RU2796447C1 - Komagataella phaffii t07/ppzl-4x-oa-xyl-asor strain capable of producing xylanase from aspergillus oryzae fungi - Google Patents

Abstract

FIELD: microbiology; biotechnology; genetic engineering.

SUBSTANCE: Komagatella phaffii T07/pPZL-4x-OA-xyl-AsOr strain was obtained as a result of genetic modification of Komagatella phaffii Т07 yeast strain. Available nutrient media are used for cultivation of the strain. The strain produces the enzyme xylanase from Aspergillus oryzae (xAOr-Fl) fungus.

EFFECT: invention makes it possible to obtain xylanase with increased resistance and stability with a high degree of efficiency from a xylanase-producing strain.

1 cl, 1 dwg, 1 tbl, 3 ex

Description

The invention relates to microbiology and biotechnology and can be used in the chemical, food, fermentation, and pharmaceutical industries.

Enzymes are used in various fields such as food production, animal feed, detergents, cosmetics, brewing and textile industries, paper industry, pharmaceuticals, and as research and development tools.

Xylanases belong to a group of various enzymes that hydrolyze the main hemicellulose of the cell wall of plant cells - xylans [1]. Xylanes are one of the main constituents of lignocellulosic biomass. The mechanism of action of xylanases is the random cleavage of β-1,4-linked D-xylopyranose units that make up the carbon skeleton of the xylan homopolymer structure. Currently, xylanases are grouped into families of glycosyl hydrolases (GH) 5, 7, 8, 10, 11, 26, 30, and 43, while the mechanisms of action of xylanases belonging to families 10 and 11 are currently the best studied [2 ].

Hemicellulases, such as xylanases, are the most important group of enzymes with wide application in industrial production, including the bioconversion of lignocellulose to biofuel, SCP (single-cell protein English microbial proteins), biobleaching, textile industry, juice clarification, wastewater treatment water and ink removal [3]. The use of xylanases in the above industries reduces the use of hazardous chemicals and thus reduces the toxic burden on the environment [4].

However, it should be noted that the large-scale production of fungal xylanase during production in direct hosts is difficult due to the low generation rate and problems of aeration in a viscous medium [5].

An urgent task today is to obtain recombinant producers with a higher efficiency of protein production in comparison with producers obtained from natural organisms.

Fungal xylanases have been shown to be more active than bacterial ones; however, most of these xylanases are effective at temperatures below 50°C and in the pH range of 4–6 [6]. Thus, for Aspergillus oryzae endo-1,4-β-xylanase, it was shown that the optimum pH is 5.0, while the optimum temperature is 60°C [7]. Expression of Aoxyn10 xylanase occurred in the extracellular space with the highest enzyme activity of 45.0 U/ml with an optimum pH of 5.5 and a temperature optimum of 60°C; however, it remained stable at pH range from 4.0 to 7.0 and at temperatures below 50 °С [8]. It is known that XynFl xylanase from Aspergillus oryzae LCl was cloned into the E. coli BL21(DE3) expression system, which showed a specific activity of 1037.3 IU/mg, which was 9.3 higher expression level than in the native organism. It was shown that the recombinant xylanase XynFl with a molecular weight of 37 kDa has activity in the pH spectrum from 3.0 to 10.0 and at temperatures from 30 to 70°C, while the pH and temperature optima were 5.0 and 30°C, respectively [9].

Known recombinant strain of yeast Penicillium canescens, producing non-inhibited endo-1,4-β-xylanase E. Known design containing the target coding sequence, including sequentially connected gene eglII, encoding a highly active endo-1,4β-glucanase II, and the gene xyIE Penicillium canescens encoding non-inhibited endo-1,4-(3-xylanase E. The invention also relates to recombinant strains of Penicillium verruculosum EX13, VKM F-4765D, and Penicillium verruculosum EX35, VKM F-4766D. These strains are intended for the production of homologous highly active endo-1 ,4-β-glucanase II and non-inhibited endo-1,4-β-xylanase E Penicillium canescens [10].

Known recombinant strain of yeast Pichia pastoris -producer of xylanase from Paenibacillus brasilensis. The invention relates to microbiology and biotechnology. A recombinant yeast strain Pichia pastoris VKPM Y-4393 producing xylanase is proposed. Said strain contains the xyl gene encoding endo-1,4-β-xylanase from Paenibacillus brasilensis. The strain produces the enzyme in the amount of 1114 units/ml of the culture liquid [11].

