention. As a result of cultivation of each strain, a culture liquid is obtained containing the secreted inventive precursor protein, which forms the target product as a result of autocatalytic processing; analysis of the activity of which proves that it is a fully functional phospholipase A2.

The invention is illustrated by the following figures:

FIG. 1 - Scheme for the construction of DNA fragments encoding the claimed precursor proteins. Legend: pINT - plasmid pInt4, pInt4Y or pInt5; INT-DNA sequence encoding intein Int4, Int4Y or Int5; PlaN and PlaC - DNA sequences encoding the N-terminal (from 1 to 75 residue) and C-terminal (from 76 to 122 residue) parts of phospholipase A2 as part of the claimed precursor proteins; pPla - plasmids pUC18x-Pla22 or pUC18x-Pla22ng; Pla-xx - DNA sequence encoding the claimed precursor protein

FIG. 2. PAGE-electrophoregram of proteins of the culture fluid of yeast strains D-Pla22ng (Int5) and D-Pla22ng (Int4Y) before (-) and after (+) processing carried out for 69 hours at room temperature. The position of protein bands corresponding to precursor proteins (Pla22ng (Int)), inteins Int5 and Int4Y, as well as mature phospholipase A2 (Pla22ng) is shown.

Example 1. Cloning of the Int4 intein structural gene

A DNA fragment encoding the P. chrysogenum mini-intein PRP8 (GeneBank AM042015.1), called Int4, was obtained using PCR. PCR was performed using Phusion DNA polymerase (ThermoFisher). The matrix is the total genomic DNA of the P. chrysogenum VKPM F-3 strain, purified according to the standard method [Kaiser et. al., 1994]. The DNA fragment encoding Int4 is amplified using primers:

After opening the ends using the restriction enzymes NcoI and XhoI, the amplified DNA fragments are cloned into the laboratory vector pUC19mx, a derivative of the standard vector pUC19 containing the modified polylinker surrounded by the recognition sites of the restriction enzymes NcoI and XhoI.

As a result of cloning, plasmid pInt4 containing the sequence of the structural gene of intein Int4 is obtained.

Example 2. Cloning of the Int4Y intein structural gene

A DNA fragment encoding a mutant variant of the P. chrysogenum mini-intein PRP8 (GeneBank AM042015.1), called Int4Y, was cloned as described in example 1, but primers were used for amplification:

As a result of cloning, plasmid pInt4Y is obtained containing the sequence of the structural gene of the mutant intein Int4Y containing the Cys11Tyr substitution.

Example 3. Synthesis of the structural gene of intein Int5

The DNA fragment SEQ ID N1 encoding the Nau PRP8 intein of the N. aurata NRRL 4378 strain, called Int5, is obtained as a synthetic BamHI / XhoI DNA fragment. This fragment is cloned into the laboratory plasmid pUC18x. The result is a plasmid pInt5 containing the sequence of the structural gene of intein Int5.

Example 4. Construction of the Pla22 gene

A DNA fragment enclosing the structural gene of phospholipase A2, called Pla22, coding for the amino acid sequence of mature phospholipase A2 of S. violaceoruber A-2688 (GenBank AEM88445) was obtained using PCR. The matrix for PCR is the sequence of the synthetic Pla2 gene [Cheperegin et al. 2019]. A DNA fragment containing this gene was cloned at the BamHI / XhoI sites in the pUC18x vector, which is a derivative of the standard pUC18 vector and contains the XhoI restriction endonuclease recognition site instead of the EcoRI site in the modified polylinker.

The desired DNA fragment is obtained in 2 stages. At the first stage, 2 overlapping DNA fragments are obtained using PCR:

и

- fragment 1 is obtained using a primer

and

и

- fragment 2 is obtained using a primer

and

The amplified DNA fragments are purified from the gel, and the next step is PCR-mediated ligation of the purified DNA fragments. For this purpose, PCR is carried out using a mixture of fragments 1 and 2 as a template. Amplification primers are N168 and N1272. The amplified DNA fragment is eluted from the agarose gel and, after opening the ends using the restriction enzymes BamHI and XhoI, is cloned into the plasmid pUC18x, digested at the same sites. As a result of cloning, the plasmid pUC18x-Pla22 is obtained, in which the nucleotide sequence of the cloned gene is confirmed by sequencing.

The resulting plasmid pUC18x-Pla22 contains a BamHI / XhoI DNA fragment encoding the Pla22 variant of phospholipase A2, the amino acid sequence of which contains a threonine residue at position 46 (Ser46Thr substitution) and phenylalanine and glycine residues at the C-terminus at positions 121 and 122, respectively (Leu121 substitution (PheGly)). Plasmid pUCT8x-Pla22 is used to generate the gene for the mutant phospholipase variant and to insert DNA sequences encoding inteins.

Example 5. Construction of the Pla22ng gene

A DNA fragment containing the Pla22ng structural gene encoding a mutant variant of phospholipase, from the amino acid sequence of which three N-glycosylation sites are excluded, is obtained by PCR. The DNA of the plasmid pUC18x-Pla22 was used as a template for PCR. PCR is carried out in 2 stages.

At the first stage, the sequence of the mutant gene is obtained in the form of 4 DNA fragments obtained by PCR and having pairwise overlapping ends:

и

- fragment 1 is obtained using a primer

and

и

- fragment 2 is obtained using a primer

and

и

- fragment 3 is obtained using a primer

and

и

- fragment 4 is obtained using a primer

and

The amplified DNA fragments are purified from the gel using a Qiagen kit (Qiagen, cat. # 28706).

The next step is PCR-mediated ligation of 4 purified DNA fragments. For this purpose, PCR is carried out using a mixture of fragments 1, 2, 3 and 4 as a template. Amplification primers are N1346 and N169. The amplified DNA fragment was eluted from the agarose gel and, after opening the ends using the restriction enzymes BamHI and XhoI, was cloned into the plasmid pUC18x (Example 4), digested at the same sites. As a result of cloning, the plasmid pUC18x-Pla22ng is obtained, in which the nucleotide sequence of the cloned gene is confirmed by sequencing.

The resulting plasmid pUC18x-Pla22ng contains a BamHI / XhoI DNA fragment encoding a mutant phospholipase containing 3 substitutions: Ser31Ala, Ser82Ala and Asn108Gln, inactivating all 3 potential N-glycosylation sites. The mutant phospholipase variant is called Pla22ng.

Plasmid pUC18x-Pla22ng is used to insert DNA sequences encoding inteins.

Example 6. Obtaining DNA fragments encoding the claimed intein-containing precursor proteins

The claimed proteins-precursors of the native, containing sites of N-glycosylation, phospholipase A2, containing insertions of inteins Int4 or Int4Y, are called Pla22 (Int4) and Pla22 (Int4Y), respectively. To obtain DNA sequences encoding these proteins, the plasmid pUC18x-Pla22 is used.

The claimed proteins-precursors of the mutant phospholipase A2 containing no N-non-glycosylation sites containing insertions of Int5 or Int4Y inteins are called Pla22ng (Int5) and Pla22ng (Int4Y), respectively. To obtain DNA sequences encoding these proteins, the plasmid pUC18x-Pla22ng is used.

и rev

To construct DNA fragments encoding the inventive precursor proteins containing intein insertions, the PCR-mediated ligation of overlapping DNA sequences is used according to the scheme of FIG. 1. To do this, use universal primers for dir amplification

and rev

и Р2

а для получения последовательности ДНК, кодирующей белок-предшественник Pla22ng(Int5) используют праймеры Р1

и Р2

Для ПЦР используют ДНК-полимеразу Phusion (ThermoFisher Scientific, F-#F530S).In addition to them, primers P1 are used to obtain DNA sequences encoding the precursor proteins Pla22 (Int4), Pla22 (Int4Y) and Pla22ng (Int4Y)

and P2

and to obtain the DNA sequence encoding the precursor protein Pla22ng (Int5) primers P1

and P2

For PCR, Phusion DNA polymerase (ThermoFisher Scientific, F- # F530S) was used.

As a result of PCR ligation, BamHI / XhoI DNA fragments are obtained containing the DNA sequences SEQ ID N1, SEQ ID N2, SEQ ID N3 and SEQ ID N4, encoding the claimed precursor proteins Pla22 (Int4), Pla22 (Int4Y), Pla22ng (Int5) and Pla22ng (Int4Y), respectively.

These DNA fragments are used to construct expression plasmids, producer strains and obtain corresponding variants of phospholipase A2.

Example 7. Construction of expression plasmids for the biosynthesis of the claimed precursor proteins in the yeast S. cerevisiae

Expression plasmids are constructed by ligating three DNA fragments.

Fragment 1 is the XhoI / HindIII DNA fragment of the laboratory bireplicon vector pPDX3 [RU 2460795]. This fragment contains, starting from the XhoI site: the region of the transcription termination of the CYC1 gene of the yeast S. cerevisiae; a DNA fragment providing replication and selection of plasmids pPDX3 in E. coli cells; a DNA fragment of an endogenous 2-μm yeast plasmid, providing the ability of pPDX3 plasmids to be maintained in S. cerevisiae yeast cells in an episomal multicopy state; structural genes URA3 and PGK1 of S. cerevisiae yeast, providing selective maintenance of pPDX3 plasmids in strains with corresponding chromosomal mutations [RU 2460795, RU 2515913, etc.].

Fragment 2 is the "regulatory" HindIII / BglII DNA fragment encoding the GAL1 promoter used for expression and the matHH leader region, directing the secretion of phospholipase A2 variants in S. cerevisiae yeast.

To construct a "regulatory" DNA fragment, the pUC18x-GAL1matHH-GH plasmid is used, which contains the mature somatropin gene fused with a DNA fragment encoding a leader polypeptide comprising the mat signal peptide sequences and the doubled pro-region of the S. cerevisiae yeast HSP150 protein [RU 2460795].

Construction is carried out by replacing the NcoI / XhoI DNA fragment of the plasmid pUC18x-GAL1matHH-GH encoding somatropin with a synthetic double-stranded DNA fragment containing the BglII restriction enzyme recognition site and obtained by annealing two oligonucleotides:

As a result of cloning, the plasmid pUC18x-GAL1matHH- (BglII) is obtained, in which, in the DNA sequence encoding the leader matHH polypeptide, the BglII restriction site is constructed, formed by the arginine codon, which is part of the Kex2 proteinase recognition site, and the serine codon immediately following the arginine codon ...

Plasmid pUC18x-GAL1matHH- (BglII) serves as the source of the "regulatory" HindIII / BglII DNA fragment (Fragment 2).

Fragment 3 is a BamHI / XhoI DNA fragment containing the DNA sequence SEQ ID N1 or SEQ ID N2 or SEQ ID N3 or SEQ ID N4 encoding the claimed precursor protein Pla22 (Int4), Pla22 (Int4Y), Pla22ng (Int5) or Pla22ng (Int4Y ), respectively.

In particular,

- plasmid pPDX3-Pla22 (Int4) is obtained by ligating fragments 1 and 2, as well as fragment 3 containing the DNA sequence SEQ ID N1;

- plasmid pPDX3-Pla22 (Int4Y) is obtained by ligating fragments 1 and 2, as well as fragment 3, containing the DNA sequence SEQ ID N2;

- plasmid pPDX3-Pla22ng (Int5) is obtained by ligating fragments 1 and 2, as well as fragment 3, containing the DNA sequence SEQ ID N3;

- plasmid pPDX3-Pla22ng (Int4Y) is obtained by ligating fragments 1 and 2, as well as fragment 3, containing the DNA sequence SEQ ID N4.

The resulting expression plasmids of the pPDX3 series contain DNA sequences encoding the inventive precursor proteins fused with DNA sequences encoding the S. cerevisiae yeast GAL1 promoter and the matHH leader polypeptide containing the doubled (HH) pro-region of the HSP150 protein of the yeast S cerevisiae. These plasmids are used for transformation of cells of recipient strains of S. cerevisiae yeast and production of corresponding samples of phospholipase A2.