Known yeast strain Pichia pastoris producing xylanase from Streptomyces sp.FAl. The invention relates to a xylanase-producing strain and its use, and relates to the field of genetic engineering. The xylanase of the invention is obtained by cloning the xylanase (xyn A) gene of Streptomyces sp. FAl and expression in P. pastoris. PPICZ alpha or pPIC9k is used to construct the P. pastoris expression and transformation vector, the high-efficiency expression of Streptomyces sp.FAl-derived xylanase in P. pastoris is realized first, the fermentation in the 3-liter fermentation tank lasts 140 hours, and the total protein content of the fermented supernatant can reach 6.5 g/ml, which is the highest expression level of xylanase derived from the tenth family of Streptomyces sp. in Pichia pastoris. Recombinant xylanase is used to improve the quality of steamed buns and has an excellent effect [12].

Known yeast strain Pichia pastoris, producing xylanase from Aspergillus sulphureus. The xynB gene from Aspergillus sulphureus, encoding endo-β-1,4-xylanase, was synthesized de novo by polymerase chain reaction splicing to extend the overlap according to the codon shift of the Pichia pastoris protein. Synthetic DNA and wild-type DNA were placed under the control of the promoter of the glyceraldehyde-3-phosphate dehydrogenase (GAP) gene in the plasmid of the pGAPzαA constitutive expression vector and electrotransformed into the P. pastoris X-33 strain, respectively. Transformants screened with Zeocin were able to constitutively secrete xylanase in liquid YPD medium. The maximum yield of recombinant xylanase produced by synthetic DNA was 105 U/ml, which was about 5 times higher than when using wild-type DNA when cultured in flasks at 28°C for 3 days. The enzyme exhibited optimal activity at 50°C and pH 5.0. The residual activity remained above 90% after pretreatment of the recombinant xylanase in Na ₂ HPO ₄ -citric acid (pH 2.4) buffer for 2 hours. The xylanase activity was significantly improved by Zn ²⁺ . These biochemical characteristics suggest that recombinant xylanase has promising applications in the feed industry as an additive [13].

The closest to the claimed strain - the prototype is a strain of Pichia pastoris GS/Xyn4-11, extracellularly expressing xylanase 10 family of A. oryzae. The transformant, designated P. pastoris GS/Xyn4-11l, exhibited the highest recombinant AoXyn10 activity (named reAoXyn10) at 45.0 U/mL. Purified reAoXyn10 showed maximum activity at pH 5.5 and 60°C. It was stable in the pH range of 4.0-7.0 at 50°C and below. Its activity is not affected by multiple metal ions or EDTA, but is inhibited by Mn ²⁺ and Ba ²⁺ . Km and Vmax reAoXyn10 were 1.7 mg/mL and 817 µmol/min/mg, respectively [14].

The disadvantage of this strain is that the enzyme it produces is stable in a narrow pH range, not stable at temperatures above 50°C.

The objective of the invention is to obtain a strain - producer of xylanase from fungi of the species Aspergillus oryzae (xAOr-Fl) based on the recipient strain Komagataella phaffii T07 4x-OA-xyl-AsOr, resistant to temperature and pH conditions.

EFFECT: expansion of the range of xylanase-producing strains, increased resistance to temperature and pH conditions, simplification of strain cultivation conditions.

This task is achieved by obtaining a strain of Komagataella phaffii T07/pPZL-4x-OA-xyl-AsOr, capable of producing xylanase from fungi of the species Aspergillus oryzae (xAOr-Fl). XAOr-Fl xylanase has the ability to be active at pH from 3.0 to 9.0, as well as at 2.5 and 10.0, while the enzyme is heat-resistant, showing high activity also at 85°C.

Yeast strain Komagatella phaffii Т07 4x-OA-xyl-AsOr was obtained as a result of genetic modification of yeast strain Komagatella phaffii Т07.

Genetic engineering methods are used to obtain plasmid pPZL-4x-OA-xyl-AsOr, carrying a gene having a size of 981 bp, with a nucleotide sequence of SEQ ID NO 1, encoding xAOr-Fl xylanase with SEQ ID NO 2, fused with a hybrid signal Saccharomyces cerevisiae alpha-factor sequence with S. cerevisiae prepeptide Ostl, under the control of the alcohol oxidase AOX1 gene promoter of the Komagataella phaffii species yeast.

The initial genetic material for constructing the recombinant plasmid pPZL-4x-OA-xyl-AsOr is:

- plasmid pPICZ(alpha)-A (Invitrogen),

- artificially synthesized xAOr-Fl xylanase gene,

- lox66 and lox71 recombination sites in oligonucleotides.