Example 8. Construction of producer strains of the claimed precursor proteins using laboratory recipient strain beta-0 S. cerervisiae

For the construction of cell-producing strains of the laboratory recipient strain S. cerervisiae beta-0 [Cheperegin et al. 2019] are transformed using the resulting expression plasmids of the pPDX3 series. The transformation is carried out using lithium acetate as described [Gietz & Schiestl 2007]. As a result of transformation, production strains of series B are obtained. In particular,

- as a result of the transformation of the cells of the laboratory recipient strain beta-0 S. cerervisiae with the plasmid pPDX3-Pla22 (Int4), the strain B-Pla22 (Int4) is obtained, which is used for biosynthesis of the precursor protein Pla22 (Int4).

- as a result of transformation of the cells of the laboratory recipient strain beta-0 S. cerervisiae with the plasmid pPDX3-Pla22 (Int4Y), the strain B-Pla22 (Int4Y) is obtained, which is used for the biosynthesis of the precursor protein Pla22 (Int4Y).

- as a result of transformation of the cells of the laboratory recipient strain beta-0 S. cerervisiae with the plasmid pPDX3-Pla22ng (Int5), the strain B-Pla22ng (Int5) is obtained, which is used for biosynthesis of the precursor protein Pla22ng (Int5).

- as a result of transformation of the cells of the laboratory recipient strain beta-0 S. cerervisiae with the plasmid pPDX3-Pla22ng (Int4Y), strain B-Pla22ng (Int4Y) is obtained, which is used for biosynthesis of the precursor protein Pla22ng (Int4Y).

Example 9. Construction of producer strains of the claimed precursor proteins using a laboratory recipient strain S. cerervisiae D721W

To construct producer strains, cells of the laboratory recipient strain S. cerervisiae D721W [RU 2460795] are transformed using the obtained expression plasmids of the pPDX3 series. The transformation is carried out according to the method of Ito [Ito et al. 1983]. As a result of transformation, producer strains of the D series are obtained.

In particular,

- as a result of transformation of the cells of the laboratory recipient strain D721W S. cerervisiae with the plasmid pPDX3-Pla22 (Int4), the strain D-Pla22 (Int4) is obtained, which is used for the biosynthesis of the precursor protein Pla22 (Int4).

- as a result of transformation of the cells of the laboratory recipient strain D721W S. cerervisiae with the plasmid pPDX3-Pla22 (Int4Y), the strain D-Pla22 (Int4Y) is obtained, which is used for biosynthesis of the precursor protein Pla22 (Int4Y).

- as a result of transformation of the cells of the laboratory recipient strain D721W S. cerervisiae with the plasmid pPDX3-Pla22ng (Int5), the strain D-Pla22ng (Int5) is obtained, which is used for the biosynthesis of the precursor protein Pla22ng (Int5).

- as a result of the transformation of the cells of the laboratory recipient strain D721W S. cerervisiae with the plasmid pPDX3-Pla22ng (Int4Y), the strain D-Pla22ng (Int4Y) is obtained, which is used for the biosynthesis of the precursor protein Pla22ng (Int4Y).

Example 10. Obtaining the inventive precursor proteins and their processing products. To obtain the inventive precursor proteins, the designed producer strains are cultivated. To this end:

- producer strains of series B are cultured in a liquid YPDG medium (yeast extract - 1%; bactopeptone - 2%; glucose - 2%; galactose - 2%; water - the rest) for 96 h until the stationary growth phase using a rotary shaker with speed of 250 rpm at a temperature of 22 ° C.

- producer strains of the D series are cultivated in a liquid medium 223 (yeast extract - 2%; bactopeptone - 2%; glucose - 3%; water - the rest) for 96 hours before the stationary growth phase using a rotary shaker at a speed of 250 rpm at a temperature of 22 ° C.

Upon completion of the cultivation of the producer strains of both series, samples of the culture fluid are obtained, which are freed from yeast cells by centrifugation for 10 minutes at an acceleration of 16000 g and subsequent transfer of the samples over the sedimentary liquid into suitable clean containers.

As a result, yeast cell-free treated culture fluid samples are obtained, each of which contains one of the claimed precursor proteins, Pla22 (Int4) or Pla22 (Int4Y) or Pla22ng (Int5) or Pla22ng (Int4Y), which is not necessarily purified and concentrated, however, they must undergo autocatalytic processing.

FIG. 2 shows the results of autocatalytic processing of the Pla22ng (Int5) and Pla22ng (Int4Y) precursor proteins produced by the D-Pla22ng (Int5) and D-Pla22ng (Int4Y) strains, respectively, which is carried out at room temperature for 69 hours.

As a result of autocatalytic processing, samples of the target product are obtained.

Example 11. Evaluation of "special" and "own" activity of samples of the target product

The determination of the “special” activity of the samples of the target product is carried out using a pH titrator according to the method of “Instrumental determination of activity under standard conditions using a pH titrator” [Cheperegin et al. 2019]. For calibration, a sample of the commercial product Pla2 Nagase is used. As a result of the measurement, data is obtained on the value of the "special" activity of each sample of the target product, expressed in units of the activity of the commercial drug "Pla2 Nagase". In other words, data is obtained on the relative activity of each sample of the target product and the control preparation "Pla2 Nagase".

On the basis of the data obtained for each sample of the target product, a control sample "Pla2 Nagase" is prepared, the calculated value of the "special" activity of which is equal to the activity of the corresponding sample of the target product. As a result, experimental pairs are formed, consisting of a sample of the target product and a diluted control sample "Pla2 Nagase.

In each experimental pair, a repeated measurement of the "special" activity is carried out. The results of measuring the activity of the samples of the target product are expressed as a fraction of the activity of the corresponding control sample "Pla2 Nagase" and entered in table. 1.

Then, in each experimental pair, the "own" activity of the diluted control sample "Pla2 Nagase" and the sample of the target product are measured. The measurement is carried out according to the method of "Instrumental determination of activity on egg yolk" [Cheperegin et al. 2019]. As in the case of measuring the "special" activity, the results of measuring the "intrinsic" activity of the sample of the target product are expressed as a fraction of the activity of the corresponding control sample "Pla2 Nagase" and entered in the table. 1.

The results are shown in table. 1 confirm that the claimed method provides for obtaining samples of the target product with indicators of "special" and "intrinsic" activity, similar to those of the commercial drug Pla2-Nagase. The presence of “special” and “intrinsic” activity indicators in the samples of the target product, similar to those of the commercial drug Pla2-Nagase, proves that the target product is a fully functional phospholipase A2.

The advantages of the proposed method:

- microbiological synthesis of the target product is carried out using the yeast Saccharomyces cerevisiae, traditionally recognized as safe for the production of enzymes for the food industry, which include phospholipase A2;

- the method allows to obtain native phospholipase A2, that is, containing N-glycosylation sites, or mutant, that is, not containing N-glycosylation sites;

- when implementing the method, different inteins can be used, including mutant ones;

- the method does not require large labor costs, since the target product is secreted, and the process of formation of the target product from its precursor proceeds spontaneously;

- the method does not require the use of proteases, since the processing of the precursor protein is carried out on the basis of an autocatalytic mechanism;

- the method ensures the receipt of the target product, the key indicators of which, characterizing not only "special", but also "own" activity, are at the level of indicators of the commercial drug "Pla2-Nagase" and, thus, correspond to the indicators of natural highly active phospholipase A2.

Literature

Baklanov K.V. Improving the technology of high-calorie mayonnaise - M: 2008, p. 23

Cheperegin S.E., Sannikova E.P., Malysheva A.V., Klebanov F.A., Kozlov D.G. (2019) Highly active modified variants of the recombinant phospholipase A2 of Streptomyces violaceoruber for efficient biosynthesis in yeast. Biotechnology, 2019, T. 35, No. 3, S. 30-41. doi: 10.21519 / 0234-2758-2019-35-3-30-41

Cesarini S. Moving towards a Competitive Fully Enzymatic Biodiesel Process / S. Cesarini, F.I.J. Pastor, P.M. Nielsen, P. Diaz. // Sustainability. - 2015. - V.7 (6). - P. 7884-7903. doi: 10.3390 / su7067884

, Svendsen A., Patkar S. (2007). Phospholipases and their industrial applications. Appl Microbiol Biotechnol, 74: 290-300. DOI 10.1007/s00253-006-0775-xDe Maria L., Vind J.,

, Svendsen A., Patkar S. (2007). Phospholipases and their industrial applications. Appl Microbiol Biotechnol 74: 290-300. DOI 10.1007 / s00253-006-0775-x

Gasser B., Prielhofer R., Marx H., et al. Pichia pastoris: protein production host and model organism for biomedical research. Future Microbiol. 2013, 8 (2), 191-208. doi: 10.2217 / fmb. 12.133.v

Gietz R.D., Schiestl R.H. Quick and easy yeast transformation using the LiAc / SS carrier DNA / PEG method. Nat Protoc. 2007,2 (1) 35-37. doi: 10.1038 / nprot.2007.14

Hoogevest P., Wendel A. (2014). The use of natural and synthetic phospholipids as pharmaceutical excipients. Eur J Lipid Sci Technol. 116 (9): 1088-1107. doi: 10.1002 / ejlt.201400219.

Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983,153 (1): 163-168.

Kaiser et. al. (1994) Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Lefkowitz L.J., Deems R.A., Dennis E.A. (1999). Expression of Group IA Phospholipase A2 in Pichia pastoris: Identification of a Phosphatidylcholine Activator Site Using Site-Directed Mutagenesis. Biochemistry, 38, 14174-14184.

Liu A., Yu X.-W., Sha C, Xu Y. (2015). Streptomyces violaceoruber phospholipase A2: expression in Pichia pastoris, properties, and application in oil degumming. Appl Biochem Biotechnol, 17'5 (6): 3195-3206. DOI 10.1007 / s 12010-015-1492-7

A. and Dennis, E.A. (1982) Acyl and phosphoryl migration in lysophospholipids: importance in phospholipids synthesis and phospholipase activity, Biochemistry, 21: 1743-1750.

A. and Dennis, EA (1982) Acyl and phosphoryl migration in lysophospholipids: importance in phospholipids synthesis and phospholipase activity, Biochemistry, 21: 1743-1750.

Raveendran S., Parameswaran B., Ummalyma S.B., Abraham A., Mathew A.K., Madhavan A., Rebello S., Pandey A. Applications of Microbial Enzymes in Food Industry. Food Technol Biotechnol, 2018, 56 (1), 16-30. doi: 10.17113 / ftb.56.01.18.5491.

Stubbs M.T., Bode W. Coagulation factors and their inhibitors. Curr Opin Struct Biol. 1994.4 (6): 823-832. doi: 10.1016 / 0959-440x (94) 90263-1

Singh R., Kumar M., Mittal A., Mehta P.K. Microbial enzymes: industrial progress in 21st century. 3 Biotech., 2016, 6 (2), 174. doi: 10.1007 / s13205-016-0485-8.

Sugiyama M., Ohtan K., Izuhara M., Koike T., Suzuki K., Imamura S., Misaki H. (2002). A Novel Prokaryotic Phospholipase A2. Characterization, gene cloning, and solution structure. The Journal of Biological Chemistry, 277 (22): 20051-20058.

Valero F. (2012). Heterologous expression systems for lipases: A review. In Lipases and Phospholipases: Methods and Protocols, Methods in Molecular Biology; Sandoval, G., Ed .; Springer Science Business Media: New York, NY, USA, 2012; Volume 861, pp. 161-178.

, Mattanovich D., Gasser B. Systems biotechnology for protein production in Pichia pastoris. FEMS Yeast Res., 2017, 17(7), fox068. doi: 10.1093/femsyr/fox068Zahrl RJ,

, Mattanovich D., Gasser B. Systems biotechnology for protein production in Pichia pastoris. FEMS Yeast Res., 2017, 17 (7), fox068. doi: 10.1093 / femsyr / fox068

--->

Sequence listing

<110> Federal State Budgetary Institution "State Research Institute of Genetics and Selection of Industrial Microorganisms" of the National Research Center "Kurchatov Institute" (NRC "Kurchatov Institute" - GosNIIgenetics)

<120> Method for producing secreted fully functional phospholipase A2 in yeast Saccharomyces cerevisiae , precursor protein for this method (variants)

<160> 4

<210> 1

<211> 852

<212> DNA

<213> Artificial sequence for the synthesis of recombinant phospholipase A2 of Streptomyces violaceoruber strain A-2688