The resulting plasmid pPZL-4x-OA-xyl-AsOr is characterized by the following features:

- has a size of 12199 bp;

- contains 4 copies of the artificial xAOr-Fl gene under the control of the AOXl gene promoter and terminator;

- consists of the following elements:

BleoR - a gene encoding a protein sequence that provides cell resistance to bleomycin, phleomycin and zeocin;

TEF1 promoter - a promoter that regulates the operation of the BleoR gene in yeast cells;

EM7 promoter - a promoter that regulates the operation of the BleoR gene in E. coli cells;

CYCl terminator - transcription terminator from the yeast S. cerevisiae;

ori - origin of replication of the plasmid construct;

lox66 and lox71 - recombination sites of the cre-lox system;

xylanase AsOr, a gene encoding the xAOr-Fl xylanase sequence [15] without the first amino acid methionine, and under the control of the K. phaffii AOXl gene promoter;

AOX1 promoter - promoter of the K. phaffii AOXl gene;

AOX1 terminator - K. phaffii AOXl gene terminator;

α-factor secretion signal - signal sequence of the S. cerevisiae alpha factor;

pre-Ostl - prepeptide signal sequence Ostl S. cerevisiae.

Functional map of the plasmid pPZL-4x-OA-xyl-AsOr is shown in Fig.1.

The nucleotide sequences of the synthetic gene encoding xAOr-Fl xylanase (SEQ ID NO 1) as well as the amino acid sequence of xAOr-Fl xylanase itself (SEQ ID NO 2) are shown in the sequence listing.

The proposed strain is obtained by transformation of yeast cells K. phaffii T07 with plasmid pPZL-4x-OA-xyl-AsOr by electroporation on an electroporator Gene Pulser Xcell (Bio-Rad). The selection of transformants is carried out on Yeast extract-Peptone-Dextrose (YPD) medium with zeocin (100 mg/l). Then, the selected clones are transformed with the pSH67 plasmid [16] carrying, in particular, the cre-recombinase gene and the geneticin resistance marker G418. Grown clones by subculture check for the absence of resistance to zeocin and geneticin.

XAOr-Fl xylanase produced by the claimed strain has the ability to be active at pH from 3.0 to 9.0, as well as at 2.5 and 10.0, while the enzyme is heat-resistant, showing high activity also at 85°C. The optimum of enzyme activity is 60°C; at 85°C and pH 9.0, the enzyme retains 40% of its activity.

Xylanase xAOr-Fl has a higher temperature optimum compared to the prototype, and is also highly active at higher temperatures (up to 85°C) and higher pH values up to 9.0. These parameters of the enzyme operation allow the use of enzyme preparations based on xAOr-Fl xylanase in technological processes requiring high temperatures (for example, extraction) or alkaline pH (for example, detergents).

The yeast strain Komagatella phaffii T07/pPZL-4x-OA-xyl-AsOr is characterized by the following features.

Cultural and morphological features: rounded cells, 3-4 microns in diameter. Cells bud, budding is true, multilateral. On agar medium YPD form large round white colonies, glossy with a smooth edge, convex middle.

Physiological and biochemical characteristics: The strain is capable of growth both in aerobic and anaerobic conditions. The optimum growth temperature is 30°C. Grows within the pH range of 6.0 to 10.0 with an optimum of 7.0. It can use methanol, ethanol, glucose, glycerol as the sole source of carbon.

The strain does not have an infectious and general toxic effect.

The strain is non-pathogenic and is not included in the lists given in the sanitary rules SP 1.3.2322-08; the strain does not carry dangerous genetic constructs.

The strain was identified based on whole genome sequencing analysis.

The strain is stored on YPD medium with glycerol at -70°C.

For cultivation of the strain, YPD, BMGY and other media of the following composition are used:

YPD medium (%): peptone - 2, yeast extract - 1, glucose - 2.

BMGY medium (%, w/v): yeast extract - 1, peptone - 2, YNB - 1.34, glycerol - 1. Also - biotin - 0.4 mg/ml, buffered 1/10 volume 0.1 M potassium phosphate buffer.

The yield of the enzyme in the bioreactor is 258240 IU/ml for the culture liquid. It was shown that xylanase xAOr-Fl can be active at pH from 3.0 to 9.0, and also has the ability to be active at 2.5 and 10.0, while the enzyme preparation is heat-resistant, showing high activity also at 85 °C. Enzyme activity, thermal and pH stability, temperature optimum and pH optimum have been proven by experimental methods.

Enzyme activity was determined according to GOST 31488-2012 “Enzymatic preparations. Methods for determining the enzymatic activity of xylanase” using oat xylan as a substrate with a mass fraction of 1% [17].