<220>

<221> CDS

<222> (1) ... (846)

<223> Sequence encodes precursor of the recombinant phospholipase A2

<221> misc_feature

<222> (1) ... (6)

<223> Sequence encodes additional dipeptide conjugated to the N-end of the recombinant phospholipase A2

<221> misc_feature

<222> (1) ... (241)

<223> Sequence encodes N-terminal part of the recombinant phospholipase A2

<221> misc_feature

<222> (242) ... (702)

<223> Sequence encodes native intein PRP8 of Penicillium chrysogenum

<221> misc_feature

<222> (703) ... (846)

<223> Sequence encodes C-terminal part of the recombinant phospholipase A2

<221> misc_feature

<222> (847) ... (852)

<223> Sequence encodes recognition site for endonuclease XhoI

<400> 1

TCC GGA GCT CCT GCC GAC AAG CCT CAG GTC CTG GCC AGT TTC ACC CAG 48

Ser Gly Ala Pro Ala Asp Lys Pro Gln Val Leu Ala Ser Phe Thr Gln

1 5 10 15

ACC AGT GCC AGT TCT CAG AAC GCC TGG CTG GCT GCC AAC CGT AAC CAG 96

Thr Ser Ala Ser Ser Gln Asn Ala Trp Leu Ala Ala Asn Arg Asn Gln

20 25 30

AGT GCC TGG GCT GCC TAC GAG TTC GAC TGG AGT ACC GAC TTG TGT ACA 144

Ser Ala Trp Ala Ala Tyr Glu Phe Asp Trp Ser Thr Asp Leu Cys Thr

35 40 45

CAG GCC CCT GAC AAC CCT TTC GGT TTC CCT TTC AAC ACC GCC TGT GCC 192

Gln Ala Pro Asp Asn Pro Phe Gly Phe Pro Phe Asn Thr Ala Cys Ala

50 55 60

CGT CAT GAC TTC GGT TAC CGT AAC TAC AAG GCT GCC GGT TGT CTC GCC 240

Arg His Asp Phe Gly Tyr Arg Asn Tyr Lys Ala Ala Gly Cys Leu Ala

65 70 75 80

AAG GGG ACC CGT CTC TTG CGA TGC GAT GGA ACC GAG ATC AAT GTG GAA 288

Lys Gly Thr Arg Leu Leu Arg Cys Asp Gly Thr Glu Ile Asn Val Glu

85 90 95

GAC GTG CGC GAA GGT GAC CTA CTT CTG GGT CCC GAT GGA GAG CCT CGC 336

Asp Val Arg Glu Gly Asp Leu Leu Leu Gly Pro Asp Gly Glu Pro Arg

100 105 110

CGT GCA TTC AAC ATA GTG AAT GGC ATC GAC CGC CTG TAC CGC ATC AAG 384

Arg Ala Phe Asn Ile Val Asn Gly Ile Asp Arg Leu Tyr Arg Ile Lys

115 120 125

ATC GGC GGT GAG AAA GAG GAC CTT GTG GTG ACG CCG AAC CAT ATT CTG 432

Ile Gly Gly Glu Lys Glu Asp Leu Val Val Thr Pro Asn His Ile Leu

130 135 140

GTG CTT TAT AGA GAG GAT GGT TCC AAG AAT GTG GAG AAG CAA ACG GTG 480

Val Leu Tyr Arg Glu Asp Gly Ser Lys Asn Val Glu Lys Gln Thr Val

145 150 155 160

GAG ATC ACT GCT GCC GAG TTT GCC GCG CTT TCT ACC GAG GAA AGA AGC 528

Glu Ile Thr Ala Ala Glu Phe Ala Ala Leu Ser Thr Glu Glu Arg Ser

165 170 175

CTC TAT AGT GCC TTT ACA TCT CCT AGG GCT GAG AAG GGC GCC GAT GAT 576

Leu Tyr Ser Ala Phe Thr Ser Pro Arg Ala Glu Lys Gly Ala Asp Asp

180 185 190

TCG GCT CAA ACG CAC AGT TTC AAG ATT GAG CAA GTT AGC CTC GAA TCC 624

Ser Ala Gln Thr His Ser Phe Lys Ile Glu Gln Val Ser Leu Glu Ser

195 200 205

GAG AAG ACA GAG TGG GCT GGT TTC CGA GTC GAC AAA GAT CAG CTT TAC 672

Glu Lys Thr Glu Trp Ala Gly Phe Arg Val Asp Lys Asp Gln Leu Tyr

210 215 220

CTG CGT CAT GAC TAC CTT GTC CTG CAC AAC AGT TTC GAC GCC AAC AAG 720

Leu Arg His Asp Tyr Leu Val Leu His Asn Ser Phe Asp Ala Asn Lys

225 230 235 240

AGT CGT ATC GAC AGT GCC TTC TAC GAG GAC ATG AAG CGT GTC TGT ACT 768

Ser Arg Ile Asp Ser Ala Phe Tyr Glu Asp Met Lys Arg Val Cys Thr

245 250 255

GGT TAC ACC GGT GAG AAG AAC ACC GCC TGT AAC AGT ACC GCC TGG ACC 816

Gly Tyr Thr Gly Glu Lys Asn Thr Ala Cys Asn Ser Thr Ala Trp Thr

260 265 270

TAC TAC CAG GCC GTC AAG ATC TTC GGT TAA CTCGAG 852

Tyr Tyr Gln Ala Val Lys Ile Phe Gly ***

275,280

<210> 2

<211> 852

<212> DNA

<213> Artificial sequence for the synthesis of recombinant phospholipase A2 of Streptomyces violaceoruber strain A-2688

<220>

<221> CDS

<222> (1) ... (846)

<223> Sequence encodes precursor of the recombinant phospholipase A2

<221> misc_feature

<222> (1) ... (6)

<223> Sequence encodes additional dipeptide conjugated to the N-end of the recombinant phospholipase A2

<221> misc_feature

<222> (1) ... (241)

<223> Sequence encodes N-terminal part of the recombinant phospholipase A2

<221> misc_feature

<222> (242) ... (702)

<223> Sequence encodes mutant intein PRP8 of Penicillium chrysogenum , enclosing mutation Cys11Tyr

<221> misc_feature

<222> (703) ... (846)

<223> Sequence encodes C-terminal part of the recombinant phospholipase A2

<221> misc_feature

<222> (847) ... (852)

<223> Sequence encodes recognition site for endonuclease XhoI

<400> 2

TCC GGA GCT CCT GCC GAC AAG CCT CAG GTC CTG GCC AGT TTC ACC CAG 48

Ser Gly Ala Pro Ala Asp Lys Pro Gln Val Leu Ala Ser Phe Thr Gln

1 5 10 15

ACC AGT GCC AGT TCT CAG AAC GCC TGG CTG GCT GCC AAC CGT AAC CAG 96

Thr Ser Ala Ser Ser Gln Asn Ala Trp Leu Ala Ala Asn Arg Asn Gln

20 25 30

AGT GCC TGG GCT GCC TAC GAG TTC GAC TGG AGT ACC GAC TTG TGT ACA 144

Ser Ala Trp Ala Ala Tyr Glu Phe Asp Trp Ser Thr Asp Leu Cys Thr

35 40 45

CAG GCC CCT GAC AAC CCT TTC GGT TTC CCT TTC AAC ACC GCC TGT GCC 192

Gln Ala Pro Asp Asn Pro Phe Gly Phe Pro Phe Asn Thr Ala Cys Ala

50 55 60

CGT CAT GAC TTC GGT TAC CGT AAC TAC AAG GCT GCC GGT TGT CTC GCC 240

Arg His Asp Phe Gly Tyr Arg Asn Tyr Lys Ala Ala Gly Cys Leu Ala

65 70 75 80

AAG GGG ACC CGT CTC TTG CGA TAC GAT GGA ACC GAG ATC AAT GTG GAA 288

Lys Gly Thr Arg Leu Leu Arg Tyr Asp Gly Thr Glu Ile Asn Val Glu

85 90 95

GAC GTG CGC GAA GGT GAC CTA CTT CTG GGT CCC GAT GGA GAG CCT CGC 336

Asp Val Arg Glu Gly Asp Leu Leu Leu Gly Pro Asp Gly Glu Pro Arg

100 105 110

CGT GCA TTC AAC ATA GTG AAT GGC ATC GAC CGC CTG TAC CGC ATC AAG 384

Arg Ala Phe Asn Ile Val Asn Gly Ile Asp Arg Leu Tyr Arg Ile Lys

115 120 125

ATC GGC GGT GAG AAA GAG GAC CTT GTG GTG ACG CCG AAC CAT ATT CTG 432

Ile Gly Gly Glu Lys Glu Asp Leu Val Val Thr Pro Asn His Ile Leu

130 135 140

GTG CTT TAT AGA GAG GAT GGT TCC AAG AAT GTG GAG AAG CAA ACG GTG 480

Val Leu Tyr Arg Glu Asp Gly Ser Lys Asn Val Glu Lys Gln Thr Val

145 150 155 160

GAG ATC ACT GCT GCC GAG TTT GCC GCG CTT TCT ACC GAG GAA AGA AGC 528

Glu Ile Thr Ala Ala Glu Phe Ala Ala Leu Ser Thr Glu Glu Arg Ser

165 170 175

CTC TAT AGT GCC TTT ACA TCT CCT AGG GCT GAG AAG GGC GCC GAT GAT 576

Leu Tyr Ser Ala Phe Thr Ser Pro Arg Ala Glu Lys Gly Ala Asp Asp

180 185 190

TCG GCT CAA ACG CAC AGT TTC AAG ATT GAG CAA GTT AGC CTC GAA TCC 624

Ser Ala Gln Thr His Ser Phe Lys Ile Glu Gln Val Ser Leu Glu Ser

195 200 205

GAG AAG ACA GAG TGG GCT GGT TTC CGA GTC GAC AAA GAT CAG CTT TAC 672

Glu Lys Thr Glu Trp Ala Gly Phe Arg Val Asp Lys Asp Gln Leu Tyr

210 215 220

CTG CGT CAT GAC TAC CTT GTC CTG CAC AAC AGT TTC GAC GCC AAC AAG 720

Leu Arg His Asp Tyr Leu Val Leu His Asn Ser Phe Asp Ala Asn Lys

225 230 235 240

AGT CGT ATC GAC AGT GCC TTC TAC GAG GAC ATG AAG CGT GTC TGT ACT 768

Ser Arg Ile Asp Ser Ala Phe Tyr Glu Asp Met Lys Arg Val Cys Thr

245 250 255

GGT TAC ACC GGT GAG AAG AAC ACC GCC TGT AAC AGT ACC GCC TGG ACC 816

Gly Tyr Thr Gly Glu Lys Asn Thr Ala Cys Asn Ser Thr Ala Trp Thr

260 265 270

TAC TAC CAG GCC GTC AAG ATC TTC GGT TAA CTCGAG 852

Tyr Tyr Gln Ala Val Lys Ile Phe Gly ***

275,280

<210> 3

<211> 852

<212> DNA

<213> Artificial sequence for the synthesis of recombinant phospholipase A2 of Streptomyces violaceoruber strain A-2688, enclosing mutations Ser31Ala, Ser82Ala and Asn108Gln

<220>

<221> CDS

<222> (1) ... (873)

<223> Sequence encodes precursor of the recombinant phospholipase A2

<221> misc_feature

<222> (1) ... (6)

<223> Sequence encodes additional dipeptide conjugated to the N-end of the recombinant phospholipase A2

<221> misc_feature

<222> (1) ... (241)

<223> Sequence encodes N-terminal part of the recombinant phospholipase A2

<221> misc_feature

<222> (242) ... (723)

<223> Sequence encodes mutant intein PRP8 of the strain Neosartorya aurata NRRL 4378

<221> misc_feature

<222> (724) ... (867)

<223> Sequence encodes C-terminal part of the recombinant phospholipase A2

<221> misc_feature

<222> (847) ... (852)