The proposed strain of yeast Komagatella phaffii T07/pPZL-4x-OA-xyl-AsOr - xAOr-Fl xylanase producer, has a number of advantages over known strains, which are as follows.

1. The proposed strain produces a highly active xylanase from the fungus Aspergillus oryzae - xAOr-Fl, suitable for use in the chemical, food, fermentation, pharmaceutical industries.

2. Yeast strain Komagatella phaffii T07/pPZL-4x-OA-xyl-AsOr produces xylanase xAOr-Fl, which can be active in a wide pH range: from 3.0 to 9.0, as well as at 2.5 and 10.0 , which allows the use of this strain and enzyme in various fields, as well as tools for research and development.

3. Yeast strain Komagatella phaffii T07/pPZL-4x-OA-xyl-AsOr produces a thermostable xylanase xAOr-Fl, capable of exhibiting high activity also at 85°C, which makes it possible to use this strain in production.

4. Yeast strain Komagatella phaffii T07/pPZL-4x-OA-xyl-AsOr produces 258240 IU/ml of enzyme in culture liquid under bioreactor conditions.

Since the proposed strain was obtained for the first time and the enzyme isolated from it is characterized by a unique set of properties, it can be concluded that the proposed strain meets the invention criteria "novelty" and "inventive step".

The invention is illustrated by examples of a specific implementation.

Example 1 Preparation of Plasmid pPZL-4x-OA-xyl-AsOr and Komagatella phaffii T07/pPZL-4x-OA-xyl-AsOr

To obtain the expression construct, a modified plasmid derived from the plasmid pPICZ(alpha)-A (Invitrogen) was used. In the original plasmid, the prepeptide region of the S. cerevisiae alpha factor signal sequence was replaced with the prepeptide of the S. cerevisiae Ostl signal sequence, and lox66 and lox71 recombination sites flanking the expression cassette were added. Then, the artificially synthesized xAOr-Fl gene sequence was fused by the Gibson method with the above signal sequence to secrete the product into the culture fluid. In this case, after cutting off the signal peptide in the lumen of the EPR, at the N-terminus of the protein, 4 amino acids (Glu-Ala-Glu-Ala) are found that are absent in the natural protein (and the first methionine residue of the natural protein is absent).

The expression cassette was then multiplied to 4 tandem copies by the restriction ligation method. The obtained plasmid pPZL-4x-OA-xyl-AsOr was used to transform the yeast strain Komagatella phaffii T07.

After the selection of clones releasing active xylanase into the culture liquid, the antibiotic resistance gene necessary for selection at the first stages was deleted from them. The gene was removed using the pSH67 helper plasmid encoding the Cre recombinase.

Example 2 Cultivation of Komagatella phaffii T07/pPZL-4x-OA-xyl-AsOr and Isolation of XAor-Fl Xylanase

The strain was cultivated in a ProLab bioreactor (GPC, France). The K. phaffii T07/pPZL-4x-OA-xyl-AsOr strain was plated on YPD agar medium to individual colonies and cultivated for 48 hours, and then inoculated into 5 ml of YPD medium with 200 μg/ml Zeocin and cultivated overnight at 30°C. The overnight culture was inoculated into the medium in a volume of 1:100 with YNB medium and cultivated in a shaker at 30°C and 250 rpm for 48 h.

The primary culture was aseptically introduced into a fermenter containing a saline medium with concentrations of 32.5 g/l glycerol, 9.375 g/l (NH ₄ ) ₂ SO ₄ , 1.875 g/l CaSO ₄ ⋅2H ₂ O, 0.9375 g/l NaCl , 3.75 g/l MgSO ₄ ⋅7H ₂ O, and 3.75 g/l KH ₂ PO ₄ . The fermenter was pre-sterilized for 45 minutes at 121°C.

At the start of cultivation, the temperature was 30°C at a constant air flow of 3 L/min and an initial stirrer speed of 400 rpm. The concentration of dissolved oxygen was maintained at a level of >20% by gradually increasing the stirring speed to 1200 rpm, and the pH of the medium was 5.8-6.0, using a 4M NaOH solution to adjust. Before inoculation, 2.5 ml/l of trace elements and 2.5 ml/l of vitamins were added to the medium (table 1).

XAor-Fl xylanase isolation was performed as follows. The culture fluid was separated from cells and other suspensions by centrifugation at 4000 rpm for 10 minutes, then the supernatant was purified and concentrated by tangential ultrafiltration on SartoJet equipment (Sartorius, Germany) using filters with pores of 10 kDa. All operations were performed at 4°C.