<223> Sequence encodes recognition site for endonuclease XhoI

<400> 3

TCC GGA GCT CCT GCC GAC AAG CCT CAG GTC CTG GCC AGT TTC ACC CAG 48

Ser Gly Ala Pro Ala Asp Lys Pro Gln Val Leu Ala Ser Phe Thr Gln

1 5 10 15

ACC AGT GCC AGT TCT CAG AAC GCC TGG CTG GCT GCC AAC CGT AAC CAG 96

Thr Ser Ala Ser Ser Gln Asn Ala Trp Leu Ala Ala Asn Arg Asn Gln

20 25 30

GCT GCC TGG GCT GCC TAC GAG TTC GAC TGG AGT ACC GAC TTG TGT ACA 144

Ala Ala Trp Ala Ala Tyr Glu Phe Asp Trp Ser Thr Asp Leu Cys Thr

35 40 45

CAG GCC CCT GAC AAC CCT TTC GGT TTC CCT TTC AAC ACC GCC TGT GCC 192

Gln Ala Pro Asp Asn Pro Phe Gly Phe Pro Phe Asn Thr Ala Cys Ala

50 55 60

CGT CAT GAC TTC GGT TAC CGT AAC TAC AAG GCT GCC GGT TGT CTC GCC 240

Arg His Asp Phe Gly Tyr Arg Asn Tyr Lys Ala Ala Gly Cys Leu Ala

65 70 75 80

AAA GGT ACT AGA TTA TTG AGA TAT GAT GGT TCT GAA ATT GAA GTT CAA 288

Lys Gly Thr Arg Leu Leu Arg Tyr Asp Gly Ser Glu Ile Glu Val Gln

85 90 95

GAT GTC AAA GAA GGT GAT TTG TTA TTG GGT CCA GAT GGA GGT CCA AGA 336

Asp Val Lys Glu Gly Asp Leu Leu Leu Gly Pro Asp Gly Gly Pro Arg

100 105 110

AGA GCT TTT AAC ATA GTT TCA GGT GAA GAT AGG TTG TAT AGA GTT AAG 384

Arg Ala Phe Asn Ile Val Ser Gly Glu Asp Arg Leu Tyr Arg Val Lys

115 120 125

ATT GAC GGT TCT GTT GAG GAT TTA GTT GTT ACT CCT AAT CAT ATC TTG 432

Ile Asp Gly Ser Val Glu Asp Leu Val Val Thr Pro Asn His Ile Leu

130 135 140

GTC TTT CAT AGA GAA CAG AAA GCT AGG GAT AAA GAA GAT GAT CAA TTG 480

Val Phe His Arg Glu Gln Lys Ala Arg Asp Lys Glu Asp Asp Gln Leu

145 150 155 160

CCA GAA TCT TAT GAC ACA GTT GAA ATG ACA GCA GCT GAG TTT GCT GCT 528

Pro Glu Ser Tyr Asp Thr Val Glu Met Thr Ala Ala Glu Phe Ala Ala

165 170 175

TTA TCT GCT GAA GAT AGA TCA AGA TAT AGA GCA TTC AGG TCT CCA TCT 576

Leu Ser Ala Glu Asp Arg Ser Arg Tyr Arg Ala Phe Arg Ser Pro Ser

180 185 190

TTC GAT TTG TCT GAA AAA GCT GTA CCT ACT AAT CAC AGA TTT GCT ATT 624

Phe Asp Leu Ser Glu Lys Ala Val Pro Thr Asn His Arg Phe Ala Ile

195 200 205

AAG GAT ATT AGA TTA GAA TTG GAG ACT ACT GAA TGG GCA GGA TTT AGA 672

Lys Asp Ile Arg Leu Glu Leu Glu Thr Thr Glu Trp Ala Gly Phe Arg

210 215 220

GTC GAT AAA GAT CAA TTG TAT TTG AGG CAT GAT TAC TTA GTT TTG CAT 720

Val Asp Lys Asp Gln Leu Tyr Leu Arg His Asp Tyr Leu Val Leu His

225 230 235 240

AAT AGT TTC GAC GCC AAC AAG GCT CGT ATC GAC AGT GCC TTC TAC GAG 768

Asn Ser Phe Asp Ala Asn Lys Ala Arg Ile Asp Ser Ala Phe Tyr Glu

245 250 255

GAC ATG AAG CGT GTC TGT ACT GGT TAC ACC GGT GAG AAG AAC ACC GCC 816

Asp Met Lys Arg Val Cys Thr Gly Tyr Thr Gly Glu Lys Asn Thr Ala

260 265 270

TGT CAG AGT ACC GCC TGG ACC TAC TAC CAG GCC GTC AAG ATC TTC GGT 864

Cys Gln Ser Thr Ala Trp Thr Tyr Tyr Gln Ala Val Lys Ile Phe Gly

275 280 285

TAA CTCGAG 873

***

<210> 4

<211> 852

<212> DNA

<213> Artificial sequence for the synthesis of recombinant phospholipase A2 of Streptomyces violaceoruber strain A-2688, enclosing mutations Ser31Ala, Ser82Ala and Asn108Gln

<220>

<221> CDS

<222> (1) ... (846)

<223> Sequence encodes precursor of the recombinant phospholipase A2

<221> misc_feature

<222> (1) ... (6)

<223> Sequence encodes additional dipeptide conjugated to the N-end of the recombinant phospholipase A2

<221> misc_feature

<222> (1) ... (241)

<223> Sequence encodes N-terminal part of the recombinant phospholipase A2

<221> misc_feature

<222> (242) ... (702)

<223> Sequence encodes mutant intein PRP8 of Penicillium chrysogenum , enclosing mutation Cys11Tyr

<221> misc_feature

<222> (703) ... (846)

<223> Sequence encodes C-terminal part of the recombinant phospholipase A2

<221> misc_feature

<222> (847) ... (852)