The concentrate was frozen to -70°C and then freeze-dried (Labconco, USA). The intermediate was then dissolved in 10 mM sodium phosphate buffer (pH 7.3), purified using Sephadex G-25 (Sigma-Aldrich, Germany). Next, we used the method of ion-exchange chromatography on a column (V=10 ml) with an anion exchange resin DEAE-Sepharose 6HF (Biotoolomics, UK). The column was washed with 10 mM sodium phosphate buffer (pH 7.5), the protein was eluted with a linear gradient of NaCl to 0.5 M in starting buffer. Fractions where the maximum xylolytic activity was found were pooled.

Example 3 Evaluation of Enzymatic Activity of Komagatella phaffii T07/pPZL-4x-OA-xyl-AsOr

The presence of xAor-Fl xylanase production was checked by inoculating the obtained colonies into a 24-well deep well plate, with each colony inoculated into a separate well containing 2 ml of YPGM (YPD medium with the addition of methanol) with 0.3% glucose and 1% methanol. The K. phaffii T07 strain without the inserted xylanase gene was used as a negative control. The plate was placed in a thermoshaker at 30°C and the cells were cultured at 360 rpm, with 200 µl of 10% methanol added every 24 h to each well of the plate. On the third day of cultivation, culture aliquots were taken and the cells were pelleted by centrifugation at 4000g for 5 minutes.

The enzymatic activity was tested by the release of the dye from the synthetic substrate 4-nitrophenyl-(3-0-xylopyranoside. To determine the activity, 1 volume of the culture liquid obtained after culturing the resulting clones was added to the well of the tablet, then 4 volumes of buffer (50 mM Tris- HCl, 1 mM CaCl ₂ , pH 9.0) and 5 volumes of a solution of 100 mg of 4-nitrophenyl-β-D-xylopyranoside in 10 ml of 100 mM PBS. The reaction was carried out at 60°C. The progress of the reaction was monitored using an Epoch plate spectrophotometer ( Biotek, USA Measurements were taken every 10 minutes at a wavelength of 405 nm Supernatant from a culture sample of the K. phaffii T07 strain without the inserted protease gene was used as a negative control.

XAor-Fl xylanase activity was calculated based on xylose calibration, while the molar absorption coefficient was ε ≈ 0.00045±0.00005 μmol ^-1 xcm ^-1 . It was shown that at pH 7.5 the enzyme activity was 258240 IU/ml for the culture liquid.

Information sources

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Peter Beely. Xylanases of glycoside hydrolase family 30 - An overview, Biotechnology Advances, Volume 47, 2021, 107704, ISSN 0734-9750, https://doi.org/10.1016/j.biotechadv.2021.107704.

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9. Bhardwaj N., Verma V.K., Chaturvedi V. et al. GH10 XynF1 and XynllA: the predominant xylanase identified in the profiling of extracellular proteome of Aspergillus oryzae LC1. Ann Microbiol 68, 731-742 (2018). https://doi.org/10.1007/sl3213-018-1378-3.

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14. Yin X, Gong YY, Wang JQ, Tang CD, Wu MC. Cloning and expression of a family 10 xylanase gene (AoxynlO) from Aspergillus oryzae in Pichia pastoris. J Gen Appl Microbiol. 2013;59(6):405-15. doi: 10.2323/jgam.59.405. PMID: 24492599.

15. https://www.ncbi.nlm.nih.gov/protein/094163.1?report=genbank&l og$=protalign&blast_rank=1&RID=EJ2S9 WF8016.

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SWVQGITDKDTLTEVIKNHITTIMQRYKGQIYAWDVVNEIFDEDGTLRDSVFSQVLGEDFVRIAFETARE

ADPNAKLYINDYNLDSADYAKTKGMVSYVKKWLDAGVPIDGIGSQSHYSANGFPVSGAKGALTALASTGV

SEVAVTELDIEGASSESYLEVVNACLDVSSCVGITVWGVSDKDSWRSSTSPLLFDSNYQAKDAYNAIIDA

L</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

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Claims (1)

Komagatella phaffii T07/pPZL-4x-OA-xyl-AsOr strain capable of producing xylanase from Aspergillus oryzae (xAOr-F1) species, obtained by transformation of Komagatella phaffii T07 strain with pPZL-4x-OA-xyl-AsOr plasmid carrying the gene , having the nucleotide sequence SHQ ID NO 1 and encoding xylanase xAOr-F1, having the amino acid sequence SEQ ID NO 2, fused with the hybrid signal sequence of the alpha factor of S. cerevisiae with the Ostl prepeptide of S. cerevisiae, under the control of the promoter of the alcohol oxidase AOX1 gene of the yeast species Komagataella phaffii.

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