<223> Sequence encodes recognition site for endonuclease XhoI

<400> 4

TCC GGA GCT CCT GCC GAC AAG CCT CAG GTC CTG GCC AGT TTC ACC CAG 48

Ser Gly Ala Pro Ala Asp Lys Pro Gln Val Leu Ala Ser Phe Thr Gln

1 5 10 15

ACC AGT GCC AGT TCT CAG AAC GCC TGG CTG GCT GCC AAC CGT AAC CAG 96

Thr Ser Ala Ser Ser Gln Asn Ala Trp Leu Ala Ala Asn Arg Asn Gln

20 25 30

GCT GCC TGG GCT GCC TAC GAG TTC GAC TGG AGT ACC GAC TTG TGT ACA 144

Ala Ala Trp Ala Ala Tyr Glu Phe Asp Trp Ser Thr Asp Leu Cys Thr

35 40 45

CAG GCC CCT GAC AAC CCT TTC GGT TTC CCT TTC AAC ACC GCC TGT GCC 192

Gln Ala Pro Asp Asn Pro Phe Gly Phe Pro Phe Asn Thr Ala Cys Ala

50 55 60

CGT CAT GAC TTC GGT TAC CGT AAC TAC AAG GCT GCC GGT TGT CTC GCC 240

Arg His Asp Phe Gly Tyr Arg Asn Tyr Lys Ala Ala Gly Cys Leu Ala

65 70 75 80

AAG GGG ACC CGT CTC TTG CGA TAC GAT GGA ACC GAG ATC AAT GTG GAA 288

Lys Gly Thr Arg Leu Leu Arg Tyr Asp Gly Thr Glu Ile Asn Val Glu

85 90 95

GAC GTG CGC GAA GGT GAC CTA CTT CTG GGT CCC GAT GGA GAG CCT CGC 336

Asp Val Arg Glu Gly Asp Leu Leu Leu Gly Pro Asp Gly Glu Pro Arg

100 105 110

CGT GCA TTC AAC ATA GTG AAT GGC ATC GAC CGC CTG TAC CGC ATC AAG 384

Arg Ala Phe Asn Ile Val Asn Gly Ile Asp Arg Leu Tyr Arg Ile Lys

115 120 125

ATC GGC GGT GAG AAA GAG GAC CTT GTG GTG ACG CCG AAC CAT ATT CTG 432

Ile Gly Gly Glu Lys Glu Asp Leu Val Val Thr Pro Asn His Ile Leu

130 135 140

GTG CTT TAT AGA GAG GAT GGT TCC AAG AAT GTG GAG AAG CAA ACG GTG 480

Val Leu Tyr Arg Glu Asp Gly Ser Lys Asn Val Glu Lys Gln Thr Val

145 150 155 160

GAG ATC ACT GCT GCC GAG TTT GCC GCG CTT TCT ACC GAG GAA AGA AGC 528

Glu Ile Thr Ala Ala Glu Phe Ala Ala Leu Ser Thr Glu Glu Arg Ser

165 170 175

CTC TAT AGT GCC TTT ACA TCT CCT AGG GCT GAG AAG GGC GCC GAT GAT 576

Leu Tyr Ser Ala Phe Thr Ser Pro Arg Ala Glu Lys Gly Ala Asp Asp

180 185 190

TCG GCT CAA ACG CAC AGT TTC AAG ATT GAG CAA GTT AGC CTC GAA TCC 624

Ser Ala Gln Thr His Ser Phe Lys Ile Glu Gln Val Ser Leu Glu Ser

195 200 205

GAG AAG ACA GAG TGG GCT GGT TTC CGA GTC GAC AAA GAT CAG CTT TAC 672

Glu Lys Thr Glu Trp Ala Gly Phe Arg Val Asp Lys Asp Gln Leu Tyr

210 215 220

CTG CGT CAT GAC TAC CTT GTC CTG CAC AAC AGT TTC GAC GCC AAC AAG 720

Leu Arg His Asp Tyr Leu Val Leu His Asn Ser Phe Asp Ala Asn Lys

225 230 235 240

GCT CGT ATC GAC AGT GCC TTC TAC GAG GAC ATG AAG CGT GTC TGT ACT 768

Ala Arg Ile Asp Ser Ala Phe Tyr Glu Asp Met Lys Arg Val Cys Thr

245 250 255

GGT TAC ACC GGT GAG AAG AAC ACC GCC TGT CAG AGT ACC GCC TGG ACC 816

Gly Tyr Thr Gly Glu Lys Asn Thr Ala Cys Gln Ser Thr Ala Trp Thr

260 265 270

TAC TAC CAG GCC GTC AAG ATC TTC GGT TAA CTCGAG 852

Tyr Tyr Gln Ala Val Lys Ile Phe Gly ***

275,280

<---

Claims (5)

1. A method of obtaining a secreted fully functional phospholipase A2, carried out by obtaining and introducing into cells of the yeast Saccharomyces cerevisiae a genetic construct encoding a precursor protein comprising a sequence of a native, containing N-glycosylation sites, or mutant, containing no N-glycosylation sites, phospholipase A2 strain Streptomyces violaceoruber A-2688 containing an intein insertion between the glycine residue at position 75 and the serine residue at position 76, as well as having an additional dipeptide at the N-terminus, consisting of serine and glycine amino acid residues, followed by biosynthesis of this precursor protein and its autocatalytic processing with the formation of the target product. 2. Protein-precursor for implementing the method according to claim 1, encoded by SEQ ID N: 1 and containing the insertion of intein PRP8 of Penicillium chrysogenum VKPM F-3 in the composition of native phospholipase A2 of Streptomyces violaceoruber A-2688. 3. A precursor protein for carrying out the method according to claim 1, encoded by the sequence SEQ ID N: 2 and containing the insertion of the intein PRP8 of the Penicillium chrysogenum VKPM F-3 strain carrying the Cys11Tyr mutation, as part of the native phospholipase A2 of the Streptomyces violaceoruber A-2688 strain. 4. A precursor protein for carrying out the method according to claim 1, encoded by the sequence SEQ ID N: 3 and containing the insertion of the intein PRP8 of the Neosartorya aurata NRRL 4378 strain as part of the mutant phospholipase A2 of the Streptomyces violaceoruber A-2688 strain carrying the Ser31Ala, Ser1082Al and Asn mutations. 5. Protein-precursor for implementing the method according to claim 1, encoded by the sequence SEQ ID N: 4 and containing the insertion of intein PRP8 of the Penicillium chrysogenum VKPM F-3 strain carrying the Cys11Tyr mutation, in the composition of the phospholipase A2 of Streptomyces violaceoruber A-2688 carrying the Ser31Ala mutation , Ser82Ala and Asn108Gln.

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